In JoVE (1)

Other Publications (70)

Articles by Yunjun Yan in JoVE

Other articles by Yunjun Yan on PubMed

[Cloning and Overexpression of Lipase Gene from Geotrichum Candidum Y162]

Wei Sheng Wu Xue Bao = Acta Microbiologica Sinica. Feb, 2008  |  Pubmed ID: 18437999

By means of bioinformatics, we aligned nucleotide sequence of reported lipase gene from Geotrichum. Primers were designed based on the conservative nucleotide sequence, and the lipase gene of G. candidum Y162 was cloned for the first time in China. Nucleotide sequencing revealed that the open reading frame has 1692 nucleotides without any introns, encoding 563 amino acid residues including a signal sequence of 19 amino acid residues, which is 86% identical to lipase I of G. fermentans. Subsequently, we cloned the lipase gene into expression vector pPIC9K, and then transformed into Pichia pastoris GS115. Cultures of recombined P. pastoris accumulated active enzyme in the supernatant to levels of 55 U/mL after induction for 96 hours in shake flasks. The purified lipase exhibited maximum activity at 50 degrees C and pH 8.0, and was stable between pH 6.0 and 10.0 and below 60 degrees C. Lipase activity was compatible with the presence of organic solvents such as methanol, n-heptane, hexane, cyclohexane, glycerol, benzene and diethyl ether. Lipase showed hydrolysis preference for triacylglycerol substrates containing cis-9 unsaturated fatty acid. The results suggest that the lipase could be a candidate for industrial applications.

Lipase-catalyzed Biodiesel Production with Methyl Acetate As Acyl Acceptor

Zeitschrift Fur Naturforschung. C, Journal of Biosciences. Mar-Apr, 2008  |  Pubmed ID: 18533477

Biodiesel is an alternative diesel fuel made from renewable biological resources. During the process of biodiesel production, lipase-catalyzed transesterification is a crucial step. However, current techniques using methanol as acyl acceptor have lower enzymatic activity; this limits the application of such techniques in large-scale biodiesel production. Furthermore, the lipid feedstock of currently available techniques is limited. In this paper, the technique of lipase-catalyzed transesterification of five different oils for biodiesel production with methyl acetate as acyl acceptor was investigated, and the transesterification reaction conditions were optimized. The operation stability of lipase under the obtained optimal conditions was further examined. The results showed that under optimal transesterification conditions, both plant oils and animal fats led to high yields of methyl ester: cotton-seed oil, 98%; rapeseed oil, 95%; soybean oil, 91%; tea-seed oil, 92%; and lard, 95%. Crude and refined cottonseed oil or lard made no significant difference in yields of methyl ester. No loss of enzymatic activity was detected for lipase after being repeatedly used for 40 cycles (ca. 800 h), which indicates that the operational stability of lipase was fairly good under these conditions. Our results suggest that cotton-seed oil, rape-seed oil and lard might substitute soybean oil as suitable lipid feedstock for biodiesel production. Our results also show that our technique is fit for various lipid feedstocks both from plants and animals, and presents a very promising way for the large-scale biodiesel production.

[Directed Evolution of Lipase of Bacillus Pumilus YZ02 by Error-prone PCR]

Sheng Wu Gong Cheng Xue Bao = Chinese Journal of Biotechnology. Mar, 2008  |  Pubmed ID: 18589821

Random mutagenesis on Bacillus pumilus lipase YZ02 gene was conducted by using error-prone PCR strategy. Through two cycles of directed evolution, two optimum mutants BpL1-7 and BpL2-1369 with lipase activity improved 2 folds and 6 folds respectively were screened. The sequence of BpL2-1369 lipase gene showed that four nucleotides substitution, T61C, C147T, A334G and T371A have occurred, and three of them caused amino acid changes. Thus, amine acid Ser21 was changed into Pro21, Arg112 to Gly112, and Leu124 to His124. According to the 3D structure of Bacillus pumilus lipase mimicked by SWISS-MODEL Repository, three mutated amino acids were located at the third amino acid of the first alpha-helix, the turn between the fourth and fifth beta fold, and the first amino acid of the fifth beta fold, respectively. The BpL and BpL2-1369 genes were ligated into pET28a vector, and transferred into E. coli BL21 (DE3). After induced by IPTG the lipases were purified and characterized. The results showed that the specific activity of the evolved lipase was 1.31-fold than that of the wild lipase, and the Km decreased from 8.24 mmol/L to 7.17 mmol/L. The pH stability of the evolved lipase was better than wild lipase when pH>8.0.

[Strategies for Exploiting Microbial Lipase Resource and Improving Lipase Biocatalyst--a Review]

Wei Sheng Wu Xue Bao = Acta Microbiologica Sinica. Sep, 2008  |  Pubmed ID: 19062657

Lipase plays a very important role in food, pharmaceutical, fine chemical and bio-energy industries. Obtaining microbial lipase resource and improving their biocatalyst are important. Based on protein engineering and molecular techniques, directed evolution, hybrid enzyme, surface display give effective approaches to obtain novel lipase biocatalysts. By physical and chemical modification approaches such as bio-imprinting, pH memory, oriented immobilization, cross-linked enzyme crystal and lipid-coated enzyme, more broad and suitable characteristics of modified lipases are achieved. In conclusion, strategies for exploitation of microbial lipase resource and improvement of lipase biocatalyst will accelerate industrial application of lipase.

[Cell Surface Display of Yarrowia Lipolytica Lipase Lip2 in Saccharomyces Cerevisiae with A-agglutinin As Carrier Protein]

Wei Sheng Wu Xue Bao = Acta Microbiologica Sinica. Nov, 2008  |  Pubmed ID: 19149173

In order to display extracellular.lipase Lip2 from Yarrowia lipolytica on the surface of yeast Saccharomyces cerevisiae for whole cell catalysts.

Cloning and Expression of Pseudomonas Fluorescens 26-2 Lipase Gene in Pichia Pastoris and Characterizing for Transesterification

Applied Biochemistry and Biotechnology. Nov, 2009  |  Pubmed ID: 19005622

Pseudomonas lipases are important biocatalysts widely used in a variety of industrial fields. An extracellular lipase gene lipA with 1,854-bp open reading frame was cloned from Pseudomonas fluorescens 26-2. The multialignment assay of the putative amino acid and the secondary structure prediction revealed this enzyme could be classified into the lipolytic subfamily I.3 and secreted via adenosine-triphosphate-binding cassette pathway. The lipA gene was integrated into Pichia pastoris GS115, and the methanol-inducible recombinants with Mut(S) and Mut(+) phenotypes were acquired. The characteristics and the transesterification capacity shown by this enzyme suggested it is a useful biocatalyst for biodiesel preparation.

Aspergillus Niger Lipase: Heterologous Expression in Pichia Pastoris, Molecular Modeling Prediction and the Importance of the Hinge Domains at Both Sides of the Lid Domain to Interfacial Activation

Biotechnology Progress. Mar-Apr, 2009  |  Pubmed ID: 19248178

Aspergillus niger lipase (ANL) is an important biocatalyst in the food processing industry. However, there is no report of its detailed three-dimensional structure because of difficulties in crystallization. In this article, based on experimental data and bioinformational analysis results, the structural features of ANL were simulated. Firstly, two recombinant ANLs expressed in Pichia pastoris were purified to homogeneity and their corresponding secondary structure compositions were determined by circular dichroism spectra. Secondly, the primary structure, the secondary structure and the three-dimensional structure of ANL were modeled by comparison with homologous lipases with known three-dimensional structures using the BioEdit software, lipase engineering database (http://www.led.uni-stuttgart.de/), PSIPRED server and SwissModel server. The predicted molecular structure of ANL presented typical features of the alpha/beta hydrolase fold including positioning of the putative catalytic triad residues and the GXSXG signature motif. Comparison of the predicted three-dimensional structure of ANL with the X-ray three-dimensional structure of A. niger feruloyl esterase showed that the functional difference of interfacial activation between lipase and esterase was concerned with the difference in position of the lid. Our three-dimensional model of ANL helps to modify lipase structure by protein engineering, which will further expand the scope of application of ANL.

[Homologous Expression of Burkholderia Cepacia G63 Lipase Gene Based on T7 RNA Polymerase Expression System]

Sheng Wu Gong Cheng Xue Bao = Chinese Journal of Biotechnology. Feb, 2009  |  Pubmed ID: 19459326

In order to realize over-expression of Burkholderia cepacia (B. cepacia) lipase, we introduced the widely used T7 RAN polymerase expression system into B. cepacia G63 to over-express the lipase gene. By using PCR technique, we amplified the T7 RNA polymerase gene (T7 RNAP) from the BL21 (DE3) and cloned it into the suicide plasmid pJQ200SK. After that, we flanked T7 RNAP with two 500 bp homologous fragments and integrated it into the genomes of B. cepacia by tri-parental mating, so that T7 RNAP was under-controlled by lipase gene (lipA) promoter. Then, we cloned the lipA and its partner gene lipB into the vector pUCPCM and pBBR22b both or separately. Therefore, we got 7 expression plasmids pBBR22blipAB, pBBR22blipA, pUCPCMlipAB, pUCPCMlipA, pUCPCMdeltalipAlipB, pUCPCMdeltalipA, pUCPCMdeltalipB, and then electroporated them into B. cepacia containing T7 RNA. After shake flask culture, we found B. cepacia containing pUCPCMlipAB produced the most quantity of lipase, and lipase activity was up to 607.2 U/mg, 2.8-folds higher than that of the wild strain. Moreover, lipase activities of all engineering strains except the one containing pUCPCMdeltalipB were enhanced to some extent. The specific activities of wild type B. cepacia and B. cepacia containing pUCPCMlipAB were respectively 29 984 U/mg and 30 875 U/mg after ammonium sulfate precipitation and gel filtration chromatography. The T7 RNA polymerase expression system could effectively enhanced lipase expression in B. cepacia, and secretion signal PelB and ribosome-binding site may promote lipase expression in engineering strain.

