Browsing by Author "Pawaskar, G.-M."

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Expression of Bacillus licheniformis chitin deacetylase in E. coli pLysS: Sustainable production, purification and characterisation
(2019) Bhat, P.; Pawaskar, G.-M.; Raval, R.; Cord-Landwehr, S.; Moerschbacher, B.; Raval, K.
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 m , V max and K cat 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. 2019 Elsevier B.V.
Expression of Bacillus licheniformis chitin deacetylase in E. coli pLysS: Sustainable production, purification and characterisation
(Elsevier B.V., 2019) Bhat, P.; Pawaskar, G.-M.; Raval, R.; Cord-Landwehr, S.; Moerschbacher, B.; Raval, K.
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. Km, Vmax and Kcat 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. © 2019 Elsevier B.V.
Screening of chitin deacetylase producing microbes from marine source using a novel receptor on agar plate
(Elsevier B.V., 2019) Pawaskar, G.-M.; Pangannaya, S.; Raval, K.; Trivedi, D.; Raval, R.
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. © 2019 Elsevier B.V.