adsorptive-bioprocess-improves-yield-melanin-from-pseudomonas

Adsorptive Bioprocess Improves Yield of Melanin from <em>Pseudomonas stutzeri</em>

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as ...

National Institute of Technology

Uricase (urate: oxygen oxidoreductase, EC 1.7.3.3), an enzyme belonging to the class of oxidoreductases, catalyzes the enzymatic oxidation of uric acid to allantoin and finds a wide variety of application as therapeutic and clinical reagent. In this study, uricase production ability of the bacterial strains isolated from deep litter poultry soil is investigated. The strain with maximum extracellular uricase production capability was identified as Xanthomonas fuscans subsp. aurantifolii based on 16S rRNA sequencing. Effect of various carbon and nitrogen sources on uricase productivity was investigated. The uricase production for this strain was optimized using statistically based experimental designs and resulted in uricase activity of 306 U/L, which is 2 times higher than initial uricase activity. Two-step purification, such as ammonium sulfate precipitation and aqueous two-phase system, was carried out and a twofold increase in yield and specific activity was observed.

Enhancement of a novel extracellular uricase production by media optimization and partial purification by aqueous three-phase system.

Naringinase bioprocess based on Bacillus methylotrophicus was successfully scaled up based on constant oxygen transfer rate (OTR) as the scale-up criterion from 5-L bioreactor to 20-L bioreactor. OTR was measured in 5 and 20-L bioreactor under various operating conditions using dynamic method. The operating conditions, where complete dispersion was observed were identified. The highest OTR of 0.035 and 0.04 mMol/L/s was observed in 5 and 20-L bioreactor, respectively. Critical dissolved oxygen concentration of novel isolated strain B. methylotrophicus was found to be 20% of oxygen saturation in optimized medium. The B. methylotrophicus cells grown on sucrose had maximum oxygen uptake rate of 0.14 mMol/L/s in optimized growth medium. The cells produced the maximum naringinase activity of 751 and 778 U/L at 34 hr in 5 and 20-L bioreactors, respectively. The maximum specific growth rate of about 0.178/hr was observed at both the scales of operations. The maximum naringinase yield of 160 and 164 U/g biomass was observed in 5 and 20-L bioreactors, respectively. The growth and production profiles at both scales were similar indicating successful scale-up strategy for B. methylotrophicus culture.

Scale-up of naringinase production process based on the constant oxygen transfer rate for a novel strain of Bacillus methylotrophicus.

Chitin, the biopolymer of the N-acetylglucosamine, is the most abundant biopolymer on the planet after cellulose. However owing to its crystalline nature, its deacetylated derivative; chitosan is industrially more potent. This conversion on an enzymatic scale can be made using chitin deacetylase. The metagenomics library constructed from the soil exposed to chitin and chitosan yielded chitin modifying enzymes, one of them being chitin deacetylase (CDA) utilized for the present study. The gene was amplified and expressed using the pET 22b vector in E. coli Rosetta cells. The effect of two additives; chitin and glycerol on the CDA activity were studied. The inclusion of glycerol in the medium improved the biomass by 50% from the initial value of 1.25g/l to 2.5g/l. The activity of CDA increased from 90μmol/min/ml to 343μmol/min/ml. The CDA activity reported in the present paper is the highest observed for any strain. The addition of glycerol to the media not only helped improve the yield of the chitin deacetylase but also imparted value addition to the waste of the biofuel industry.

Expression studies of Bacillus licheniformis chitin deacetylase in E. coli Rosetta cells.

