Process Evaluation and Kinetics of Recombinant Chitin Deacetylase Expression in E. coli Rosetta pLysS Cells Using a Statistical Technique

Summary

In the current protocol, a statistical technique, central composite design (CCD), was applied to optimize the process conditions for the expression of recombinant bacterial chitin deacetylase (BaCDA) in E. coli Rosetta pLysS cells. The employment of CCD resulted in a ~2.39-fold increase in the expression and activity of BaCDA.

Abstract

In recent years, the greener route of the deacetylation of chitin to chitosan using the enzyme chitin deacetylase has gained importance. Enzymatically converted chitosan with emulating characteristics has a broad range of applications, particularly in the biomedical field. Several recombinant chitin deacetylases from various environmental sources have been reported, but there are no studies on process optimization for the production of these recombinant chitin deacetylases. The present study used the central composite design of response surface methodology to maximize the recombinant bacterial chitin deacetylase (BaCDA) production in E. coli Rosetta pLysS. The optimized process conditions were 0.061% glucose concentration, 1% lactose concentration, an incubation temperature of 22 °C, an agitation speed at 128 rpm, and 30 h of fermentation. At optimized conditions, the expression due to lactose induction was initiated after 16 h of fermentation. The maximum expression, biomass, and BaCDA activity were recorded 14 h post-induction. At the optimized condition, the BaCDA activity of expressed BaCDA was increased ~2.39-fold. The process optimization reduced the total fermentation cycle by 22 h and expression time by 10 h post-induction. This is the first study to report the process optimization of recombinant chitin deacetylase expression using a central composite design and its kinetic profiling. Adapting these optimal growth conditions could result in cost-effective, large-scale production of the lesser-explored moneran deacetylase, embarking on a greener route for biomedical-grade chitosan production.

Introduction

Chitin, a structural β, 1-4 glycosidic linked natural polymer, is the second-most abundant polysaccharide in nature after cellulose. Despite this fact, chitin has limited industrial applications due to its insolubility¹. This bottleneck is addressed by subjecting chitin to N-deacetylation, which imparts a positive charge and increases the solubility of the resulting polymer, chitosan¹. Chitin can be modified to chitosan through two different routes: chemical and enzymatic. The biomedical application of chitosan requires controlled and defined deacetylation, which is restricted in chemical routes

Protocol

1. Expression media and culture condition

1. Transform the pET-22b vector containing the BaCDA gene into E. coli Rosetta pLysS competent cells using the heat-shock method, as described in¹⁵.
   NOTE: Care has to be taken while working with microorganisms. All microbiological work has to be performed inside a biosafety cabinet hood to avoid contamination.
2. Perform the preliminary expression study in TB media containing 0.05% (w/v) glucose and 0.2% (w/v) lactose at 16 °C and 180 rpm. Grow 6.792 x 10⁷ E. coli Rosetta pLysS cells in 100 mL of media containing

Results

Process optimization of expression of periplasmic recombinant enzyme chitin deacetylase in E. coli using central composite design (CCD)
The pET22b-BaCDA construct, when grown in unoptimized conditions, gave a maximum biomass yield and BaCDA activity of 22.26 ± 0.98 g/L and 84.67 ± 0.56 U/L, respectively¹⁵. In the current study, a statistical approach CCD was adopted to find the optimal process conditions for expressing periplasmic recombinant enzyme .......

Discussion

Deacetylated chitin, chitosan, has many applications, especially in the biomedical field³⁰. However, the reproducibility of chitosan concerning its degree of acetylation (DA) and pattern of acetylation (PA) is a major concern in addition to other environmental apprehensions. The greener route, using enzymes, has thus been exploited. The array of CDAs can be employed to create chitosan with a unique pattern of deacetylation, which would increase their biomedical applications⁴

