Isolation and screening of a chitin deacetylase producing Bacillus cereus and its potential for chitosan preparation

Abstract

Chitosan is a biopolymer material extracted from marine biomass waste such as shrimp and crab shells, which has good biocompatibility and degradability with great potential for application in the field of wastewater treatment and soil remediation. The higher the degree of deacetylation (DD), the better the adsorption performance of chitosan. Chitin deacetylase (CDA) can specifically catalyze the deacetylate of chitin in a green reaction that is environmentally friendly. However, the scarcity of high yielding chitin deacetylase strains has been regarded as the technical bottleneck of chitosan green production. Here, we screened a natural chitin degrading bacterium from coastal mud and identified it as Bacillus cereus ZWT-08 by re-screening the chitin deacetylase activity and degree of deacetylation values. By optimizing the medium conditions and enzyme production process, ZWT-08 was cultured in fermentation medium with 1% (m/V) glucose and yeast extract at pH 6.0, 37°C, and a stirring speed of 180 r/min. After fermenting in 5 L fermenter for 48 h, the deacetylation activity of the supernatant reached 613.25 U/mL. Electron microscopic examination of the chitin substrate in the fermentation medium revealed a marshmallow-like fluffy texture on its structural surface. Meanwhile, 89.29% of the acetyl groups in this chitin substrate were removed by enzymatic digestion of chitin deacetylase produced by ZWT-08, resulting in the preparation of chitosan a degree of deacetylation higher than 90%. As an effective strain for chitosan production, Bacillus cereus ZWT-08 plays a positive role in the bioconversion of chitin and the upgrading of the chitosan industry.

Keywords: Bacillus cereus; chitin deacetylase; chitosan; degree of deacetylation; green preparation.

FIGURE 1

Morphological identification and staining results of Bacillus cereus ZWT-08. (A) A yellow chromogenic circle appeared around the ZWT-08 in the selective chitin plates, whose shade represents the CDA-producing capacity of the strain. (B) Single colony morphology of ZWT-08 in the chitin plates indicated it to be an irregular colony, soft texture, molten wax-like, and no pigment. (C) Gram staining results were positive. (D) The spore staining was positive.

FIGURE 2

Effect of medium and culture conditions on the CDA activity of Bacillus cereus ZWT-08. The effect of carbon sources (A) and nitrogen sources (B) on the activity of CDA was compared at a concentration of 1% (m/V). The effect of pH (C) and temperature (D) on the CDA activity was studied in the optimized culture medium. The CDA activity determined in the basal fermentation medium at pH 7.0°C and 37°C with shaking at 180 rpm for 48 h was designated as 100% while others were expressed against this value.

FIGURE 3

Time courses of biomass content, CDA activity, and reducing sugar in the fermentation broth. The strain ZWT-08 was cultured in a chitin medium containing 1% (m/V) glucose and 1% (m/V) yeast extract at a pH of 6.0, a temperature of 37°C in 5 L fermenters. Error bars represent ± standard deviations (n = 3).

FIGURE 4

SEM of chitin and chitosan surface structures at ×1,000 magnification. (A) Untreated chitin. (B) The treated chitin after cultivation with B. cereus ZWT-08 for 48 h, which has been deacetylated to chitosan.