The difficulty in removal of heavy metals at concentrations below 10 mg/L has led to the exploration of efficient adsorbents for removal of heavy metals. The adsorption capacity of biosynthesized melanin for Mercury (Hg(II)), Chromium (Cr(VI)), Lead (Pb(II)) and Copper (Cu(II)) was investigated at different operating conditions like pH, time, initial concentration and temperature. The heavy metals adsorption process was well illustrated by the Lagergren's pseudo-second-order kinetic model and the equilibrium data fitted excellently to Langmuir isotherm. Maximum adsorption capacity obtained from Langmuir isotherm for Hg(II) was 82.4 mg/g, Cr(VI) was 126.9 mg/g, Pb(II) was 147.5 mg/g and Cu(II) was 167.8 mg/g. The thermodynamic parameters revealed that the adsorption of heavy metals on melanin is favorable, spontaneous and endothermic in nature. Binding of heavy metals on melanin surface was proved by Fourier Transform Infrared Spectroscopy (FT-IR) and X-ray Photoelectron Spectroscopy (XPS). Contemplating the results, biosynthesized melanin can be a potential adsorbent for efficient removal of Hg(II), Cr(VI), Pb(II) and Cu(II) ions from aqueous solution.

Kinetic and thermodynamic studies on the adsorption of heavy metals from aqueous solution by melanin nanopigment obtained from marine source: Pseudomonas stutzeri.

Heavy metals are one of deadly contaminants in ground water across the globe. Thus, herein, this data set comprises experimental and modelled data on the removal of heavy metals from ground water using melanin synthesized by the marine bacteria . Characterization of biosynthesized melanin and modelling of the kinetic and the thermodynamic study on adsorption of heavy metals such as mercury (Hg(II)), lead (Pb(II)), chromium (Cr(VI)), and copper (Cu(II)) are included in this article. Apart from the study of parameters involved in adsorption such as pH, temperature, concentration and time; the data from these studies are modelled to analyze the nature and characteristic of heavy metals adsorbing to melanin nanoparticles. The figures from models, results from models as tables, characterization and analytical figures are depicted in this work.

Data on the removal of heavy metals from aqueous solution by adsorption using melanin nanopigment obtained from marine source: .

Chitosan is a deacetylated form of naturally occurring polymer; chitin. On an industrial scale, the deacetylation of chitin to chitosan is performed using harsh chemicals like sodium hydroxide. This not only adds to the environmental pollution but the product is also random in terms of its deacetylation. This shortcoming can be addressed by using enzymes like chitin deacetylase (CDA). The screening of these organisms would require a reliable, fast and sensitive screening method. The deacetylation of chitin into chitosan, releases acetate as the byproduct of the reaction. A receptor which specifically binds to the acetate ion was synthesized chemically. The receptor upon binding with the acetate ion emitted a fluorescence which could be viewed using the gel documentation unit. The receptor was optimized for the screening of CDA producing microbes with the positive fungal control as Penicillium sp. and bacterial control as Bacillus megaterium. A parallel study with the 4-Nitroacetanilide, the reported screening indicator for CDA was performed. The results obtained with the receptor in the present study were concordant with the 4-Nitroacetanilide. Upon standardization, the protocol was extended for the screening of CDA producing microbes from the marine crustacean dumped soil and water samples. The CDA activity of these microbes was further confirmed using spectrophotometric MBTH assay. This is the first report using this receptor for the screening of CDA producers. The method is not only sensitive but also reproducible and can be extended for a high throughput screening of CDA producers.

Screening of chitin deacetylase producing microbes from marine source using a novel receptor on agar plate.

Chitosan obtained by enzymatic deacetylation of chitin using chitin deacetylase (CDA) holds promise primarily due to the possibility to yield chitosan with non-random patterns of acetylation and more environmentally friendly process compared to chemical deacetylation. In the present study, a sustainable bioprocess is reported for over-expression of a bacterial CDA in E. coli pLysS cells. A Bacillus licheniformis CDA gene is identified in the genome of the bacterium, cloned, and expressed, yielding enzymatically active recombinant protein. For enzyme production, a growth medium is formulated using carbon and nitrogen sources, which do not compete with the human food chain. The maximum enzyme activity of 320 ± 20 U/mL is achieved under optimized conditions. The CDA productivity is improved by about 23 times in shake flask culture by optimizing operating conditions and medium components. The CDA is purified and the enzyme kinetic values i.e. K, V and K are reported. Also the effect of cofactors, temperature, and pH on the enzyme activity is reported. Further, economic yield is proposed for production of CDA through this bioprocess.

