An Exochitinase with N-Acetyl-β-Glucosaminidase-Like Activity from Shrimp Head Conversion by Streptomyces speibonae and Its Application in Hydrolyzing β-Chitin Powder to Produce N-Acetyl-d-Glucosamine

Abstract

Marine chitinous byproducts possess significant applications in many fields. In this research, different kinds of fishery chitin-containing byproducts from shrimp (shrimp head powder (SHP) and demineralized shrimp shell powder), crab (demineralized crab shell powder), as well as squid (squid pen powder) were used to provide both carbon and nitrogen (C/N) nutrients for the production of an exochitinase via Streptomyces speibonae TKU048, a chitinolytic bacterium isolated from Taiwanese soils. S. speibonae TKU048 expressed the highest exochitinase productivity (45.668 U/mL) on 1.5% SHP-containing medium at 37 °C for 2 days. Molecular weight determination analysis basing on polyacrylamide gel electrophoresis revealed the mass of TKU048 exochitinase was approximately 21 kDa. The characterized exochitinase expressed some interesting properties, for example acidic pH optima (pH 3 and pH 5-7) and a higher temperature optimum (60 °C). Furthermore, the main hydrolysis mechanism of TKU048 exochitinase was N-acetyl-β-glucosaminidase-like activity; its most suitable substrate was β-chitin powder. The hydrolysis experiment revealed that TKU048 exochitinase was efficient in the cleavage of β-chitin powder, thereby releasing N-acetyl-d-glucosamine (GlcNAc, monomer unit of chitin structure) as the major product with 0.335 mg/mL of GlcNAc concentration and a yield of 73.64% after 96 h of incubation time. Thus, TKU048 exochitinase may have potential in GlcNAc production due to its N-acetyl-β-glucosaminidase-like activity.

Keywords: N-acetyl-β-glucosaminidase; Streptomyces speibonae; exochitinase; shrimp heads; β-chitin powder.

Figure 1

Effect of different chitinous byproducts on S. speibonae TKU048 exochitinase production. The medium was prepared by adding 1 g of each different fishery byproducts, including demineralized crab shell powder (deCSP), demineralized shrimp shell powder (deSSP), shrimp head powder (SHP), and squid pen powder (SPP) to 250 mL glass Erlenmeyer flasks containing 100 mL of basal salt medium. The cultivation conditions were conducted with 1% (v/v) of stock solution of S. speibonae TKU048, at an incubation temperature of 37 °C, and 150 rpm of agitation. An aliquot of culture (1 mL) was withdrawn every 24 h for testing exochitinase activity.

Figure 2

A typical ion-exchange chromatography profile of S. speibonae TKU048 exochitinase on Macro-Prep High Q. The elution was performed using Tris-HCl buffer system (20 mM, pH 7) with a NaCl gradient from 0 to 0.5 M and 2.5 mL of flow rate. Fifty microliters of each tube were withdrawn to test the exochitinase activity. The exochitinase activity fraction was found from tubes 80 to 91.

Figure 3

SDS-PAGE analysis of TKU048 exochitinase. 1, protein markers; 2, purified exochitinase after HPLC; *, location of purified TKU048 exochitinase.

Figure 4

Effects of temperature (A) and pH (B) on the activity and stability TKU048 exochitinase: (—) optimum; (…) stability; (⚫) glycine HCl buffer; (△) sodium acetate buffer; (▼) sodium phosphate buffer; and (⚪) sodium bicarbonate–carbonate buffer.

Figure 5

Effect of ion metals on the activity of TKU048 exochitinase. TKU048 exochitinase was pre-incubated with each of chemicals for 30 min. The activity of TKU048 exochitinase in the absence of treatment chemicals was used as a control to estimate relative activity (%).

Figure 6

HPLC analysis of the hydrolysis products from (GlcNAc)_2–6 by TKU048 exochitinase. A–E: (GlcNAc)₂–(GlcNAc)₆, respectively. The reaction was conducted by adding 500 µL substrate solution (0.5 mg/mL) with 500 µL enzyme solution (2 U, approximately) and incubated at 50 °C. Twenty microliters of sample was used for a single HPLC analysis.

Figure 7

The hydrolysis of β-chitin powder by TKU048 exochitinase: A: HPLC analysis of chitin hydrolysis pattern; B, the time course of the chitin hydrolysis of chitin.