[Two-step Synthesis of the Full Length Aspergillus Niger Lipase Gene LipA Leads to High-level Expression in Pichia Pastoris]

Sheng Wu Gong Cheng Xue Bao = Chinese Journal of Biotechnology. Mar, 2009  |  Pubmed ID: 19621578

Aspergillus niger lipases are important biocatalysis widely used in industries for food processing and pharmaceutical preparation. High-level expression recombinants can lead to cost effective lipase large scale production. Full length gene synthesis is an efficient measure to enhance the expression level of the gene. In order to reduce the non-specific binding between oligonucleotides and bases mutation caused by the complicate secondary structure of DNA and excessive PCR amplification, a frequently phenomenon in one-step gene synthesis, we used a two-step method including assembly PCR (A-PCR) and digestion-ligation step to synthesis Aspergillus niger lipase gene lipA. Assisted by DNA2.0 and Gene2Oliga software, we optimized the codon usage and secondary structure of RNA and induced enzyme sites Cla I (237 site) and Pst I (475 site) into the gene. In the first step, fragments F1 (237 bp), F2 (238 bp) and F3 (422 bp) were separately synthesized by assembly PCR. In the second step, fragments F1, F2 and F3 were separately digested by Cla I and Pst I, and then ligated into a full length lipA gene. Two-step method efficiently enhanced successful ratio for full-length gene synthesis and dispersed the risk for gene redesign. The synthesized gene was cloned into pPIC9K vector and transferred into Pichia pastoris. After methanol inducement, the expression level of the codon optimized lipA-syn gene reached 176.0 U/mL, 10.8-fold of the original lipA gene (16.3 U/mL) in Pichia pastoris GS1115. The recombinant offers the possibility for lipase large-scale production.

[Cloning, Expression and Characterization of a Novel Lipase Gene LipB from Aspergillus Niger F044]

Wei Sheng Wu Xue Bao = Acta Microbiologica Sinica. Aug, 2009  |  Pubmed ID: 19835173

We cloned, expressed and characterized a novel lipase gene lipB from Aspergillus niger F044, to facilitate the large scale production and application of that enzyme.

Optimization of Lipase-catalyzed Transesterification of Lard for Biodiesel Production Using Response Surface Methodology

Applied Biochemistry and Biotechnology. Jan, 2010  |  Pubmed ID: 18931953

Biodiesel, an alternative diesel fuel made from renewable biological resources, has become more and more attractive recently. Combined use of two immobilized lipases with complementary position specificity instead of one lipase is a potential way to significantly reduce cost of lipase-catalyzed biodiesel production. In this study, the process of biodiesel production from lard catalyzed by the combined use of Novozym435 (non-specific) and Lipozyme TLIM (1,3-specific) was optimized by response surface methodology. The optimal reaction conditions were 0.04 of amount of lipase/oil (w/w), 0.49 of proportion of Novozym435/total lipases (w/w), 0.55 of quantity of tert-butanol/oil (v/v), 5.12 of quantity of methanol/oil (mol/mol), and 20 h of reaction time, by which 97.2% of methyl ester (ME) yield was attained, very close to the predicted value (97.6%). This optimal reaction condition could be true of other similar reactions with plant and animal oil resources; their ME yield could be higher than 95%. The lipases regenerated by washing with organic solvent after each reaction cycle could be continuously reused for 20 cycles without any loss of activity, exhibiting very high manipulation stability.

Surface Display of Active Lipase in Saccharomyces Cerevisiae Using Cwp2 As an Anchor Protein

Biotechnology Letters. Feb, 2010  |  Pubmed ID: 19821073

Lipase Lip2 from Yarrowia lipolytica was displayed on the cell surface of Saccharomyces cerevisiae using Cwp2 as an anchor protein. Successful display of the lipase on the cell surface was confirmed by immunofluorescence microscopy and halo assay. The length of linker sequences was further examined to confirm that the correct conformation of Lip2 was maintained. The results showed that the displayed Lip2 exhibited the highest activity at 7.6 +/- 0.4 U/g (dry cell) when using (G(4)S)(3) sequence as the linker, with an optimal temperature and pH at 40 degrees C and pH 8.0. The displayed lipase did not lose any activity after being treated with 0.1% Triton X-100 and 0.1% Tween 80 for 30 min, and it retained 92% of its original activity after incubation in 10% DMSO for 30 min. It also exhibited better thermostability than free Lip2 as reported previously.

Cloning of a Novel Lipase Gene, LipJ08, from Candida Rugosa and Expression in Pichia Pastoris by Codon Optimization

Biotechnology Letters. Feb, 2010  |  Pubmed ID: 19841868

A novel lipase gene, lipJ08, was cloned from Candida rugosa ATCC14830, along with the already reported five lipase genes (lip1-lip5). Nucleotide sequencing indicated that the lipJ08 gene contains a 1650 bp open reading frame (ORF) without introns. The deduced amino acid sequence corresponds to 534 amino acid residues, including a putative signal sequence of 15 amino acid residues. Seventeen of the non-universal serine codons (CTG) of lipJ08 were converted into universal serine codons (TCT) by PCR-based mutagenesis. The native and codon-optimized lipJ08 genes were expressed in Pichia pastoris. The hydrolytic activity of the recombinant LIPJ08 was 4.7 U/ml, whereas the activity of the recombinant wild-type lipase could not be detected.

Lip2, a Novel Lipase Gene Cloned from Aspergillus Niger Exhibits Enzymatic Characteristics Distinct from Its Previously Identified Family Member

Biotechnology Letters. Jul, 2010  |  Pubmed ID: 20213520

We have cloned a novel lipase gene, lip2, from Aspergillus niger and expressed it in Escherichia coli. Upon purification of the recombinant Lip2 protein, its properties were characterized. In comparison with a previously identified lipase Lip1, both enzymes are acid lipases (optimal pH <6.5), Ca(2+)-dependent and PMSF-sensitive, but have different molecular weights (35 and 43 kDa), optimal substrate spectra (C10 and C8), optimal reaction temperatures (45 and 50 degrees C) and thermal stability. Circular dichroism spectroscopy revealed that Lip2 contains a typical Ca(2+)-active site. This first report on the cloning of the Lip2 gene and its enzymatic characteristics may greatly facilitate its potential industrial application.

[Cloning, Codon Optimization and Expression of Mature Lipase Gene Penicillium Expansum]

Wei Sheng Wu Xue Bao = Acta Microbiologica Sinica. Feb, 2010  |  Pubmed ID: 20387466

To clone Penicillum expansum CICC 40356 lipase (PEL) gene cDNA and to over-express active lipase in Pichia pastoris GS115.

Esterification Activity and Conformation Studies of Burkholderia Cepacia Lipase in Conventional Organic Solvents, Ionic Liquids and Their Co-solvent Mixture Media

Bioresource Technology. Dec, 2010  |  Pubmed ID: 20713309

In this work, experiments were carried out to evaluate the esterification activity and conformation of lipase from Burkholderia cepacia in the selected conventional organic solvents, ionic liquids and their co-solvent mixture media. The results revealed that the activity of esterification of B. cepacia lipase was mostly highest in co-solvent mixture of ionic liquids-organic solvents, followed by conventional organic solvents and ionic liquids. Hence, co-solvent mixture was a high-effective strategy to enhance the activity of B. cepacia lipase for non-aqueous enzymology reaction. Conformational studies via circular dichroism spectroscopy indicated that the secondary structure of B. cepacia lipase was variant in the above-mentioned media, especially the content of alpha-helix, which was probably responsible for lipase activity difference.

[Gene Cloning, Codon Optimization and Functional Expression of Yarrawia Lipolytica Lipase Lip1]

Wei Sheng Wu Xue Bao = Acta Microbiologica Sinica. Jul, 2010  |  Pubmed ID: 20815247

To implement inducible and constitutive over-expression of Yarrowia lipolytica lipase gene lipl in Pichia pastoris using codon optimization.

Preparation of a Whole-cell Biocatalyst of Aspergillus Niger Lipase and Its Practical Properties

Journal of Agricultural and Food Chemistry. Oct, 2010  |  Pubmed ID: 20828152

Aspergillus niger lipase (ANL), a widely used hydrolase, was displayed for the first time on the surface of Saccharomyces cerevisiae using a-agglutinin as an anchor protein. Localization of ANL on the cell surface was confirmed by immunofluorescence microscopy. The displayed ANL was confirmed to be active toward tributyrin and p-nitrophenyl caprylate (pNPC). The hydrolytic activity toward pNPC reached 43.8 U/g of dry cell weight after induction by galactose for 72 h. The ANL-displaying cells were characterized for their use as whole-cell biocatalysts. The optimum temperature was 45 °C, and the pH was 7.0. The cells had good thermostability, retaining almost 80% of the full activity after incubation at 60 °C for 1 h, and >80% of the full activity at 50 °C for 6 h. The displayed lipase showed a preference for medium-chain fatty acid p-nitrophenyl esters. Therefore, the produced whole-cell catalyst is likely to have a wide range of applications.

[Burkholderia Cepacia Lipase Gene Modification and Its Constitutive and Inducible Expression in Pichia Pastoris]

Wei Sheng Wu Xue Bao = Acta Microbiologica Sinica. Sep, 2010  |  Pubmed ID: 21090259

To achieve fast, safe and stable expression of Burkholderia cepacia lipase in Pichia pastoris.

Preparation of Cross-linked Lipase-coated Micro-crystals for Biodiesel Production from Waste Cooking Oil

Bioresource Technology. Apr, 2011  |  Pubmed ID: 21277775

A dual modification procedure composed of cross-linking and protein coating with K(2)SO(4) was employed to modify Geotrichum sp. lipase for catalyzing biodiesel production from waste cooking oil. Compared to single modification of protein coating with K(2)SO(4), the dual modification of cross-linking and lipase coating improved catalytic properties in terms of thermostable stability, organic solvent tolerance, pH stability and operational stability in biodiesel production process, although biodiesel yield and initial reaction rate for CLPCMCs were not improved. After five successive batch reactions, CLPCMCs could still maintain 80% of relative biodiesel yield. CLPCMCs retained 64% of relative biodiesel yield after incubation in a pH range of 4-6 for 4 h, and 85% of relative biodiesel yield after incubation in a range of 45-50 °C for 4 h. CLPCMCs still maintained 83% of relative biodiesel yield after both treated in polar organic solvent and non-polar organic solvent for 4 h.

Enzymatic Enrichment of Polyunsaturated Fatty Acids Using Novel Lipase Preparations Modified by Combination of Immobilization and Fish Oil Treatment

Bioresource Technology. Jul, 2011  |  Pubmed ID: 21565494

Novel modification methods for lipase biocatalysts effective in hydrolysis of fish oil for enrichment of polyunsaturated fatty acids (PUFAs) were described. Based on conventional immobilization in single aqueous medium, immobilization of lipase in two phase medium composed of buffer and octane was employed. Furthermore, immobilization (in single aqueous or in two phase medium) coupled to fish oil treatment was integrated. Among these, lipase immobilized in two phase medium coupled to fish oil treatment (IMLAOF) had advantages over other modified lipases in initial reaction rate and hydrolysis degree. The hydrolysis degree increased from 12% with the free lipase to 40% with IMLAOF. Strong polar and hydrophobic solvents had negative impact on immobilization-fish oil treatment lipases, while low polar solvents were helpful to maintain the modification effect of immobilization-fish oil treatment. After five cycles of usage, the immobilization-fish oil treatment lipases still maintained more than 80% of relative hydrolysis degree.