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Kits
Acetate assay kit	Megazyme, Ireland	K-ACETAK	The protocol has been slightly modified and optimized to perform the assay in 96 well plate
Glucose estimation kit	Agappe diagnosis Ltd., India	12018013	The protocol has been slightly modified and optimized to perform the assay in 96 well plate
Chemicals
Acetic acid	Hi-media, India	AS001	Used for preparing SDS-PAGE staining and destaing solution
Acrylamide	Hi-media, India	MB068	Used for preparing SDS-PAGE gel
Ammonium pursulphate	Hi-media, India	MB003	Used for preparing SDS-PAGE gel
Bis-acrylamide	Hi-media, India	MB005	Used for preparing SDS-PAGE gel
Coomassie briliiant blue G-250	Hi-media, India	MB092	Used for preparing SDS-PAGE staining and destaing solution
Coomassie briliiant blue R-250	Hi-media, India	MB153	Used for preparing Bardford's assay
Ethylene glycol chitosan	Sigma-aldrich, USA	E1502	Used to prepare Ethylene glycol chitin and Ethylene glycol chitin was used as substrate for enzymatic reaction
D-glucose	Hi-media, India	MB037	Used as an media component.
Imidazole	Hi-media, India	GRM1864	Used in lysis buffer
Lactose	Hi-media, India	GRM017	Used as an media component.
Methanol	Finar, India	30930LC250	Used for preparing SDS-PAGE staining and destaing solution
Sodium chloride (NaCl)	Hi-media, India	MB023	Used in lysis buffer
Phosphoric acid	Hi-media, India	MB157	Used for preparing Bardford's assay
sodium dodecyl sulfate (SDS)	Hi-media, India	GRM6218	Used for preparing SDS-PAGE gel
Sodium phosphate dibasic anhydrous	Hi-media, India	MB024	Used to prepare TB sald for media and buffer for enzymatic reaction.
Sodium phosphate monobasic anhydrous	Hi-media, India	GRM3964	Used to prepare TB sald for media and buffer for enzymatic reaction.
Tetramethylethylenediamine (TEMED)	Hi-media, India	MB026	Used for preparing SDS-PAGE gel
Tris base	Hi-media, India	MB029	Used for preparing SDS-PAGE gel
Tryptone	Hi-media, India	RM7707	Used as an media component.
Yeast extract	Hi-media, India	RM027	Used as an media component.
Equipment
AlphaImager HP gel documentation unit	ProteinSimple, USA	92-13823-00	Used to capture SDS-PAGE photographs
Benchtop mixer	Eppendorf, Germany	9.776 660	Used to keep for enzymatic reaction with 2 mL adaptor
Bioincubator shaker	Trishul instruments, India	13410622	Used to incubate bacterial culture at different temparature and RPM
Biospectrophotometer	Eppendorf, Germany	6135000009	Used to take all spectroscopic readings
Cooling centrifuge	Eppendorf, Germany	5805000017	Used to centrifuge culture, lysate and all other centrifuging protocols
Dry bath	Labnet International, USA	S81522039	Used to denature protein sample for SDS-PAGE
Micropipettes	Eppendorf, Germany	3123000900	Used throghout the protocol for volume measurements
Rocker shaker	Trishul instruments, India	11770719	Used to shake SDS-PAGE gel for staining and destaining
SDS-PAGE unit	Bio-Rad, USA	1658001FC	Used to cast and run SDS-PAGE gel
Ultra sonicator	Sonics & Materials, Inc., USA	VCX 130	Used to lyse the bacterial cell by ultra sonication
Weighing balance	Sartorius, Germany	BSA124 S	Used to measure weight throughout the protocol
Devices
Nanosep Centrifugal Devices with Omega Membrane (3 kDa)	PALL life sciences, USA	OD003C33	Used to separate enzyme after substrate treatment
Softwares	Version	Developed at
MINITAB	17.0  (Trial version)	The Pennsylvania State University	Used to design the experimental model and analyse the data
ImageJ	1.53o	National Institutes of Health (NIH)	Used to analyse the expression level using SDS-PAGE image
Plasmid
pET22b (+) DNA—Novagen	Merck- Millipore, USA	69744	Stored at − 20 °C
Cells
E. coli Rosetta pLysS—Novagen	Merck- Millipore, USA	70956	Maintained in Luria–Bertani (LB) broth containing 25% glycerol at − 80 °C