Expression of Bacillus licheniformis chitin deacetylase in E. coli pLysS: Sustainable production, purification and characterisation.

Heavy metals like mercury, chromium, lead and copper present in groundwater at lower concentrations cause severe health issues and can even be fatal when consumed. The biopigment/biopolymer melanin can be reaped from different sources like bacterium, fungus, and human hair. It has excellent heavy metal ion scavenging property and can be exploited for non-biological applications, substantially including water purification. In this work, melanin nanoparticles were derived from the marine bacterium Pseudomonas stutzeri and were coated onto hydrophobic polyvinylidene fluoride (PVDF) membrane as a support, for batch and continuous removal of heavy metal studies. Batch studies on the effect of pH, temperature and adsorbate dose and continuous adsorption studies on the effect of flow rate, adsorbate and adsorbent mass loadings were carried out by using biosynthesised melanin-coated PVDF membranes for the removal of Hg(II), Cr(VI), Pb(II) and Cu(II). Scanning electron microscope (SEM) images revealed the surface morphology, Fourier-transform infrared spectroscopy (FTIR) and energy-dispersive X-ray spectroscopy (EDS) deciphered the chemical characteristics of melanin-coated PVDF membranes before and after adsorption. Contact angle measurement confirmed the improvement in hydrophilicity of PVDF membrane upon coating with melanin. The maximum removal percentages of heavy metals achieved by melanin-coated PVDF membranes under batch mode operation were 87.6%, 88.45%, 91.8% and 95.8% for mercury, chromium, lead and copper, respectively optimised at 318 K and pH of 3 for chromium and 5 for other metals. However, the continuous mode of operation with a flow rate of 0.5 mL/min having 1 mg/L of heavy metal solution concentration exposed to 50 mg of melanin loading with a working volume of 200 mL showed better removal efficiencies compared with batch mode. The dynamic studies using Thomas and Yoon-Nelson models described the transient stage of the breakthrough curve and the model constants were calculated for column design and scale-up.

Batch and continuous studies on the removal of heavy metals from aqueous solution using biosynthesised melanin-coated PVDF membranes.

The metalloid arsenic is one of the most conspicuous groundwater contaminants in the Indian subcontinent and its removal from aqueous medium is the main focus of this study. The study aims at functionalising melanin using iron and copper for the efficient removal of arsenic and rendering water fit for consumption. Melanin obtained from the marine bacteria Pseudomonas stutzeri was functionalised by iron impregnation (Fe-melanin) and copper impregnation (Cu-melanin). Morphological studies using FESEM portrayed the impregnated iron and copper granules on the surface of melanin, while XRD analysis confirmed the presence of FeO and CuO on melanin. Adsorption studies on As (V) and As (III) were conducted using Fe-melanin and Cu-melanin for different operating variables like pH, temperature and contact time. More than 99% per cent of As (III) and As (V) from water was removed at a pH range between 4 and 6 within 50 min in the case of Fe-melanin and 80 min for Cu-melanin. Adsorption equilibrium studies showed better fit with Langmuir adsorption isotherm and had good agreement with Redlich-Peterson's three-parameter model. The maximum adsorption capacities of Fe-melanin and Cu-melanin obtained from Langmuir adsorption model are 50.12 and 20.39 mg/g, respectively, for As (V) and similarly 39.98 and 19.52 mg/g, respectively, for As (III). Arsenic-binding to the functionalised melanin was confirmed using FT-IR and the XPS analysis. Reuse of the adsorbent was effectively done by desorbing the iron and copper together with the bound As (III) and As (V) and further re-impregnation of iron and copper in melanin. Re-functionalised melanin showed 99% adsorption efficiency up to four cycles of adsorption/desorption.

Adsorptive removal of trivalent and pentavalent arsenic from aqueous solutions using iron and copper impregnated melanin extracted from the marine bacterium Pseudomonas stutzeri.

