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

Abstract

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 Bacillus aryabhattai B8W22 by 16S rRNA sequencing. The CDA gene from the isolate was cloned and overexpressed in E. coli 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 Mg²⁺. 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 K _m, V _max, and K _cat were 3.06E-05 µg mL^-1, 3.06E + 01 µM mg^-1 min^-1 and 3.27E + 04 s^-1, respectively, with EGC as the substrate and 7.14E-07 µg mL^-1, 7.14E + 01 µM mg^-1 min^-1 and 1.40E + 06 s^-1, 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 BaCDA extremozyme could be used for industrial production of chitosan polymer as well as chitosan oligosaccharides for biomedical application.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-021-03073-3.

Keywords: Bacillus aryabhattai B8W22; Chitin deacetylase extremozyme; Halotolerant; Lactose induction; Receptor plate assay; Thermostable.

Fig. 1

The Phylogenetic analysis. The phylogenetic tree showing the isolate MS7 was highly homologous with B. aryabhattai B8W22 with a 99% match. The Bacillus niacini was taken as an out-group. The phylogenetic tree was constructed by a maximum parsimony algorithm. The names of the culture collection centres of the type isolates compared for the phylogenetic tree are American Type Culture Collection (ATCC), Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Institute for Fermentation, Osaka (IFO), Microbial Type Culture Collection (MTCC)

Fig. 2

Expression study in TB lactose-induced media. Glucose concentration (●), cell biomass (♦), and enzyme activity (▲) plotted against time (X-axis). All experiments were performed in triplicates and error bars represent the standard deviation of the mean

Fig. 3

A Recombinant BaCDA enzyme velocity study. B Recombinant BaCDA activity in buffer system covering the pH range 4–10, when EGC was used as substrate. (Citrate buffer—●, Bis–tris buffer—■, Phosphate buffer—♦, Tris–HCl buffer—▲, Boric acid buffer—○, Carbonate buffer—□). C Recombinant BaCDA activity in buffer system covering the pH range 4–10, when COS was used as substrate. (Citrate buffer—●, Bis–tris buffer—■, Phosphate buffer—♦, Tris–HCl buffer—▲, Boric acid buffer—○, Carbonate buffer—□). D The optimum temperature for BaCDA, the enzyme activity was determined at the temperature ranging from 20 to 60 ºC. E The salt tolerance of BaCDA, the enzyme activity was investigated in presence of NaCl concentration ranging from 0.001 to 2 M. F The thermo-stability of BaCDA, the enzyme activity was tested by incubating the enzyme at a different temperature ranging from 4 to 80 °C for 24 h. All experiments were performed in triplicates and error bars represent the standard deviation of the mean

Fig. 4

A Michaelis–Menten kinetic curve generated using EGC as a substrate. B Michaelis–Menten kinetic curve generated using COS as a substrate. All experiments were performed in triplicates and error bars represent the standard deviation of the mean