Welcome to Chinese Agricultural Science Bulletin,

Chinese Agricultural Science Bulletin ›› 2023, Vol. 39 ›› Issue (32): 15-21.doi: 10.11924/j.issn.1000-6850.casb2023-0276

Previous Articles     Next Articles

Mannanase Gene in Lactobacillus casei HDS-01: Heterologous Expression and Functional Verification

YANG Ruoxi1(), ZHANG Xin2, ZHAO Dan1,3()   

  1. 1 School of Life Sciences, Heilongjiang University/Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education/Heilongjiang Provincial Key Laboratory of Plant Genetic Engineering and Biological Fermentation Engineering for Cold Region/Key Laboratory of Microbiology, College of Heilongjiang, Harbin 150080
    2 Changchun Kinsey Pharmaceutical Co., Ltd, Changchun 130012
    3 Hebei Key Laboratory of Agro-ecological Safety, Qinhuangdao, Hebei 066102
  • Received:2023-04-11 Revised:2023-06-15 Online:2023-11-15 Published:2023-11-10

Abstract:

In order to realize heterologous expression of mannanase and verify its functions, the mannanase-producing Lactobacillus casei HDS-01 was used as the test strain in this study. The M1 predicted to be mannosidase was expressed in Escherichia coli. M1 gene engineering strains were constructed using pET-28a as the vector and E. coli BL21 (DE3) as the host. The recombinant protein was purified and the degradation function of mannan was verified. The results indicated that the recombinant protein M1 existed as inclusion body during expression. After purification by Ni affinity chromatography, the size of the protein was 98 kDa. After renaturation, mannanase activity and protein concentration were 19.24±0.55 U/mL and 0.51±0.01 mg/mL, respectively. High pressure liquid chromatography (HPLC) was used to detect the degradation product of Konjac powder. The concentration of mannotriose or tetrasaccharide was significantly higher in the first 3 hours, and mannose and mannobiose gradually increased as the reaction progressing. This study laid the foundation for the direct application of mannanase-producing lactic acid bacteria in food-grade field and provided the basis for the selective modification of mannanase in lactic acid bacteria.

Key words: β-1,4-mannanase, Lactobacillus casei, heterologous expression, recombinant protein, functional verification