Homology Modeling, Molecular Docking and Spectra Assay Studies of Sterol 14α-demethylase from Penicillium Digitatum

Biotechnology Letters. Oct, 2011  |  Pubmed ID: 21660575

Sterol 14α-demethylase from Penicillium digitatum (PdCYP51) is a prime target of antifungal drugs for citrus disease in plants. To design novel antifungal compounds, a homology model of PdCYP51 was constructed using the recently reported crystal structure of human CYP51 as the template. Molecular docking was performed to investigate the interaction of four commercial fungicides with the modeled enzyme. The side chain of these compounds interplayed with PdCYP51 mainly through hydrophobic and van der Waals interactions. Biochemical spectra analysis of inhibitors combined with PdCYP51 are also compatible with the docking results. This is the first molecular modeling for PdCYP51 based on the eukaryotic crystal structure of CYP51. The structural information and binding site mapping of PdCYP51 for different inhibitors obtained from this study could aid in screening and designing new antifungal compounds targeting this enzyme.

[Phylogeny Diversity of the Nitrite Reductase Gene (nirS) in the Sediments of the Eutrophic East Lake, Wuhan]

Wei Sheng Wu Xue Bao = Acta Microbiologica Sinica. May, 2011  |  Pubmed ID: 21800630

The study aims to investigate the phylogeny diversity of the denitrification bacteria communities in the sediments of the eutrophic East Lake, Wuhan based on nitrite reductase gene (nirS) restriction fragment length polymorphism (RFLP) method and sequencing analysis, and to analyse community variation according to the environment parameters.

Cloning, Expression and Characterization of a New Lipase from Yarrowia Lipolytica

Biotechnology Letters. Dec, 2011  |  Pubmed ID: 21805187

Bioinformatic analysis of the Yarrowia lipolytica CLIB122 genome has revealed 18 putative lipase genes all of which were expressed in Escherichia coli and screened for hydrolyzing activities against p-nitrophenyl-palmitate. One positive transformant containing an ORF of 1,098 bp encoding a protein of 365 amino acids was obtained. To characterize its enzymatic properties, the lipase gene was functionally expressed in Pichia pastoris. The resulting lipase exhibited the highest activity towards p-NP-decanoate at pH 7 and 35 °C. In addition, the new lipase had a lower optimal temperature and pH compared to other Y. lipolytica lipases. It was noticeably enhanced by Ca(2+), but was inhibited by PMSF, Hg(2+) and Ni(2+). The new lipase displayed the 1,3-specificity for triolein.

Biodiesel Synthesis and Conformation of Lipase from Burkholderia Cepacia in Room Temperature Ionic Liquids and Organic Solvents

Bioresource Technology. Nov, 2011  |  Pubmed ID: 21955878

Biodiesel synthesis and conformation of Burkholderia cepacia lipase (BCL) were studied in 19 different room temperature ionic liquids (RTLLs) with a range of cation and anion structures. Overall, anion selection had a greater influence on biodiesel conversion than cation choice. RTILs containing Tf2N- and PF6- anions were suitable reaction media, while RTIL of [OmPy][BF4] was the best reaction medium with a biodiesel yield of 82.2±1.2%. RTILs with strong water miscible properties showed very low biodiesel yields. Conformational analysis by FT-IR revealed that higher biodiesel conversion in RTILs was correlated with a low tendency in α-helix content of BCL. An ultrasound-assisted biocatalysis process in RTILs was used to improve mass transfer rate, leading to 83% reduction of the reaction time for biodiesel production.

[Heterologous Expression and Characterization of Yarrowia Lipolytica Lipase 4 and Lipase 5 in Pichia Pastoris]

Wei Sheng Wu Xue Bao = Acta Microbiologica Sinica. Oct, 2011  |  Pubmed ID: 22233059

To clone cDNA sequences of lipase 4 (LIP4) and lipase 5 (LIPS), analyze gene structures and express them in Pichia pastoris so as to investigate their enzymatic characteristics.

[Expression of Vitreoscilla Hemoglobin Improves Recombinant Lipase Production in Pichia Pastoris]

Sheng Wu Gong Cheng Xue Bao = Chinese Journal of Biotechnology. Dec, 2011  |  Pubmed ID: 22506416

Yarrowia lipolytica lipase Lip2 (YlLip2) is an important industrial enzyme with many potential applications. To alleviate the dissolved oxygen (DO) limitation and improve YlLip2 production during high-cell density fermentation, the YlLip2 gene lip2 and Vitreoscilla hemoglobin (VHb) gene vgb were co-expressed in Pichiapastoris under the control of AOX1 and PsADH2 promoter, respectively. The PsADH2 promoter from Pichia stipitis could be activated under oxygen limitation. The SDS-PAGE and CO-difference spectrum analysis indicated that VHb and YlLip2 had successfully co-expressed in recombinant strains. Compared with the control cells (VHb-, GS115/9Klip2), the expression levels of YlLip2 in VHb-expressing cells (VHb+, GS115/9Klip2-pZPVT) under oxygen limitation were improved 25% in shake-flask culture and 83% in a 10 L fermentor. Moreover, the VHb+ cells displayed higher biomass than VHb- cells at lower DO levels in a 10 L fermentor. In this study, we also achieved a VHb-expressing clone harboring multicopy lip2 gene (GS115/9Klip2-pZPVTlip2 49#), which showed the maximum lipolytic activity of 33 900 U/mL in a 10 L fermentor under lower DO conditions. Therefore, it can be seen that expression of VHb with PsADH2 promoter in P. pastoris combined with increasing copies of lip2 gene is an effective strategy to improve YlLip2 production.

Intracellular Expression of Vitreoscilla Hemoglobin Improves Production of Yarrowia Lipolytica Lipase LIP2 in a Recombinant Pichia Pastoris

Enzyme and Microbial Technology. Jan, 2012  |  Pubmed ID: 22133436

The Yarrowia lipolytica lipase LIP2 (YlLIP2) gene lip2 and Vitreoscilla hemoglobin gene vgb were co-expressed in Pichia pastoris, both under the control of AOX1 promoter, in order to alleviate respiration limitation under conditions of high cell-density fermentation and enhance YlLIP2 production. The results showed that recombinant P. pastoris strains harboring the lip2 and vgb genes (VHb(+)) displayed higher biomass and YlLIP2 activity than control strains (VHb(-)). Compared with VHb(-) cells, the expression levels of YlLIP2 in VHb-expressing cells when oxygen was not a limiting factor were improved 31.5% in shake-flask culture and 22% in a 10-L fermentor. Under non-limiting dissolved oxygen (DO) conditions, the maximum YlLIP2 activity of VHb(+) in a 10-L fermentor reached 33,000 U/mL. Oxygen limitation had a more negative effect on YlLIP2 productivity in VHb(-) cells than in VHb(+) cells. The highest YlLIP2 activity of VHb(+) cells was approximately 1.84-fold higher than that of VHb(-) cells at lower DO levels. Moreover, the recombinant strain VHb(+) exhibited a higher specific oxygen uptake rate and achieved higher cell viability under oxygen limiting and non-limiting conditions compared with VHb(-) cells. Therefore, the above results suggest that intracellular expression of VHb in recombinant P. pastoris has the potential to improve cell growth and industrial enzyme production.

Improving Stability and Activity of Cross-linked Enzyme Aggregates Based on Polyethylenimine in Hydrolysis of Fish Oil for Enrichment of Polyunsaturated Fatty Acids

Applied Biochemistry and Biotechnology. Feb, 2012  |  Pubmed ID: 22167690

Cross-linking of enzyme aggregates from recombinant Geotrichum sp. lipase based on polyethylenimine (PEI) was applied to hydrolyze fish oil for enrichment of polyunsaturated fatty acids successfully. Through acetone precipitation and cross-linking of physical aggregates using glutaraldehyde in the presence of PEI, firmly cross-linked enzyme aggregates (PEI-CLEAs) were prepared. They could maintain more than 65% of relative hydrolysis degree after incubation in the range of 50-55 °C for 4 h and maintain more than 85% of relative hydrolysis degree after being treated by acetone, tert-butyl alcohol and octane for 4 h. PEI-CLEAs increased hydrolysis degree to 42% from 12% by free lipase. After five batch reactions, PEI-CLEAs still maintained 72% of relative hydrolysis degree. Hydrolysis of fish oil by PEI-CLEAs produced glycerides containing concentrated EPA and DHA in good yield. PEI-CLEAs had advantages over general CLEAs and free lipase in initial reaction rate, hydrolysis degree, thermostability, organic solvent tolerance and reusability.

Constitutive Expression of Yarrowia Lipolytica Lipase LIP2 in Pichia Pastoris Using GAP As Promoter

Applied Biochemistry and Biotechnology. Mar, 2012  |  Pubmed ID: 22246727

A gene encoding Yarrowia lipolytica lipase LIP2 (YlLIP2) was cloned into a constitutive expression vector pGAPZαA and electrotransformed into the Pichia pastoris X-33 strain. The high-yield clones obtained by high copy and enzyme activity screening were chosen as the host strains for shaking flask and fermentor culture. The results showed that glucose was the optimum carbon source for YlLIP2 production, and the maximum hydrolytic activity of recombinant YlLIP2 reached 1,315 U/ml under the flask culture at 28 °C, pH 7.0, for 48 h. The fed-batch fermentation was carried out in 3- and 10-l bioreactors by continuously feeding glucose into the growing medium for achieving high cell density and YlLIP2 yields. The maximum hydrolytic activity of YlLIP2 and cell density obtained in the 3-l bioreactor were 10,300 U/ml and 116 g dry cell weight (DCW)/l, respectively. The peak hydrolytic activity of YlLIP2 and cell density were further improved in the 10-l fermentor where the values respectively attained were 13,500 U/ml and 120 g DCW/l. The total protein concentration in the supernatant reached 3.3 g/l and the cell viability remained approximately 99% after 80 h of culture. Furthermore, the recombinant YlLIP2 produced in P. pastoris pGAP and pAOX1 systems have similar content of sugar (about 12%) and biochemical characteristics. The above results suggest that the GAP promoter-derived expression system of P. pastoris is effective for the expression of YlLIP2 by high cell density culture and is probably an alternative to the conventional AOX1 promoter expression system in large-scale production of industrial lipases.

