Isolation of a Chitinase-producing Strain Brevibacillus laterosporus and Its Enzymatic Properties

doi:10.13560/j.cnki.biotech.bull.1985.2016.06.025

Abstract

Abstract: This study aims to screen and identify Bacillus strain producing high yield of chitinase and to study its enzymatic properties for laying groundwork in the efficient utilization of chitinase resources. Firstly，the Bacillus producing chitinase was isolated and purified，and then chitinase gene in the target strain was heterologously expressed，and enzymatic parameters of the purified chitinase were assayed at optimal conditions. Considering the bio-safety of the strain，Brevibacillus laterosporus CDY64 was selected as study object，and its chitinase(Chi72A)was expressed in Escherichia coli BL21(DE3). Enzymatic studies revealed that the purified Chi72A by Ni-NTA affinity presented solid activity at pH range 6.0-8.0，and the optimal temperature of its activity was 60℃；and the Km and k cat of Chi72A were 5.85 μmol/L and 29.27 S^-1，respectively while using colloidal chitin as substrate. In conclusion，Chi72A from CDY64 had high catalytic ability at high temperature and exhibited high antifungal activity against many phytopathogenic fungi in vitro.

Key words: chitinase, Brevibacillus laterosporus, enzymatic properties

LIU Pu-lin, CHENG De-yong, MIAO Li-hong. Isolation of a Chitinase-producing Strain Brevibacillus laterosporus and Its Enzymatic Properties[J]. Biotechnology Bulletin, 2016, 32(6): 174-180.

References

[1]Morimoto K, Karita S, Kimure T, et al. Cloning sequencing and expression of the gene encoding Clostrium paraputrificum chitinase ChiB and analysis of the functions of novel cadherin-like domains and a chitin-binding domain[J]. Journal of Bcteriology, 1997, 179：7306-7314.
[2]Manjeet K, Purushotham P, Neeraja C, et al. Bacterial chitin binding proteins show differential substrate binding and synergy with chitinases[J]. Micorbiological Reaearch, 2013, 168：461-468.
[3]Ding X, Luo Z, Gao B, et al. Improving the insecticidal activity by expression a recombinant cry1A gene with chitinase-encoding gene in a crystalliferous Bacillus thuringiensis[J]. Current Microbiology, 2008, 56：442-446.
[4]Regev A, Keller M, Strizhov N, et al. Synergistic activity of a Bacillus thuringensis delta-endotoxin and a bacterial endochitinase against Spodoptera littoralis larvae[J]. Applied Environmental Microbiology, 1996, 62：3581-3586.
[5]Nanjo F, Sakai K, Ishikawa M, et al. Properties and transglycosylation reaction of a chitinase from Nocardia orientalis[J]. Agricultural and Biological Chemistry, 1989, 53：2189-2196.
[6]Usui T, Matsui H, Isobe K. Enzymic synthesis of chitooligosaccharides utilizing transglycosylation by chitinolytic enzymes in a buffer containing ammonium sulfate[J]. Carbohydrate Research, 1990, 203：65-77.
[7]赵斌, 何绍江. 微生物学实验[M]. 北京：科学出版社, 2002：85-88.
[8]Miller GL. Use of dinitrisalicylic acid reagent for determination of reducing sugar[J]. Analytical Chemistry, 1959, 31：426-428.
[9]Roberts WK, Selitrennikoff CP. Isolation and partial characterization of two antifungal proteins from barley[J]. Biochimica et Biophysica acta, 1986, 880：161-170.
[10]Bradford MM. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 1976, 72：248-254.
[11]Trivedi P, Spann T, Wang N. Isolation and characterization of beneficial bacteria associated with Citrus roots in Florida[J]. Microbial Ecology, 2011, 62：324-336.
[12]Ruiu L. Brevibacillus laterosporus, a pathogen of invertebrates and a broad-spectrum antimicrobial species[J]. Insects, 2013, 4：476-492.
[13]Terahara T, Ikeda S, Noritake C, et al. Molecular diversity of bacterial chitinase in arable soils and the effects of environmental factors on the chitinolytic bacterial community[J]. Soil Biology and Biochemistry, 2009, 41：473-480.
[14]Kudan S, Pichyanqkura R. Purification and characterization of thermostable chitinase from Bacillus licheniformis SK-1[J]. Applied Biochemistry and Biotechnology, 2009, 157：23-35.
[15]Pradeep GC, Choi YH, Choi YS, et al. An extremely alkaline novel chitinase from Streptomyces sp. CS495[J]. Process Biochemistry, 2014, 49：223-229.