Chitin deacetylase (CDA) (EC 3.5.1.41) is a hydrolytic enzyme that belongs to carbohydrate esterase family 4 as per the CAZY database. The CDA enzyme deacetylates chitin into chitosan. As the marine ecosystem is a rich source of chitin, it would also hold the unexplored extremophiles. In this study, an organism was isolated from 40 m sea sediment under halophilic condition and identified as  B8W22 by 16S rRNA sequencing. The CDA gene from the isolate was cloned and overexpressed in  Rosetta pLysS and purified using a Ni-NTA affinity chromatography. The enzyme was found active on both ethylene glycol chitin (EGC) and chitooligosaccharides (COS). The enzyme characterization study revealed, maximum enzyme velocity at one hour, optimum pH at 7 with 50 mM Tris-HCl buffer, optimum reaction temperature of 30 ºC in standard assay conditions. The co-factor screening affirmed enhancement in the enzyme activity by 142.43 ± 7.13% and 146.88 ± 4.09% with substrate EGC and COS, respectively, in the presence of 2 mM Mg. This activity was decreased with the inclusion of EDTA and acetate in the assay solutions. The enzyme was found to be halotolerant; the relative activity increased to 116.98 ± 3.87% and 118.70 ± 0.98% with EGC and COS as substrates in the presence of 1 M NaCl. The enzyme also demonstrated thermo-stability, retaining 87.27 ± 2.85% and 94.08 ± 0.92% activity with substrate EGC and COS, respectively, upon treatment at 50 ºC for 24 h. The kinetic parameters  ,  , and   were 3.06E-05 µg mL, 3.06E + 01 µM mg min and 3.27E + 04 s, respectively, with EGC as the substrate and 7.14E-07 µg mL, 7.14E + 01 µM mg min and 1.40E + 06 s, respectively, with COS as the substrate. The enzyme was found to be following Michaelis-Menten kinetics with both the polymeric and oligomeric substrates. In recent years, enzymatic conversion of chitosan is gaining importance due to its known pattern of deacetylation and reproducibility. Thus, this CDA extremozyme could be used for industrial production of chitosan polymer as well as chitosan oligosaccharides for biomedical application.

Article	Year
Enhancement of a novel extracellular uricase production by media optimization and partial purification by aqueous three-phase system. Preparative biochemistry & biotechnology\| PubMed ID: 25674929	2015
Scale-up of naringinase production process based on the constant oxygen transfer rate for a novel strain of Bacillus methylotrophicus. Preparative biochemistry & biotechnology\| PubMed ID: 27340886	2017
Expression studies of Bacillus licheniformis chitin deacetylase in E. coli Rosetta cells. International journal of biological macromolecules\| PubMed ID: 28223214	2017
Kinetic and thermodynamic studies on the adsorption of heavy metals from aqueous solution by melanin nanopigment obtained from marine source: Pseudomonas stutzeri. Journal of environmental management\| PubMed ID: 29533829	2018
Data on the removal of heavy metals from aqueous solution by adsorption using melanin nanopigment obtained from marine source: . Data in brief\| PubMed ID: 30112433	2018
Screening of chitin deacetylase producing microbes from marine source using a novel receptor on agar plate. International journal of biological macromolecules\| PubMed ID: 30902716	2019
Expression of Bacillus licheniformis chitin deacetylase in E. coli pLysS: Sustainable production, purification and characterisation. International journal of biological macromolecules\| PubMed ID: 30910681	2019
Batch and continuous studies on the removal of heavy metals from aqueous solution using biosynthesised melanin-coated PVDF membranes. Environmental science and pollution research international\| PubMed ID: 31602598	2020
Adsorptive removal of trivalent and pentavalent arsenic from aqueous solutions using iron and copper impregnated melanin extracted from the marine bacterium Pseudomonas stutzeri. Environmental pollution (Barking, Essex : 1987)\| PubMed ID: 31744681	2020
Cloning, expression, purification and characterization of chitin deacetylase extremozyme from halophilic B8W22. 3 Biotech\| PubMed ID: 34917446	2021

Keyur Raval

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