Lipase-coated K2SO4 Micro-crystals: Preparation, Characterization, and Application in Biodiesel Production Using Various Oil Feedstocks

Bioresource Technology. Apr, 2012  |  Pubmed ID: 22330591

This study investigated the preparation and characteristics of protein-coated microcrystals (PCMCs) from Pseudomonas cepacia lipase (PS) and K(2)SO(4), and their application in biodiesel synthesis, via single factorial experiments and response surface methodology (RSM), the optimized PCMC-PS exhibited high activity and stability; the optimal temperature was 60°C (which gave 99.83% conversion), although fairly high activity was exhibited after incubation at different temperatures (25-70°C). The organic solvents stability of the PCMC-PS was improved, and it significantly reduced ethanol toxicity. Circular dichroism (CD) analysis revealed the correlation between the conformation and the enzyme activity. The morphology of the PCMC-PS was also confirmed via scanning electron microscopy (SEM). When catalyzed by PCMC-PS, above 83% biodiesel yield was obtained for most of the seven oils tested. The PCMC-PS (washed with hexane) activity remained relatively stable after eight batch reactions, with only a 15.73% reduction in the conversion (from 99.02% to 83.29%).

Evaluation of Structure and Hydrolysis Activity of Candida Rugosa Lip7 in Presence of Sub-/super-critical CO₂

Enzyme and Microbial Technology. Dec, 2012  |  Pubmed ID: 23040391

This work aimed to assess the effect of sub-/super-critical CO₂ on the structure and activity of Candida rugosa Lip7 (CRL7) in its solution form. The structure was examined by SDS-PAGE gel electrophoresis, circular dichroism (CD) and fluorescence spectra photometry. Results revealed that the primary structure remained intact after sub-/super-critical CO₂ treatment, and the secondary structure altered at the pressure of 10 MPa and temperature 40°C for 30 min incubation, but it was reflex to its native form with increasing incubation time up to 150 min under 10 MPa and 40 °C. Meanwhile, the tertiary structure via fluorescence spectra analysis showed that the intensity of the maximal emission wavelength at 338 nm decreased under the conditions of 10 MPa and 40°C for 150 min. Furthermore, the residue hydrolysis activity and kinetics constants (V(max) and K(m)) of CRL7 treated with sub-/super-critical CO₂ were also investigated. In cases of 6 MPa and 35°C, or 10 MPa and 40°C for 30 min, activity variance of CRL7 was maybe caused by its secondary structure alteration. But in case of 10 MPa and 40°C for 150 min, the tertiary structure change was perhaps responsibility for CRL7 activity enhancement.

[Cell Surface Display of Thermomyces Lanuginosus Lipase in Pichia Pastoris and Its Characterization]

Wei Sheng Wu Xue Bao = Acta Microbiologica Sinica. Jul, 2012  |  Pubmed ID: 23115970

To construct a novel cell-surface display system of Thermomyces lanuginosus lipase (TLL) based on an efficient anchor protein Sedlp in Pichia pastoris, to screen recombinant strains with high enzyme activity and displaying rate, and further to characterize the enzyme.

Enhanced Catalysis of Yarrowia Lipolytica Lipase LIP2 Immobilized on Macroporous Resin and Its Application in Enrichment of Polyunsaturated Fatty Acids

Bioresource Technology. Mar, 2013  |  Pubmed ID: 23347925

An immobilization strategy was employed to improve activity and operational stability of Yarrowia lipolytica lipase LIP2 (YlLIP2) by using macroporous resins as carrier. D152H, a cation-exchange resin, was the best support. Under the optimized conditions, the immobilization efficiency was 89.81% and the specific activity was 809,751 U/g, being 2.1-fold higher than that of the free lipase. Bioimprinting and interfacial activation were used to further boost the catalytic activity of YlLIP2, respectively enhanced 21.5-fold, 231.2% and 107.2% compared to the free, non-bioimprinted and non-interfacial-activated lipases. The immobilized lipase exhibited much better thermal and pH stability and broader substrate specificity; when used to enrich docosahexaenoic acid (DHA) from Chlorella protothecoides oil, it could increase 1.66-fold of DHA content and show good operational stability. These indicate that the immobilized YlLIP2 offers a promising approach for the enrichment of DHA.

A Novel Oriented Immobilized Lipase on Magnetic Nanoparticles in Reverse Micelles System and Its Application in the Enrichment of Polyunsaturated Fatty Acids

Bioresource Technology. Mar, 2013  |  Pubmed ID: 23395761

A novel oriented immobilized lipase was derived from Yarrowia lipolytica lipase LIP2 covalently immobilized on functionalized Fe3O4 magnetic nanoparticles (MNPs) in reverse micelles system (RMS). The activity recovery reached 382% compared with 29% in aqueous phase, and further ran up to 1425% under optimum conditions. (3-Aminopropyl) triethoxysilane (APTES) coated Fe3O4 nanoparticles were characterized by Fourier transform infrared (FT-IR) and X-ray diffraction (XRD). A significant alteration in the secondary structure of the lipase in RMS with a 15.5% increase of α-helix content and a 12.5% decrease of β-sheet content was detected by circular dichroism (CD). The immobilized lipase was employed to enrich polyunsaturated fatty acids in fish oil, a 90% increase of DHA content was obtained after 12h, and after 20 cycles of successive usage, it still remained over 80% of relative hydrolysis degree, which shows a good recyclability.

Assessment of Activities and Conformation of Lipases Treated with Sub- and Supercritical Carbon Dioxide

Applied Biochemistry and Biotechnology. Apr, 2013  |  Pubmed ID: 23417391

In order to illustrate the underlining mechanism of the effect of high pressure on lipases from different resources, the influence of compressed carbon dioxide treatment on the esterification activities and conformation of the three lipases Candida rugosa lipase (CRL), Pseudomonas fluorescens lipase, and Rhizopus oryzae lipase was investigated in the present work. The results showed that the lipases activities were significantly enhanced in most of high-pressure treatments, except the pressure had a negative effect on CRL activity in supercritical condition. Mild depressurization rate could remain the lipase's activity by protecting its rigid structure under supercritical fluid. Conformational analysis by Fourier transform-infrared spectrometry and fluorescence emission spectra revealed that the variances of lipase activity after high-pressure treatment were correlated with the changes of its α-helix content and fluorescence intensity. Additionally, transesterification catalyzed by three lipases in supercritical carbon dioxide were conducted, and 87.2 % biodiesel conversion was obtained by CRL after 3 h, resulting in a great reduction of reaction time.

Structural Analysis of HmtT and HmtN Involved in the Tailoring Steps of Himastatin Biosynthesis

FEBS Letters. Apr, 2013  |  Pubmed ID: 23611984

Himastatin is a novel antibiotic featuring a bicyclohexadepsipeptide structure. On the himastatin biosynthesis pathway, three cytochrome P450s (HmtT, HmtN, HmtS) are responsible for the post-tailoring of the cyclohexadepsipeptide backbone. Here we report the crystal structures of HmtT and HmtN. The overall structures of these two proteins are homologous to other cytochrome P450s. However, the exceptionally long F-G loop in HmtT has a highly unusual conformation and extends deep into the active site. As a result, the F/G helices of HmtT are both kinked. In contrast, the F/G helices of HmtN are straight. Also, the F/G helices in HmtT and HmtN take distinctive orientations, which may be a contributing factor for the substrate specificity of these two enzymes.

Effect of Sub- and Supercritical CO2 Treatment on the Properties of Pseudomonas Cepacia Lipase

Enzyme and Microbial Technology. Jul, 2013  |  Pubmed ID: 23769311

In this work, we have investigated the influences of sub- and supercritical CO2 treatment on the properties of Pseudomonas cepacia lipase (PCL), including its esterification and transesterification activities, structural changes and stability. Results demonstrated that exposure time to subcritical CO2 treatment had a negative effect on PCL transesterification activity whereas exposure time to supercritical CO2 treatment had a positive effect. But generally, most compressed treatments significantly enhanced PCL esterification activity. Conformational analysis by FT-IR and fluorescence emission spectra revealed that enhanced activities after supercritical CO2 treatment were correlated with the secondary and tertiary structural changes of PCL. Secondary structure changes also appeared to be responsible for enhancement of PCL activities by subcritical CO2 treatment. Compared to native PCL, treated PCL's esterification activity significantly decreased in hydrophilic organic media, while transesterification activity significantly increased in tert-amyl alcohol and acetone. After supercritical treatment, the thermal stability of PCL significantly decreased in esterification reactions, however, there was no significant difference in transesterification reactions.

Isolation and Characterization of a Thermostable Esterase from a Metagenomic Library

Journal of Industrial Microbiology & Biotechnology. Nov, 2013  |  Pubmed ID: 23934105

A novel esterase gene was isolated by functional screening of a metagenomic library prepared from an activated sludge sample. The gene (est-XG2) consists of 1,506 bp with GC content of 74.8 %, and encodes a protein of 501 amino acids with a molecular mass of 53 kDa. Sequence alignment revealed that Est-XG2 shows a maximum amino acid identity (47 %) with the carboxylesterase from Thermaerobacter marianensis DSM 12885 (YP_004101478). The catalytic triad of Est-XG2 was predicted to be Ser₁₉₂-Glu₃₁₃-His₄₁₂ with Ser₉₂ in a conserved pentapeptide (GXSXG), and further confirmed by site-directed mutagenesis. Phylogenetic analysis suggested Est-XG2 belongs to the bacterial lipase/esterase family VII. The recombinant Est-XG2, expressed and purified from Escherichia coli, preferred to hydrolyze short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate (K(m) and k(cat) of 0.33 mM and 36.21 s⁻¹, respectively). The purified enzyme also had the ability to cleave sterically hindered esters of tertiary alcohols. Biochemical characterization of Est-XG2 revealed that it is a thermophilic esterase that exhibits optimum activity at pH 8.5 and 70 °C. Est-XG2 had moderate tolerance to organic solvents and surfactants. The unique properties of Est-XG2, high thermostability and stability in the presence of organic solvents, may render it a potential candidate for industrial applications.

