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

In recent years, the greener route of the deacetylation of chitin to chitosan using the enzyme chitin deacetylase has gained importance. Enzymatically ...

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 ...

manipal institute of technology

Manipal Academy of Higher Education (MAHE)

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.

γ-Polyglutamic acid (γ-PGA) is a biosynthetic outcome of glutamic acid polymerization by microbes. In the current study, we have isolated Bacillus methylotrophicus on solid differential media containing methylene blue. This is the first report mentioning the use of methylene blue to distinguish the monomeric and polymeric form of glutamic acid in the liquid medium using UV-Vis spectrophotometer. Our method can simplify the analytical process of γ-PGA confirmation using the aforementioned studies. This screening protocol is sensitive to the detection of γ-PGA quantities as low as 3 μg/mL; thus, the potent producers can be effectively screened. Furthermore, we have carried out process optimization of the present strain for γ-PGA production wherein we could obtain 1.4-fold improvement in the yield with respect to utilization of carbon source and 2.6-fold increase with respect to nitrogen source under submerged fermentation at a shake flask level. We have shown an increase in γ-PGA titer from 1.5 to 36 g/L using mannitol, monosodium glutamate, peptone, and tween 20.

Selection of an Effective Indicator for Rapid Detection of Microorganisms Producing γ-Polyglutamic Acid and Its Biosynthesis Under Submerged Fermentation Conditions Using Bacillus methylotrophicus.

Poly-γ-glutamic acid (PGA) is biosynthesized by various Bacillus species through PGA synthetase, encoded by the PGA operon comprised of the ywsC and ywtABC genes. Due to the minimal available knowledge, understanding the expression pattern of PGA operon genes is pivotal. In this study, the effect of glucose and glutamic acid on the global gene expression profile of Bacillus subtilis Natto3 was investigated using high throughput microarray, with an emphasis on the PGA operon and genes influencing PGA production. Two treatment groups (set1-in the presence of glutamic acid and set2-in the presence of glutamic acid + glucose) were analyzed against the control (in the presence of glucose). In the microarray, both the groups showed a trend of up-regulation for ywsC and ywtA genes (log2 fold change of 0.55, P = 0.0194, 0.92, P = 0.0069 in set1 and 0.78, P = 0.0023, 0.59, P = 0.0172 in set2, respectively) and down-regulation of ywtB and ywtC genes (log2 fold change of -1.83, P = 0.0001, -1.42, P = 0.0017 in set1 and -1.52, P = 0.0012, -0.55, P = 0.1112 in set2, respectively), supporting the indispensability of the ywsC and ywtA genes in PGA production. Interestingly, the ywtB and ywtC genes, belonging to the same operon, were down-regulated in both the conditions (set1 and set2). To the best of our knowledge, this expression pattern of PGA operon genes is a unique observation.

Expression dynamics of the poly-γ-glutamic acid biosynthesis genes of Bacillus subtilis in response to glucose and glutamic acid-a pilot study.

Chitosan, the deacetylated form of chitin, a natural polysaccharide, is known for its various biomedical applications. The present study aimed at exploring the immunomodulatory properties of chitosan (CSNP) and gallic acid-grafted chitosan (cGANP) nanoparticles in mice model of cyclophosphamide (CPA)-induced immunosuppression. In addition, chitooligosaccharides, the hydrolysed form of chitin and chitosan, were also evaluated for its potential against immunosuppression in mice. CPA (80 mg/kg/ip) induced significant immunosuppression, which was reversed with cGANP treatment as indicated by a significant increase in the thymus and spleen indices compared to the CPA-treated group. The CSNP and chitooligosaccharides (chitin and chitosan) failed to reverse CPA-induced changes. ELISA revealed an elevation in the levels of IL-6 and a reduction in IFN-γ levels with CPA treatment. All the test compounds reduced the IL-6 levels, whereas only the nanoparticle formulations (CSNP and cGANP) exhibited a significant augmentation in the IFN-γ levels. Both the cytokines, IL-6 and IFN-γ, are secreted separately by two different types of T helper cells (Th cells), which mediate cellular and humoral immune responses in a coordinated manner. Th-1 cells release IFN-γ, facilitating cell-mediated immunity, whereas IL-6 is released by Th-2 cells, expediting humoral immune response. The nanoparticles (CSNP and cGANP) seemed to be better immune enhancers than the chitooligosaccharides owing to their ability to reverse the cytokine changes induced by CPA. Overall, it was evident that the nanoparticles, most likely, boosted the cell-mediated immunity through the induction of the Th-1 branch of the immune response.

Immunomodulatory role of chitosan-based nanoparticles and oligosaccharides in cyclophosphamide-treated mice.

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.

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.

Cloning, expression, purification and characterization of chitin deacetylase extremozyme from halophilic  B8W22.

Chitinases convert chitin into chitin oligomers and are also known antifungal agents. Chitin oligomers have numerous industrial applications. However, chitin's crystalline nature requires pretreatment before breakdown into oligomers. In the study, a novel marine bacterium  is isolated from the Arabian Sea. Bacterial growth in different crystalline chitin substrates like chitin powder, chitin flakes, and colloidal chitin confirmed the chitinase presence in bacterium could act upon insoluble crystalline chitin with the fractional release of oligomers. The domain architecture analysis of the chitinase confirmed the presence of two N-terminal LysM domains which help enzyme action on crystalline chitin. Statistical optimization of media and Process parameters revealed glycerol, yeast extract, magnesium chloride, and manganese sulfate as significant media components along with colloidal chitin. The optimum process parameters such as pH 7, temperature 40 °C, inoculum size 12.5% (v/v), and inoculum age 20 hours enhanced the specific enzyme activity to ±146.2 U/mL, ±114.9 U/mL and ±175.4 U/mL against chitin powder, chitin flakes and colloidal chitin respectively, which is five to six times higher than basal level activity. The antifungal activity of chitinase against plant pathogenic fungi like  and  revealed a zone of inhibition with 14 mm diameter.

A marine chitinase from  with antifungal activity and broad specificity toward crystalline chitin degradation.

The role of NLRP3 in the tumour microenvironment is elusive. In some cancers, the activation of NLRP3 causes a worse prognosis and in some cancers, NLRP3 increases chances of survivability. However, in many cases where NLRP3 has a protumorigenic role, inhibition of NLRP3 would be a crucial step in therapy. Consequently, activation of NLRP3 would be of essence when inflammation is required. Although many ways of inhibiting and activating NLRP3 in cancers have been discussed before, not a lot of focus has been given to chitin and chitosan in this context. The availability of these marine compounds and their versatility in dealing with inflammation needs to be investigated further in relation with cancers, along with other natural extracts. In this review, the effects of NLRP3 on gastrointestinal and gynaecological cancers and the impact of different natural extracts on NLRP3s with special emphasis on chitin and chitosan is discussed. A research gap in using chitin derivatives as anti/pro-inflammatory agents in cancer treatment has been highlighted.

Ritu Raval

Department of Biotechnology

manipal institute of technology

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

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Ritu Raval

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biotechnology

manipal institute of technology

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

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Department of Biotechnology