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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摘要玉米(Zea mays)秸秆作为牛羊等家畜的主要饲料来源,其中含有大量的纤维素,外源纤维二糖水解酶(cellobiohydrolase, CBH)可促进纤维素在青贮过程中的降解。乳酸菌(Lactococcus lactis)作为益生菌,可提高青贮饲料的品质。本研究粗提黄牛(Bos taurus domestica)瘤胃微生物混合DNA,以其为模板扩增cbh基因并克隆至食品级载体pMG36e,构建分泌型表达载体pMG36e::CBH;将其导入乳酸菌NZ9000中,利用3,5-二硝基水杨酸(3,5-dinitrosalicylic acid, DNS)法测定重组纤维二糖水解酶的酶活,并对重组酶的酶学性质进行分析。结果显示,从牛瘤胃中克隆获得约1 600 bp的重组酶序列,其编码蛋白分子量约为56 kD,滤纸酶活为(2.218 1±0.834 3) U/mL,外切葡聚糖酶的酶活为(10.499 2±1.113 5) U/mL;重组酶对再生无定型纤维素(regenerated amorphous cellulose, RAC)的特异性最高,微晶纤维素次之,对滤纸和脱脂棉的特异性较低,而对羧甲基纤维素钠(CMC-Na)几乎没有活性;重组纤维二糖水解酶的最适pH为6,最适温度为60 ℃;1 mmol/L Mn²⁺、Zn²⁺、Fe²⁺、Cu²⁺、Co²⁺和1% Tween-20对重组CBH的酶活有促进作用,1 mmol/L Ba²⁺和Hg²⁺几乎对酶活无影响,1 mmol/L K⁺、Mg²⁺和EDTA对酶活有抑制作用,5 mmol/L Mn²⁺、Fe²⁺、Ca²⁺、Cu²⁺、Co²⁺对酶活有促进作用,10% Tween-20和5 mmol/L Zn²⁺、Ba²⁺、Hg²⁺、K⁺、Mg²⁺和EDTA对酶活有抑制作用。本研究构建的牛瘤胃微生物cbh基因的重组乳酸菌,有利于促进青贮过程中秸秆纤维素酶解及提高青贮饲料的营养价值。

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	孙康永杰
	杨宇泽
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	马春娟
	曹磊
	邹爱爱
	王川

关键词 ：瘤胃微生物, 纤维二糖水解酶(CBH), 乳酸菌, 分泌表达

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

收稿日期: 2021-03-29

ZTFLH:

S816.79

基金资助:甘肃省科技计划项目重点研发计划(18YF1NA077)

通讯作者: *wangchuan@gsau.edu.cn

引用本文:

孙康永杰, 杨宇泽, 魏亚琴, 万学瑞, 张钊, 马春娟, 曹磊, 邹爱爱, 王川. 牛瘤胃微生物纤维二糖水解酶基因(cbh)在乳酸菌中的表达[J]. 农业生物技术学报, 2021, 29(12): 2375-2386.
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. Expression of Cellobiohydrolase Gene (cbh) from Cattle (Bos taurus domestica) Rumen Microbes in Lactic Acid Bacteria (Lactococcus lactis). 农业生物技术学报, 2021, 29(12): 2375-2386.

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

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