Improving Activity and Enantioselectivity of Lipase Via Immobilization on Macroporous Resin for Resolution of Racemic 1- Phenylethanol in Non-aqueous Medium

BMC Biotechnology. Oct, 2013  |  Pubmed ID: 24168516

Burkholderia cepacia lipase (BCL) has been proved to be capable of resolution reactions. However, its free form usually exhibits low stability, bad resistance and no reusability, which restrict its further industrial applications. Therefore, it is of great importance to improve the catalytic performance of free lipase in non-aqueous medium.

Conformation and Catalytic Properties Studies of Candida Rugosa Lip7 Via Enantioselective Esterification of Ibuprofen in Organic Solvents and Ionic Liquids

TheScientificWorldJournal. 2013  |  Pubmed ID: 24381516

Enantioselective esterification of ibuprofen was conducted to evaluate the enzyme activity and ees of lipase from Candida rugosa (CRL7) in ten conventional organic solvents and three ionic liquids. Different alcohols were tested for selecting the most suitable acyl acceptor due to the fact that the structure of alcohols (branch and length of carbon chains; location of -OH functional group) could affect the enzyme activity and ees. The results of alcohol and solvent selection revealed that 1-isooctanol and isooctane were the best substrate and reaction medium, respectively, because of the highest enzyme activity and ees. Compared with the control, conformational studies via FT-IR indicate that the variations of CRL7's secondary structure elements are probably responsible for the differences of enzyme activity and ees in the organic solvents and ionic liquids. Moreover, the effects of reaction parameters, such as molar ratio, water content, temperature, and reaction time, in the selected reaction medium, were also examined.

[Enhanced Biohydrogen Production by Homologous Over-expression of Fnr, PncB, FdhF in Klebsiella Sp. HQ-3]

Sheng Wu Gong Cheng Xue Bao = Chinese Journal of Biotechnology. Sep, 2013  |  Pubmed ID: 24409691

To enhance biohydrogen production of Klebsiella sp. HQ-3, the global transcriptional factor (Fnr), formate dehydrogenase H (FDH1) and the pncB gene encoding the nicotinic acid phosphoribosyltransferase (NAPRTase) were for the first time over-expressed in Klebsiella sp. HQ-3. The fnr, fdhF, pncB genes were cloned from the genomic DNA of Klebsiella sp. HQ-3 by 3 pairs of universal primers, and introduced into the corresponding sites of the modified pET28a-Pkan, resulting in the plasmids pET28a-Pkan-fnr, pET28a-Pkan-fdhF and pET28a-Pkan-pncB. The 4 plasmids were then electroported into wild Klebsiella sp. HQ-3 to create HQ-3-fnr, HQ-3-fdhF, HQ-3-pncB and HQ-3-C, respectively. Hydrogen production was measured using a gas chromatograph and the metabolites were analyzed with a high-performance liquid chromatograph (HPLC). The results indicate that over-expression of fnr, fdhF and pncB significantly enhanced hydrogen production in the three recombinant strains. Hydrogen production per mol glucose for HQ-3 fnr, HQ-3 pncB, HQ-3 fdhF was 1.113, 1.106 and 1.063 mol of hydrogen/mol glucose, which was respectively increased by 12.26%, 11.62% and 7.28% compared with that of the control strain HQ-3-C (0.991 mol of hydrogen/mol glucose). Moreover, the analysis of HPLC showed that the concentrations of formate and lactate were markedly decreased, but succinate remained unchanged in culture media compared with those of the control strain HQ-3-C.

Thermostable Lipases from Extremely Radioresistant Bacterium Deinococcus Radiodurans: Cloning, Expression, and Biochemical Characterization

Journal of Basic Microbiology. Sep, 2014  |  Pubmed ID: 24002979

A search for lipases was conducted in the radiophile of Deinococcus radiodurans R1. Four putative lipase genes, encoding DR0334, DR2078, DR1485, and DR2522, were cloned and expressed in Escherichia coli. The recombinant enzymes were subsequently purified and characterized. The results showed DR0334 and DR2078 had the ability to hydrolyze long-chain length p-nitrophenyl esters (C12-C16), while DR1485 and DR2522 hydrolyzed short- and medium-chain length p-nitrophenyl esters (C2-C10). DR0334, DR1485, DR2078, and DR2522 showed optimum pH at 8.5, and optimum temperature at 40, 50, 60, and 60 °C, respectively. DR0334 almost lost its whole activity after 60 min pretreatment at 60 °C, while DR1485, DR2078, and DR2522 retained more than 70% of their original activities after 6 h incubation at 80 °C. The activities of DR2078, DR1485, and DR2522 were enhanced by Mg(2+) , Ba(2+) , and Mn(2+) , but strongly inhibited by EDTA. Nevertheless, DR2078, DR1485, and DR2522 showed moderate stability in organic solvents and detergents. Phylogenetic analysis revealed that DR0334 and DR2078, respectively belong to family IV and family IX, while each of DR1485 and DR2522 forms a new separate branch. The unique properties of DR2078, DR1485, and DR2522, thermostability and organic solvent tolerance, make them useful in industrial applications.

Molecular Identification of Lipase LipA from Pseudomonas Protegens Pf-5 and Characterization of Two Whole-cell Biocatalysts Pf-5 and Top10lipA

Journal of Microbiology and Biotechnology. May, 2014  |  Pubmed ID: 24548931

To identify lipase LipA (PFL_0617) from Pseudomonas protegens Pf-5, a lipA deletion mutant (Pf0617) and a complementary strain (Pf0617lipA) were constructed, and their effects on the lipase production were examined. Pf0617 remarkably decreased its whole-cell lipase activity, whereas Pf0617lipA made its whole-cell lipase activity not only restore to wild-type level but also get a further increment. However, the deletion and overexpression of lipA did not affect the extracellular lipase activity. In addition, the unbroken whole cells of these strains were able to catalyze the hydrolysis of membrane-permeable p-nitrophenyl esters, but could not hydrolyze the membrane-impermeable olive oil. These results confirmed that LipA was an intracellular lipase and Pf-5 could also be used as a natural whole-cell biocatalyst. To evaluate the potential of Pf-5 as a whole-cell biocatalyst and separately characterize the whole-cell LipA, the properties of the whole-cell lipases from Pf-5 and Top10lipA were characterized. The results demonstrated that both Pf-5 and Top10lipA exhibited high tolerance to alkaline condition, high temperature, heavy metal ions, surfactants, and organic solvents. Taken together, lipA can realize functional expression in E. coli Top10, and Pf-5 and Top10lipA as whole-cell biocatalysts may have enormous potential in applications.

Structural Insight into the Tetramerization of an Iterative Ketoreductase Siam Through Aromatic Residues in the Interfaces

PloS One. 2014  |  Pubmed ID: 24901639

In the biosynthesis of polyketides, ketoreductases (KRs) are an important group of enzymes that determine the chiralities of the carbon backbones. SiaM is a special member of this group that can recognize substrates with different lengths and can be used iteratively. Here we report the crystal structure of SiaM. Structural analysis indicates that the overall structure resembles those of other KRs. However, significant disparity can be found in the conserved LDD motif that is replaced with IRD motif in SiaM. The isoleucine and aspartic acid residues take similar orientations as leucine and aspartic acid in the conserved LDD motif, while the arginine residue points out towards the solvent. PISA analysis shows that SiaM forms a tetramer. Several aromatic residues are found in the interfaces, which have aromatic stacking interactions with the aromatic residues in the neighboring protomers. Mutagenesis studies performed on the aromatic residues show that these sites are important for maintaining the structural integrity of SiaM. However, the aromatic residues contribute differently to the enzymatic activity. In the N-terminal interface, the aromatic residues can be replaced with leucine without affecting the enzymatic activity while, in the other interface, such mutations abolish the enzymatic activity.

N-terminal Transmembrane Domain of Lipase LipA from Pseudomonas Protegens Pf-5: A Must for Its Efficient Folding into an Active Conformation

Biochimie. Oct, 2014  |  Pubmed ID: 25038570

LipA from Pseudomonas protegens Pf-5 has been proven not to be secreted into the extracytoplasmic space, proposing that it is a membrane protein in virtue of its N-terminal transmembrane domain predicted by the TMHMM 2.0. However, LipA was confirmed to be an intracellular protein through determining the effects of lipA deletion or overexpression on the lipase activities in the whole-cell, lysis supernatant and lysis pellet, even through its transmembrane domain being able to make heterologous LacZ locate on the cytoplasmic membrane via construction of β-galactosidase reporter strains. Subsequently, lipase activity assays showed that the transmembrane domain played an indispensable role for the catalytic function of LipA through construction of the markerless deletion mutant of transmembrane domain sequence of lipA and the expression and purification of LipA and LipAΔTMD. To further investigate why the transmembrane domain lost its membrane localization function and significantly affected the catalytic function of LipA, the 3D structures of LipA and LipAΔTMD were constructed. The results indicated that the transmembrane domain, located in the interior of LipA, helped the α-helical lid to form an open conformation by the mediation of α5 helix. It seems to act as a kind of intramolecular chaperone like the β-roll motif of subfamily I.3 lipases, which is novel and is the first to notify the intramolecular chaperone of a subfamily I.1 lipase.

Enhancing Catalytic Performance of β-glucosidase Via Immobilization on Metal Ions Chelated Magnetic Nanoparticles

Enzyme and Microbial Technology. Sep, 2014  |  Pubmed ID: 25039060

A novel magnetic Fe3O4 nanoparticles (MNPs) coupled with agarose (AMNPs) was synthesized using co-precipitation via alkaline condition and span-80 surfactants in organic solvent. Iminodiacetate was first attached to the MNPs through epichlorohydrin agent and then chelated with metal ions. The morphology and chemical properties of these prepared supports were characterized by scanning electron microscopy (SEM), X-ray power diffraction (XRD), vibrating sample magnetometer (VSM), and Fourier transform infrared spectroscopy (FT-IR). Among them, the Co(2+)-chelated AMNPs (AMNPs-ECH-IDA-Co(2+)) showed the second highest enzyme adsorption capacity of 1.81 mg/g particles, and achieved the largest activity recovery of 117% per protein gram in immobilization of β-glucosidase (BGL). The Michaelis constant (Km) and Vmax of the immobilized BGL were 0.904 mM and 0.057 μmol/min, respectively, and its activation energy was much lower than the free form. Moreover, the immobilized enzyme exhibited enhanced thermostability and operational stability. It still retained more than 90% of its initial activity after being operated for 15 successive batches. This study demonstrates that the immobilized β-glucosidase has a good prospect in industrial applications.

