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.
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.
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 routes2,3. This limitation can be addressed using chitin deacetylases (CDAs), a green enzymatic approach for the deacetylation process4,5.
Chitin deacetylase belongs to the carbohydrate esterase 4 (CE-4) family, defined in the carbohydrate-active enzymes (CAZY) database. The enzymes of the CE-4 family share the NodB homology or polysaccharide deacetylase domain as the conserved region. The central composite design (CCD), a statistical tool, is used for the optimization of several wild-type chitin-modifying enzymes6,7,8,9. However, the downstream steps in the usage of wild-type organisms becomes tedious, hence the shift toward recombinant enzymes10,11,12,13,14,15,16,17. In recent years, halophilic recombinant CDA from marine sources have gained importance due to their ease in the industrial application and production of biomedical-grade chitosan18,19.
Recombinant enzyme production in E. coli has a limitation on the process, and media optimization is needed as its expression in E. coli varies depending on the gene and plasmid used20. Thus, screening of a suitable process and nutrient parameters becomes important. One factor at a time (OFAT), the commonly employed optimization method, requires tremendous resources and time to perform step-by-step experiments. This method suffers from a lack of statistical information regarding the interaction among the parameters20,21,22,23. Therefore, the CCD of response surface methodology (RSM) was adopted to study the halophilic bacterial chitin deacetylase (BaCDA) expression yield and BaCDA activity in E. coli Rosetta pLysS. The parameters considered for expression optimization in the E. coli host were lactose concentration, glucose concentration, incubation temperature, agitation rate, and incubation time. In most E. coli expression studies, Luria Bertani (LB) media with Isopropyl β-d-1-thiogalactopyranoside (IPTG) was used as an inducer. This addition of IPTG required regular growth monitoring24. These recurrent mediations during the fermentative process also open avenues for contamination. Hence, research groups have shifted to terrific broth (TB) with lactose as the inducer. The inclusion of lactose in the media instead of IPTG addresses this concern; E. coli consumes this lactose and produces allo-lactose as a by-product, resulting in an auto-induction condition. This auto-inducer media includes glycerol, which has exhibited improved yields of recombinant protein25. This overexpression of recombinant proteins in TB media was further improved by optimizing the process parameters. In the present study, a central composite design was applied to optimize the heterologous expression of halophilic BaCDA in E. coli Rosetta pLysS cells. The process parameters chosen were incubation temperature, agitation rate, and incubation time, and the nutrient parameters evaluated were glucose and lactose concentration. The halophilic BaCDA expression was evaluated with the predicted optimized condition and cross-validated using SDS-PAGE.
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</sup…
The authors have nothing to disclose.
The authors would like to thank Manipal Academy for Higher Education (MAHE) for the MAHE UNSW fund, and the authors would like to thank the Council of Scientific & Industrial Research – Human Resource Development Group (CSIR-MHRD), Govt. of India for a senior research fellowship, award letter-number 09/1165(0007)2K19 EMR-I dated 31.3.2019.
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 |
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