牛瘤胃微生物纤维二糖水解酶基因(<em>cbh</em>)在乳酸菌中的表达

doi:10.3969/j.issn.1674-7968.2021.12.011

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Abstract Corn (Zea mays) stalk is the main source of feed for cattle, sheep and other livestock, and contains a large amount of cellulose. Exogenous cellobiohydrolase (CBH) can promote the degradation of cellulose during silage. As probiotics, lactic acid bacteria (Lactococcus lactis) can improve the quality of silage. In present study, mixed DNA of the cattle (Bos taurus domestica) rumen microbes was obtained by crude extraction, and the cbh gene was amplified and cloned into food-grade vector pMG36e, which was used to construct a secreted expression vector pMG36e::CBH. The expression vector was transformed into L. lactis NZ9000, and the corresponding enzyme activity was determined by 3,5-dinitrosalicylic acid (DNS) method, and the enzymatic properties of the recombinant enzyme were analyzed. The results showed that a sequence with 1 600 bp was cloned from cattle rumen, the molecular weight of the recombinant enzyme was about 56 kD, the filter paper enzyme activity was (2.218 1±0.834 3) U/mL, and the exoglucanase activity was (10.499 2±1.113 5) U/mL. The recombinant enzyme had the highest specificity for regenerated amorphous cellulose (RAC), followed by microcrystalline cellulose, lower specificity for filter paper and absorbent cotton, but almost no activity for sodium carboxymethyl cellulose (CMC-Na); the optimal pH and temperature were pH 6 and 60 ℃; 1 mmol/L Mn²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Co²⁺, and 1% Tween-20 promoted the enzyme activity of recombinant CBH, while 1 mmol/L Ba²⁺ and Hg²⁺ had almost no effect on the enzyme activity, but 1 mmol/L K⁺, Mg²⁺, and EDTA inhibited the enzyme activity, 5 mmol/L Mn²⁺, Fe²⁺, Ca²⁺, Cu²⁺, and Co²⁺ promoted the enzyme activity, 10% Tween-20 and 5 mmol/L Zn²⁺, Ba²⁺, Hg²⁺, K⁺, Mg²⁺ and EDTA inhibited the enzyme activity. The recombinant lactobacillus with cbh gene from cattle rumen microbes constructed in this study is conducive to promoting the enzymatic hydrolysis of straw during silage and improving the nutritional value of silage.

Key words： Rumen microbes Cellobiohydrolase (CBH) Lactic acid bacteria Secretory expression

Received: 29 March 2021

ZTFLH:

S816.79

Corresponding Authors: *wangchuan@gsau.edu.cn

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	SUN Kang-Yong-Jie
	YANG Yu-Ze
	WEI Ya-Qin
	WAN Xue-Rui
	ZHANG Zhao
	MA Chun-Juan
	CAO Lei
	ZOU Ai-Ai
	WANG Chuan

Cite this article:

SUN Kang-Yong-Jie,YANG Yu-Ze,WEI Ya-Qin, et al. Expression of Cellobiohydrolase Gene (cbh) from Cattle (Bos taurus domestica) Rumen Microbes in Lactic Acid Bacteria (Lactococcus lactis)[J]. 农业生物技术学报, 2021, 29(12): 2375-2386.

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http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2021.12.011 OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2021/V29/I12/2375

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