Enhanced Production of Thermomyces Lanuginosus Lipase in Pichia Pastoris Via Genetic and Fermentation Strategies

Journal of Industrial Microbiology & Biotechnology. Oct, 2014  |  Pubmed ID: 25074457

This study attempted to enhance the expression level of Thermomyces lanuginosus lipase (TLL) in Pichia pastoris using a series of strategies. The tll gene was first inserted into the expression vector pPIC9 K and transformed into P. pastoris strain GS115. The maximum hydrolytic activity of TLL reached 4,350 U/mL under the optimal culture conditions of a 500 mL shaking flask containing 20 mL culture medium with the addition of 1.2 % (w/v) methanol, cultivation for 144 h at pH 7.0 and 27 °C. To further increase the TLL expression and copy number, strains containing two plasmids were obtained by sequential electroporation into GS115/9k-TLL #3 with a second vector, either pGAPZαA-TLL, pFZα-TLL, or pPICZαA-TLL. The maximum activity of the resultant strains GS115/9KTLL-ZαATLL #40, GS115/9KTLL-FZαATLL #46 and GS115/9KTLL-GAPTLL #45 was 6,600 U/mL, 6,000 U/mL and 4,800 U/mL, respectively. The tll copy number in these strains, as assessed by real-time quantitative PCR, was demonstrated to be seven, five, and three, respectively, versus two copies in GS115/9k-TLL #3. When a co-feeding strategy of sorbitol/methanol was adopted in a 3-L fermenter, the maximum TLL activity of GS115/9k-TLL #3 increased to 27,000 U/mL after 130 h of fed-batch fermentation, whereas, the maximum TLL activity was 19,500 U/mL after 145 h incubation when methanol was used as the sole carbon source.

The Two-component GacS-GacA System Activates LipA Translation by RsmE but Not RsmA in Pseudomonas Protegens Pf-5

Applied and Environmental Microbiology. Nov, 2014  |  Pubmed ID: 25128345

In Pseudomonas spp., the Gac-Rsm signal transduction system is required for the production of lipases. The current model assumes that the system induces lipase gene transcription mediated through the quorum-sensing (QS) system. However, there are no reports of a QS system based upon N-acyl homoserine lactones or the regulation of lipase gene expression in Pseudomonas protegens. In this study, we investigated the regulatory mechanism acting on lipA expression activated by the Gac-Rsm system in P. protegens Pf-5 through deletion and overexpression of gacA, overexpression of rsmA or rsmE, expression of various lacZ fusions, reverse transcription-PCR analysis, and determination of whole-cell lipase activity. The results demonstrated that the GacS-GacA (GacS/A) system activates lipA expression at both the transcriptional and the translational levels but that the translational level is the key regulatory pathway. Further results showed that the activation of lipA translation by the GacS/A system is mediated through RsmE, which inhibits lipA translation by binding to the ACAAGGAUGU sequence overlapping the Shine-Dalgarno (SD) sequence of lipA mRNA to hinder the access of the 30S ribosomal subunit to the SD sequence. Moreover, the GacS/A system promotes lipA transcription through the mediation of RsmA inhibiting lipA transcription via an unknown pathway. Besides the transcriptional repression, RsmA mainly activates lipA translation by negatively regulating rsmE translation. In summary, in P. protegens Pf-5, the Gac-RsmE system mainly and directly activates lipA translation and the Gac-RsmA system indirectly enhances lipA transcription.

Aromatic Amino Acid Mutagenesis at the Substrate Binding Pocket of Yarrowia Lipolytica Lipase Lip2 Affects Its Activity and Thermostability

TheScientificWorldJournal. 2014  |  Pubmed ID: 25197700

The lipase2 from Yarrowia lipolytica (YLLip2) is a yeast lipase exhibiting high homologous to filamentous fungal lipase family. Though its crystal structure has been resolved, its structure-function relationship has rarely been reported. By contrast, there are two amino acid residues (V94 and I100) with significant difference in the substrate binding pocket of YLLip2; they were subjected to site-directed mutagenesis (SDM) to introduce aromatic amino acid mutations. Two mutants (V94W and I100F) were created. The enzymatic properties of the mutant lipases were detected and compared with the wild-type. The activities of mutant enzymes dropped to some extent towards p-nitrophenyl palmitate (pNPC16) and their optimum temperature was 35°C, which was 5°C lower than that of the wild-type. However, the thermostability of I100F increased 22.44% after incubation for 1 h at 40°C and its optimum substrate shifted from p-nitrophenyl laurate (pNPC12) to p-nitrophenyl caprate (pNPC10). The above results demonstrated that the two substituted amino acid residuals have close relationship with such enzymatic properties as thermostability and substrate selectivity.

Biochemical Characterization of a Carboxylesterase from the Archaeon Pyrobaculum Sp. 1860 and a Rational Explanation of Its Substrate Specificity and Thermostability

International Journal of Molecular Sciences. Sep, 2014  |  Pubmed ID: 25250909

In this work, genome mining was used to identify esterase/lipase genes in the archaeon Pyrobaculum sp. 1860. A gene was cloned and functionally expressed in Escherichia coli as His-tagged protein. The recombinant enzyme (rP186_1588) was verified by western blotting and peptide mass fingerprinting. Biochemical characterization revealed that rP186_1588 exhibited optimum activity at pH 9.0 and 80 °C towards p-nitrophenyl acetate (K(m): 0.35 mM, k(cat): 11.65 s⁻¹). Interestingly, the purified rP186_1588 exhibited high thermostability retaining 70% relative activity after incubation at 90 °C for 6 h. Circular dichroism results indicated that rP186_1588 showed slight structure alteration from 60 to 90 °C. Structural modeling showed P186_1588 possessed a typical α/β hydrolase's fold with the catalytic triad consisting of Ser97, Asp147 and His172, and was further confirmed by site-directed mutagenesis. Comparative molecular simulations at different temperatures (300, 353, 373 and 473 K) revealed that its thermostability was associated with its conformational rigidity. The binding free energy analysis by MM-PBSA method revealed that the van der Waals interaction played a major role in p-NP ester binding for P186_1588. Our data provide insights into the molecular structures of this archaeal esterase, and may help to its further protein engineering for industrial applications.

Microcystin-LR Impairs Zebrafish Reproduction by Affecting Oogenesis and Endocrine System

Chemosphere. Feb, 2015  |  Pubmed ID: 25014902

Previous studies have shown that microcystins (MCs) are able to exert negative effects on the reproductive system of fish. However, few data are actually available on the effects of MC-LR on the reproductive system of female fish. In the present study, female zebrafish were exposed to 2, 10, and 50 μg L(-1) of MC-LR for 21 d, and its effects on oogenesis, sex hormones, transcription of genes on the hypothalamic-pituitary-gonad (HPG) axis, and reproduction were investigated for the first time. It was observed that egg production significantly declined at ⩾ 10 μg L(-1) MC-LR. MC-LR exposure to zebrafish increased the concentrations of 17β-estradiol (E2) and vitellogenin (VTG) at 10 μg L(-1) level, whereas concentrations of E2, VTG and testosterone declined at 50 μg L(-1) MC-LR. The transcriptions of steroidogenic pathway gene (cyp19a, cyp19b, 17βhsd, cyp17 and hmgra) changed as well after the exposure and corresponded well with the alterations of hormone levels. A number of intra- and extra-ovarian factors, such as gnrh3, gnrhr1, fshβ, fshr, lhr, bmp15, mrpβ, ptgs2 and vtg1 which regulate oogenesis, were significantly changed with a different dose-related effect. Moreover, MC-LR exposure to female zebrafish resulted in decreased fertilization and hatching rates, and may suggest the possibility of trans-generational effects of MC-LR exposure. The results demonstrate that MC-LR could modulate endocrine function and oogenesis, eventually leading to disruption of reproductive performance in female zebrafish. These data suggest there is a risk for aquatic population living in MC polluted areas.

Microcystin-LR Induced Thyroid Dysfunction and Metabolic Disorders in Mice

Toxicology. Feb, 2015  |  Pubmed ID: 25497113

There is growing evidence that microcystins (MCs) act as hazardous materials and can disrupt the endocrine systems of animals. However, the response of thyroid function and the related energy metabolism following MCs exposure is still unknown. In the present study, mice were injected intraperitoneally (i.p.) with doses of either 5 or 20 μg/kg MC-LR for 4 weeks. We report, for the first time, that mice exposed to 20 μg/kg MC-LR showed disrupted glucose, triglyceride and cholesterol metabolism with obvious symptoms of hyperphagia, polydipsia, and weight loss. The circulating thyroid hormone (TH) levels in mice following MC-LR exposure were detected. Significantly increased free triiodothyronine (FT3) and decreased free thyroxin (FT4) were largely responsible for the physiological aberrations and metabolic disorders observed in mice after the 20 μg/kg MC-LR exposure. Increased expression of TH receptor (Trα) and mTOR expression in the brain after the 20 μg/kg MC-LR exposure suggests that the increased FT3 enhanced mTOR signaling subsequently led to hyperphagia and elevated energy expenditure in mice. Furthermore, several genes involved in glucose homeostasis and lipid metabolism, which have been identified affected by TH, were also differentially expressed after MC-LR exposure. The above results clearly showed that mice exposed to MC-LR experienced thyroid dysfunction and its downstream functional changes, and are useful to better understand the endocrine toxicity of MC-LR to mammals or even humans.

Protein-Coated Microcrystals from Candida Rugosa Lipase: Its Immobilization, Characterization, and Application in Resolution of Racemic Ibuprofen

Applied Biochemistry and Biotechnology. Sep, 2015  |  Pubmed ID: 26137875

In this study, an economical heterogeneous biocatalyst, protein-coated microcrystals (PCMCs), was prepared from a commercial Candida rugosa lipase (CRL) and used for catalyzing esterification of (R, S)-ibuprofen enantiomers with isooctanol in isooctane. The main variables controlling the process (precipitating solvents, pH, saturated K2SO4 solution, and water content) were optimized via single-factorial experiments. Under optimum conditions, the enantiomeric excess of active S(+)-ibuprofen and total conversion rate were 97.34 and 49.83 %, respectively, and the corresponding enzyme (PCMC-CRL) activity attained 387.29 μmol/min/g protein, a 5.78-fold enhancement over the free lipase powder. Additionally, the thermostability, organic-solvent tolerance, and operational stability of PCMC-CRL were greatly improved as compared to the free enzyme. Fourier transform infrared (FTIR) spectroscopy was employed to reveal the correlation between conformation and enzyme activity enhancement. Moreover, the PCMC-CRL retained most of its original activity following use in more than 15 successive batches, suggesting that it exhibits adequate operational stability. These results indicate that PCMC-CRL is of great potential use in industrial applications.

