Study on Enzyme Production of a Chitinase-producing Strain Achromobacter sp. ZWW8 by Fermentation and Its Enzymatic Characterization

doi:10.13560/j.cnki.biotech.bull.1985.2020-0887

Abstract

Abstract:

A extracellular chitinase-secreting bacterium ZWW8 was isolated from shrimp shell-accumulated soil in Hubei and identified as Achromobacter spp. via 16S rDNA sequence analysis. The fermentation conditions for chitinase production by the strain ZWW8 were optimized using the single-factor experiment,and the optimal fermentation conditions were as follows：0.5% colloidal chitin,0.5% ammonium chloride,initial medium pH 7.0,and 37℃ for 2 d. Under the optimized fermentation conditions,the highest chitinase activity in the supernatant reached 33.9 U/mL,which was 8 times of the activity before optimization. By detecting the enzymatic properties of extracellular chitinase secreted by ZWW8 strain,it was revealed that the optimal temperature of chitinase produced by Achromobacter sp. ZWW8 was 50℃ and the optimal pH was 5. Ni ²⁺and Ca ²⁺ increased chitinase activity,but Hg ²⁺,Cu ²⁺ and Zn ²⁺ significantly inhibited the enzymatic activity. Meanwhile,the chitinase not only exhibited activity and stability at high salt concentrations,but also showed chitin binding capacity. The extracellular chitinase from the fermented broth purified by chitin resin column demonstrated high activity after urea removed by dialysis. The chitinase by Achromobacter sp. ZWW8 possesses excellent enzymatic properties,making it a highly efficient and stable biocatalyst,which lays a foundation for exploring its application potential in industry,agriculture and medicine,as well as the development and utilization of chitin resources.

Key words: Achromobacter, chitinase, fermentation optimization, enzymatic property, salt-tolerance

ZHANG Yao-xin, WANG Liang-jie, ZHENG Wen, XU Han-qin, ZHENG Lian, ZHONG Jing. Study on Enzyme Production of a Chitinase-producing Strain Achromobacter sp. ZWW8 by Fermentation and Its Enzymatic Characterization[J]. Biotechnology Bulletin, 2021, 37(4): 96-106.

Figures/Tables 14

Figure 1 Phylogenetic tree of strain ZWW8 based on 16S rDNA sequences Numbers in parentheses refer to the sequence’ accession number in GenBank. The number at each branch point is the percentage supported by bootstrap. Bar: 0.5% sequence divergence

Table 1 Effect of different carbon sources on chitinase production by Achromobacter sp. ZWW8

碳源Carbon source	酶活Enzyme activity/（U·L^-1）
葡萄糖 Glucose	0.2±0.8
胶体几丁质 Colloidal chitin	20.0±1.9
粉末几丁质 Chitin powder	4.2±0.3
胶体壳聚糖 Colloidal chitosan	0.0±0.1
水溶性壳聚糖 Soluble chitosan	0.2±0.1
N-乙酰氨基葡萄糖 N-Acetyl-D-glucosamine	0.4±0.6

Table2 Effect of different nitrogen sources on chitinase production by Achromobacter sp. ZWW8

氮源Nitrogen source	酶活Enzyme activity（U·L^-1）
氯化铵 Ammonium chloride	20.7±1.3
硫酸铵 Ammonium sulfate	17.8±1.5
硝酸钠 Sodium nitrate	6.4±1.9
尿素 Urea	13.3±1.0
蛋白胨 Peptone	12.1±1.8
硝酸铵Ammonium nitrate	19.2±0.2

Fig. 2 Effect of initial medium pH on chitinase production by Achromobacter sp. ZWW8

Fig.3 Effect of temperatures on chitinase production by Achromobacter sp. ZWW8

Fig.4 Effect of fermentation time on chitinase production by strain ZWW8

Fig.5 Effect of temperature on chitinase activity by strain ZWW8

Fig.6 Effect of pH on chitinase activity by strain ZWW8

Fig.7 Effect of NaCl concentration on chitinase activity by strain ZWW8

Table 3 Effect of metal ions on chitinase activity by strain ZWW8

金属离子Metal ion	相对酶活Relative activity/%
Hg²⁺	3.1±3.3
Zn²⁺	84.4±8.5
Mg²⁺	96.1±7.1
Ca²⁺	103.9±9.3
Ag⁺	99.2±6.8
Cu²⁺	75.9±0.1
Ni²⁺	114.7±10.3

Fig.8 Effect of temperature on residual activity of chitinase by strain ZWW8

Fig.9 Effect of NaCl concentration on residual activity of chitinase

Fig.10 Effect of NaCl concentration on chitin binding ability of chitinase by strain ZWW8

Fig.11 SDS-PAGE analysis of strain ZWW8 chitinase purified by chitin affinity chromatography M: Low molecular weight marker. 1: Crude chitinase. 2: Effluent. 3,4: Scrub solution, 5: Purified chitinase

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