Production of Thermophilic Chitinase by Paenibacillus sp. TKU052 by Bioprocessing of Chitinous Fishery Wastes and Its Application in N-acetyl-D-glucosamine Production

Abstract

The bioprocessing of chitinous fishery wastes (CFWs) to chitinases through fermentation approaches has gained importance owing to its great benefits in reducing the enzyme production cost, and utilizing chitin waste. In this work, our study of the chitinase production of Paenibacillus sp. TKU052 in the presence of different kinds of CFWs revealed a preference for demineralized crab shells powder (deCSP); furthermore, a 72 kDa chitinase was isolated from the 0.5% deCSP-containing medium. The Paenibacillus sp. TKU052 chitinase displayed maximum activity at 70 °C and pH 4-5, while Zn²⁺, Fe³⁺, Triton X-100, Tween 40, and SDS exerted a negative effect on its activity, whereas Mn²⁺ and 2-mercaptoethanol were found to potentially enhance the activity. Among various kinds of polysaccharide, Paenibacillus sp. TKU052 chitinase exhibited the best catalytic activity on colloidal chitin (CC) with K_m = 9.75 mg/mL and V_max = 2.43 μmol/min. The assessment of the hydrolysis of CC and N-acetyl chitooligosaccharides revealed that Paenibacillus sp. TKU052 chitinase possesses multiple catalytic functions, including exochitinase, endochitinase, and N-acetyl-β-D-glucosaminidase activities. Finally, the combination of Paenibacillus sp. TKU052 chitinase and Streptomyces speibonae TKU048 N-acetyl-β-D-glucosaminidase could efficiently convert CC to N-acetyl-D-glucosamine (GlcNAc) with a production yield of 94.35-98.60% in 12-24 h.

Keywords: N-acetyl-D-glucosamine; Paenibacillus; chitinase; chitinous fishery wastes; crab shells.

Figure 1

The chitinase-producing ability of Paenibacillus sp. TKU052 on CC agar medium.

Figure 2

Screening of suitable C/N source for chitinase production by Paenibacillus sp. TKU052: (a) different kinds of CFWs and (b) different amounts of deCSP were examined. The error bar is the standard deviation of three replicates.

Figure 3

A typical cation exchange chromatography profile of the crude enzyme obtained from the supernatant of Paenibacillus sp. TKU052 culture medium supplemented with 0.5% deCSP (a); HPLC profiles of chitinase fraction obtained from the cation exchange chromatography step (b) and the HPLC step (c).

Figure 4

SDS-PAGE (a) and chitin zymography (b) profile of Paenibacillus sp. TKU052 chitinase. M, protein markers; E, the purified enzyme. The arrow indicates the enzyme location.

Figure 5

Effect of temperature (a) and pH (b) on the activity of Paenibacillus sp. TKU052 chitinase. The error bar is the standard deviation of three replicates.

Figure 6

Hydrolysis pattern of Paenibacillus sp. TKU052 chitinase toward colloidal chitin (CC) (a) and N-acetyl COSs (b).

Figure 7

Time courses of GlcNAc and (GlcNAc)₂ production generated from the hydrolysis of colloidal chitin (CC) catalyzed by a mixture of Paenibacillus sp. TKU052 chitinase and Streptomyces speibonae TKU048 N-acetyl-β-D-glucosaminidase (a), and independently by Paenibacillus sp. TKU052 chitinase (b), and S. speibonae TKU048 N-acetyl-β-D-glucosaminidase (c). The error bar indicates the standard deviation of three replicates.

Figure 7

Time courses of GlcNAc and (GlcNAc)₂ production generated from the hydrolysis of colloidal chitin (CC) catalyzed by a mixture of Paenibacillus sp. TKU052 chitinase and Streptomyces speibonae TKU048 N-acetyl-β-D-glucosaminidase (a), and independently by Paenibacillus sp. TKU052 chitinase (b), and S. speibonae TKU048 N-acetyl-β-D-glucosaminidase (c). The error bar indicates the standard deviation of three replicates.

Figure 8

HPLC (a) and ¹H-NMR (b) profiles of the obtained and commercial GlcNAc (1, obtained GlcNAc; 2, commercial GlcNAc).