Characterizing LipR from Pseudomonas Sp. R0-14 and Applying in Enrichment of Polyunsaturated Fatty Acids from Algal Oil

Journal of Microbiology and Biotechnology. Nov, 2015  |  Pubmed ID: 26215266

In this study, Pseudomonas R0-14, which was isolated from Arctic soil samples, showed a clear halo when grown on M9 medium agarose plates containing olive oil-rhodamine B as substrate, suggesting that it expressed putative lipase(s). A putative lipase gene, lipR, was cloned from R0-14 by genome walking and Touchdown PCR. lipR encodes a 562-amino-acid polypeptide showing a typical α/β hydrolase structure with a catalytic triad consisting of Ser153-Asp202-His260 and one α-helical lid (residues 103-113). A phylogenetic analysis revealed that LipR belongs to the lipase subfamily I.3. LipR was successfully expressed in Escherichia coli, purified, and biochemically characterized. Recombinant LipR exhibited its maximum activity towards p-nitrophenyl butyrate at pH 8.5 and 60°C with a Km of 0.37 mM and a kcat of 6.42 s(-1). It retained over 90% of its original activity after incubation at 50°C for 12 h. In addition, LipR was activated by Ca(2+), Mg(2+), Ba(2+), and Sr(2+), while strongly inhibited by Cu(2+), Zn(2+), Mn(2+), and ethylenediaminetetraacetic acid. Moreover, it showed a certain tolerance to organic solvents, including acetonitrile, isopropanol, acetone, methanol, and tert-butanol. When algal oil was hydrolyzed by LipR for 24 h, there was an enrichment of n-3 long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (1.22%, 1.65-fold), docosapentaenoic acid (21.24%, 2.04-fold), and docosahexaenoic acid (36.98%, 1.33-fold), and even a certain amount of diacylglycerols was also produced. As a result, LipR has great prospect in industrial applications, especially in food and/or cosmetics applications.

Probing Role of Key Residues in the Divergent Evolution of Yarrowia Lipolytica Lipase 2 and Aspergillus Niger Eruloyl Esterase A

Microbiological Research. Sep, 2015  |  Pubmed ID: 26302844

Yarrowia lipolytica lipase 2 (YLLip2) and Aspergillus niger feruloyl esterase A (AnFaeA) are enzymes of similar structures but with different functions. They are both classified into the same homologous family in Lipase Engineering Database (LED). The major difference between the two enzymes is that YLLip2 exhibits interfacial activity while AnFaeA does not. In order to better understand the interfacial activation mechanisms of YLLip2, structure guided site-directed mutagenesis were performed, mutants were constructed, kinetics parameters and lipase properties were detected. Mutant enzymes showed enhanced catalytic efficiency towards p-nitrophenyl butyrin (pNPB) but their catalytic efficiency decreased towards p-nitrophenyl palmitate (pNPP), their catalysis behavior was more close to feruloyl esterase. Moreover, the mutant enzymes exhibited enhanced thermostability compared with their wild type. These results indicate that I100 and F129 are probably cut-off point of divergent functions between the two enzymes during evolution.

A Novel Eurythermic and Thermostale Lipase LipM from Pseudomonas Moraviensis M9 and Its Application in the Partial Hydrolysis of Algal Oil

BMC Biotechnology. Oct, 2015  |  Pubmed ID: 26463643

Lipases are regularly used in biotechnology to catalyse the hydrolysis of triglycerides and the synthesis of esters. Microbial lipases in particular have been widely used in a variety of industrial applications. However, the current commercial microbial lipases cannot meet industrial demand due to rapid inactivation under harsh conditions. Therefore, in order to identify more stable enzymes, we isolated novel eurythermic and thermostable lipase(s) from Pseudomonas moraviensis M9.

[Progress in Expression Regulation of Bacterial Lipase Genes--A Review]

Wei Sheng Wu Xue Bao = Acta Microbiologica Sinica. Nov, 2015  |  Pubmed ID: 26915218

Microbial lipases are major sources of commercial ones, which have been extensively used in a wide variety of industrial fields, such as foods, beverages, lipids, detergents, feeds, textiles, leathers, advanced materials, fine chemicals, medicines, cosmetics, papermaking, pollution treatment, and bioenergy. Compared with fungal lipases, bacterial lipases have more types of reactions and exhibit higher activity and better stability in aqueous or organic phases. Amongst bacterial lipases, the most excellent ones are those originating from the genus Pseudomonas. So far, the conventional strategies, such as traditional breeding, optimization of medium and fermentation conditions, cannot fundamentally solve the problem of low production of bacterial lipases. Construction of genetically engineered strains to efficiently overexpress their own lipases is an effective solution. But it must base on clarifying molecular regulation mechanism of lipase gene expression and further finding out key regulators. In this article, we reviewed the progress in expression regulation of bacterial lipase genes from the aspects of direct regulators, quorum sensing system, Gac/Rsm signal transduction system, regulators controlling the Gac/Rsm system, and other regulators. To provide a useful reference for the construction of genetically engineered strains, we also discussed a research prospect in this field based on our ongoing research.

Enhanced H2 Production and Redirected Metabolic Flux Via Overexpression of FhlA and PncB in Klebsiella HQ-3 Strain

Applied Biochemistry and Biotechnology. Mar, 2016  |  Pubmed ID: 26590848

Genetic modifications are considered as one of the most important technologies for improving fermentative hydrogen yield. Herein, we overexpress fhlA and pncB genes from Klebsiella HQ-3 independently to enhance hydrogen molar yield. HQ-3-fhlA/pncB strain is developed by manipulation of pET28-Pkan/fhlA Kan(r) and pBBR1-MCS5/pncB Gm(r) as expression vectors to examine the synchronous effects of fhlA and pncB. Optimization of anaerobic batch fermentations is achieved and the maximum yield of biohydrogen (1.42 mol H2/mol of glucose) is produced in the range of pH 6.5-7.0 at 33-37 °C. Whole cell H2 yield is increased up to 40 % from HQ-3-fhlA/pncB, as compared with HQ-3-fhlA 20 % and HQ-3-pncB 12 % keeping HQ-3-C as a control. Mechanism of improved H2 yield is studied in combination with metabolic flux analysis by measuring glucose consumption and other metabolites including formate, succinate, 2,3 butanediol, lactate, acetate, ethanol, and hydrogen. The results suggest that under transient conditions, the increase in the total level of NAD by NAPRTase can enhance the rate of NADH-dependent pathways, and therefore, final distribution of metabolites is changed. Combined overexpression of fhlA and pncB eventually modifies the energy and carbon balance leading to enhanced H2 production from FHL as well as by NADH pathway.

Mutational Analysis of the Hyc-operon Determining the Relationship Between Hydrogenase-3 and NADH Pathway in Enterobacter Aerogenes

Enzyme and Microbial Technology. Jan, 2016  |  Pubmed ID: 26672442

In this study, the hydrogenase-3 gene cluster (hycDEFGH) was isolated and identified from Enterobacter aerogenes CCTCC AB91102. All gene products were highly homologous to the reported bacterial hydrogenase-3 (Hyd-3) proteins. The genes hycE, hycF, hycG encoding the subunits of hydrogenase-3 were targeted for genetic knockout to inhibit the FHL hydrogen production pathway via the Red recombination system, generating three mutant strains AB91102-E (ΔhycE), AB91102-F (ΔhycF) and AB91102-G (ΔhycG). Deletion of the three genes affected the integrity of hydrogenase-3. The hydrogen production experiments with the mutant strains showed that no hydrogen was detected compared with the wild type (0.886 mol/mol glucose), demonstrating that knocking out any of the three genes could inhibit NADH hydrogen production pathway. Meanwhile, the metabolites of the mutant strains were significantly changed in comparison with the wild type, indicating corresponding changes in metabolic flux by mutation. Additionally, the activity of NADH-mediated hydrogenase was found to be nil in the mutant strains. The chemostat experiments showed that the NADH/NAD(+) ratio of the mutant strains increased nearly 1.4-fold compared with the wild type. The NADH-mediated hydrogenase activity and NADH/NAD(+) ratio analysis both suggested that NADH pathway required the involvement of the electron transport chain of hydrogenase-3.

Overexpression of Candida Rugosa Lipase Lip1 Via Combined Strategies in Pichia Pastoris

Enzyme and Microbial Technology. Jan, 2016  |  Pubmed ID: 26672457

In this study, combined strategies were employed to heterologously overexpress Candida rugosa lipase Lip1 (CRL1) in a Pichia pastoris system. The LIP1 gene was systematically codon-optimized and synthesized in vitro. The Lip1 activity of a recombinant strain harboring three copies of the codon-optimized LIP1 gene reached 1200 U/mL in a shake flask culture. Higher lipase activity, 1450 U/mL, was obtained using a five copy number construct. Co-expressing one copy of the ERO1p and BiP chaperones with Lip1p, the CRL1 lipase yield further reached 1758 U/mL, which was significantly higher than that achieved by expressing Lip1p alone or only co-expressing one molecular chaperone. When cultivated in a 3 L fermenter under optimal conditions, the recombinant strain GS115/87-ZA-ERO1p-BiP #7, expressing the molecular chaperones Ero1p and BiP, produced 13,490 U/mL of lipase activity at 130 h, which was greater than the 11,400 U/mL of activity for the recombinant strain GS115/pAO815-α-mCRL1 #87, which did not express a molecular chaperone. This study indicates that a strategy of combining codon optimization with co-expression of molecular chaperones has great potential for the industrial-scale production of pure CRL1.

First Identification of the Toxicity of Microcystins on Pancreatic Islet Function in Humans and the Involved Potential Biomarkers

Environmental Science & Technology. Mar, 2016  |  Pubmed ID: 26859764

Microcystins (MCs) produced by cyanobacteria have been recognized as a major public health threat. However, the toxicity of MCs to humans is still largely unknown. In this study, we examined the changes in pancreatic islet function in fishers exposed to ambient levels of MCs at Lake Taihu and, using a mouse model, explored the molecular mechanisms involved in toxicity. MCs content in the serum of fishers tested positive, with a range from 0.10 to 0.64 μg/L. Both lower blood insulin levels (2.26 ± 0.96 μIU/mL) and impaired fasting glucose were found in participants from the Meiliang Bay area in Lake Taihu, where MC-LR levels were substantially greater than the MC threshold established by WHO for drinking water. Animal experiments showed that glucose level increased by 27.9% in mice exposed to 5 μg/kg bw and decreased by 41.5% in mice exposed to 20 μg/kg bw. Blood insulin levels declined by 21.9% and 56.2% in mice exposed to 5 and 20 μg/kg bw MC-LR, respectively, which was consistent with the results observed in fishers. Furthermore, the diabetes gene pdx1 and several other proteins (such as Ppp3ca, Ide, Marcks, Pgk1, Suclg1, Ndufs4) involved in insulin secretion were identified for the first time in mice following MC-LR exposure; these biomarkers were considered responsible for MC-LR induced islet dysfunction. This study suggests that subchronic exposure to environmental levels of MCs may increase the risk of the occurrence of diabetes in humans.

