Cloning Endo-Glucanase Gene of Cellulolytic Microorganisms from Apriona germari and its Expression in Lactobacillus

XIE Aolichang; YANG Yuxin; WANG Xiaolong; CHEN Yulin

doi:10.3969/j.issn.1006-267x.2020.03.042

Chinese Journal of Animal Nutrition >

2020 , Vol. 32 >Issue 3: 1344 - 1352

DOI: https://doi.org/10.3969/j.issn.1006-267x.2020.03.042

Cloning Endo-Glucanase Gene of Cellulolytic Microorganisms from Apriona germari and its Expression in Lactobacillus

XIE Aolichang ,
YANG Yuxin ,
WANG Xiaolong ,
CHEN Yulin

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College of Animal Science and Technology, Northwest A & F University, Yangling 712100, China

Received date: 2019-09-03

Online published: 2020-03-13

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Abstract

The recombinant Lactobacillus containing cellulase gene was constructed to study its degradation effect on straw plant feed, which laid a foundation for the later construction of engineering bacteria that efficiently decompose cellulose. This study amplified a endo-β-1,4-glucanase gene from the Bacillus subtilis which was isolated from the Apriona germari in vivo. Through the restriction enzyme digestion and connection method, the Lactobacillus constitutive promoter element P₃₂, signal peptide SP_lp0373 gene and cellulase gene are connected with Lactobacillus expression vector pLEM-415. After secretion and expression of the target gene, the enzymatic properties and the conditions of producing enzyme were studied, as well as the fermentation test of crude enzyme to roughage. The results showed that the length of the cellulase gene fragment was 1 490 bp, the molecular weight of the expressed protein was 54 ku. The optimum enzyme producing time and temperature of recombinant Lactobacillus were 24 h and 37℃. Under the optimum enzyme producing conditions the activity of CMCase was 2.06 U/mL. The optimum temperature and pH of enzymatic reaction were 60℃, 6.0, respectively. Under the condition of pH 5.5, 50 to 70℃, the stability of the enzyme were the best. Metal ions Na⁺, K⁺ and Mn²⁺ had a promoting effect on the enzymatic reaction, while the Mg²⁺ and Cu²⁺ had a strong inhibitory effects. The fermentation test showed that recombinant Lactobacillus had degradation effect on corn straw, alfalfa hay and wheat straw, and the relative degradation rates of neutral detergent fiber and acid detergent fiber were 10.42% and 21.12%, 4.99% and 7.85%, 4.57% and 9.53%, respectively. Thus, the recombinant Lactobacillus constructed in this experiment has certain degradation ability to common straw plant feed.

Key words： Bacillus subtilis; endo-glucanase; Lactobacillus reuteri; genetic engineering; fermented feed

Cite this article

XIE Aolichang , YANG Yuxin , WANG Xiaolong , CHEN Yulin . Cloning Endo-Glucanase Gene of Cellulolytic Microorganisms from Apriona germari and its Expression in Lactobacillus[J]. Chinese Journal of Animal Nutrition, 2020 , 32(3) : 1344 -1352 . DOI: 10.3969/j.issn.1006-267x.2020.03.042

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