Microcystin-LR Induces Dysfunction of Insulin Secretion in Rat Insulinoma (INS-1) Cells: Implications for Diabetes Mellitus

Journal of Hazardous Materials. Aug, 2016  |  Pubmed ID: 27107231

Microcystins (MCs) are the most frequent cyanobacterial toxins observed in freshwater systems. Accumulating evidence suggests that MCs pose a serious threat to public health. However, the contributions of the exposure of MCs to the occurrence of human diseases remain largely unknown. This study provides the evidence of the effects of MC-LR on pancreatic β-cell function through the exposure of rat insulinoma (INS-1) cells to 0, 10, 20, or 40μM MC-LR for 72h and explores the underlying molecular mechanisms. Our results demonstrate that exposure to MC-LR for 72h suppresses cell viability, disturbs glucose-stimulated insulin secretion (GSIS), and decreases the expression of insulin protein. Moreover, MC-LR disrupts the cell cycle distribution and increases cell apoptosis at 20 or 40μM for 72h, respectively, indicating that the β-cell mass would be decreased by MC-LR exposure. A transcriptomic analysis revealed several key genes (e.g., Pdx-1, Neurod1, and Abcc8) involved in insulin secretion are significantly differentially expressed in INS-1 cells in response to MC-LR exposure. In addition, several signal transduction pathways associated with diabetes (e.g., type 1 and 2 diabetes) were also identified compared with the control cells. We recommend that MC be considered as a new environmental factor that promotes diabetes development. The identified key genes or pathways may potentially contribute to the future therapies in the environmental contaminants induced β-cell damage.

Enhanced Performance of Lipase Via Microcapsulation and Its Application in Biodiesel Preparation

Scientific Reports. Jul, 2016  |  Pubmed ID: 27424490

In the present study, a surface-active enzyme, lipase was immobilized in polyethyleneimine (PEI) microcapsules and then modified with oxidized multiwall carbon nanotubes (MWCNTs). The resulting lipase microcapsules exhibited higher activity and stability, since the activity of microcapsules was 21.9 folds than that of the free counterpart. Numerous interfaces which were created in polycondensation enhanced the performance of lipases. Illustrated by confocal laser scanning microscope (CLSM), it was found that microcapsules, whose barrier properties against molecules with diameter >4.6 nm, were with a semipermeable and porous membrane structure. The lipases preferred to locate in the wall of the microcapsules. The oxidized multiwall carbon nanotubes (MWCNTs) were further added to modify microcapsules, resulting in even higher activity. The nanocomposites were examined by scanning electron microscope (SEM) and zeta-potential analyzer. The results indicated the superior catalytic performances were attributed to the augmented interface and decreased positive charge. Finally, the MWCNTs modified microcapsules were utilized in producing biodiesel with a 97.15% yield and retained nearly 90% yield after running 10 cycles. This approach of microcapsulation will be highly beneficial for preparing various bio-active microcapsules with excellent catalytic performance.

A Living Eukaryotic Autocementation Kit from Surface Display of Silica Binding Peptides on Yarrowia Lipolytica

ACS Synthetic Biology. Dec, 2016  |  Pubmed ID: 27461158

With the development of civil engineering, the demand for suitable cementation materials is increasing rapidly. However, traditional cementation methods are not eco-friendly enough and more sustainable approach such as biobased cementation is required. To meet such demand, Euk.cement, a living eukaryotic cell-based biological autocementation kit, was created in this work. Through the surface display of different silica binding peptides on the fungus Yarrowia lipolytica, Euk.cement cells can immobilize onto any particles with a silica containing surface with variable binding intensity. Meanwhile, recombinant MCFP3 released from the cells will slowly consolidate this binding of cells to particles. The metabolism of immobilized living cells will finally complete the carbonate sedimentation and tightly stick the particles together. The system is designed to be initiated by blue light, making it controllable. This autocementation kit can be utilized for industrial and environmental applications that fit our concerns on making the cementation process eco-friendly.

New Species and New Records of Genus Rhyacophila Pictet (Trichoptera: Rhyacophilidae) from Dabie Mountains, East-central China

Zootaxa. Sep, 2016  |  Pubmed ID: 27701228

Three new species of Rhyacophila, Rhyacophila longiramata n. sp., Rhyacophila haplostephanodes n. sp., and Rhyacophila brevitergata n. sp., from the Dabie Mountains of east-central China, are described and illustrated. Rhyacophila longiramata n. sp. and R. haplostephanodes n. sp. both are highly specialized, which makes them very difficult to be put in any existing species group. Possible positions for these two species are discussed. Rhyacophila brevitergata n. sp. belongs to the R. nigrocephala Species Group. Rhyacophila spp. from Dabie Mountains are listed.

Harnessing Biodiesel-producing Microbes: from Genetic Engineering of Lipase to Metabolic Engineering of Fatty Acid Biosynthetic Pathway

Critical Reviews in Biotechnology. Feb, 2017  |  Pubmed ID: 26526353

Microbial production routes, notably whole-cell lipase-mediated biotransformation and fatty-acids-derived biosynthesis, offer new opportunities for synthesizing biodiesel. They compare favorably to immobilized lipase and chemically catalyzed processes. Genetically modified whole-cell lipase-mediated in vitro route, together with in vivo and ex vivo microbial biosynthesis routes, constitutes emerging and rapidly developing research areas for effective production of biodiesel. This review presents recent advances in customizing microorganisms for producing biodiesel, via genetic engineering of lipases and metabolic engineering (including system regulation) of fatty-acids-derived pathways. Microbial hosts used include Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and Aspergillus oryzae. These microbial cells can be genetically modified to produce lipases under different forms: intracellularly expressed, secreted or surface-displayed. They can be metabolically redesigned and systematically regulated to obtain balanced biodiesel-producing cells, as highlighted in this study. Such genetically or metabolically modified microbial cells can support not only in vitro biotransformation of various common oil feedstocks to biodiesel, but also de novo biosynthesis of biodiesel from glucose, glycerol or even cellulosic biomass. We believe that the genetically tractable oleaginous yeast Yarrowia lipolytica could be developed to an effective biodiesel-producing microbial cell factory. For this purpose, we propose several engineered pathways, based on lipase and wax ester synthase, in this promising oleaginous host.

Impairment of Endoplasmic Reticulum is Involved in β-cell Dysfunction Induced by Microcystin-LR

Environmental Pollution (Barking, Essex : 1987). Apr, 2017  |  Pubmed ID: 28162804

Microcystins (MCs) widely distributed in freshwaters have posed a significant risk to human health. Previous studies have demonstrated that exposure to MC-LR impairs pancreatic islet function, however, the underlying mechanisms still remain unclear. In the present study, we explored the role of endoplasmic reticulum (ER) impairment in β-cell dysfunction caused by MC-LR. The result showed that MC-LR modified ER morphology evidenced by increased ER amount and size at low doses (15, 30 or 60 μM) and vacuolar and dilated ER ultrastructure at high doses (100 or 200 μM). Also, insulin content showed increased at 15 or 30 μM but declined at 60, 100, or 200 μM, which was highly accordant with ER morphological alteration. Transcriptomic analysis identified a number of factors and several pathways associated with ER protein processing, ER stress, apoptosis, and diabetes mellitus in the cells treated with MC-LR compared with non-treated cells. Furthermore, MC-LR-induced ER stress significantly promoted the expression of PERK/eIF2α and their downstream targets (ATF4, CHOP, and Gadd34), which indicates that PERK-eIF2α-ATF4 pathway is involved in MC-LR-induced insulin deficiency. These results suggest that ER impairment is an important contributor to MC-LR-caused β-cell failure and provide a new insight into the association between MCs contamination and the occurrence of human diseases.

Preparation of Biodiesel with Liquid Synergetic Lipases from Rapeseed Oil Deodorizer Distillate

Applied Biochemistry and Biotechnology. Mar, 2017  |  Pubmed ID: 28353044

To reduce industrial production cost, cheap and easily available rapeseed oil deodorizer distillates were used as feedstock to prepare biodiesel in this study. As a result, liquid forms of Candida rugosa lipase and Rhizopus oryzae lipase (ROL) were functioned as new and effective catalysts with biodiesel yield of 92.63% for 30 h and 94.36% for 9 h, respectively. Furthermore, the synergetic effect between the two lipases was employed to enhance biodiesel yield with a result of 98.16% in 6 h under optimized conditions via response surface methodology. The obtained conversion rate surpassed both yields of the individual two lipases and markedly shortened the reaction time. The resultant optimal conditions were ROL ratio 0.84, water content 46 wt% (w/w), reaction temperature 34 °C, and reaction time 6 h.

Carbon Nanotube Filled with Magnetic Iron Oxide and Modified with Polyamidoamine Dendrimers for Immobilizing Lipase Toward Application in Biodiesel Production

Scientific Reports. Mar, 2017  |  Pubmed ID: 28358395

Superparamagnetic multi-walled carbon nanotubes (mMWCNTs) were prepared by filling multi-walled carbon nanotubes (MWCNTs) with iron oxide, and further modified by linking polyamidoamine (PAMAM) dendrimers (mMWCNTs-PAMAM) on the surface. Then, mMWCNTs-PAMAM was employed as the carrier and successfully immobilized Burkholderia cepacia lipase (BCL) via a covalent method (BCL-mMWCNTs-G3). The maximum activity recovery of the immobilized lipase was 1,716% and the specific activity increased to 77,460 U/g-protein, 17-fold higher than that of the free enzyme. The immobilized lipase displayed significantly enhanced thermostability and pH-resistance, and could efficiently catalyze transesterification to produce biodiesel at a conversion rate of 92.8%. Moreover, it possessed better recycling performance. After 20 cycles of repeated used, it still retained ca. 90% of its original activity, since the carbon nanotube-enzyme conjugates could be easily separated from the reaction mixture by using a magnet. This study provides a new perspective for biotechnological applications by adding a magnetic property to the unique intrinsic properties of nanotubes.

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