桑天牛肠道微生物内切葡聚糖酶基因的克隆及在乳酸杆菌中的表达

谢骜李畅; 杨雨鑫; 王小龙; 陈玉林

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

动物营养学报 >

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

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

分子与细胞营养 MOLECULAR AND CELLULAR NUTRITION

桑天牛肠道微生物内切葡聚糖酶基因的克隆及在乳酸杆菌中的表达

谢骜李畅 ,
杨雨鑫 ,
王小龙 ,
陈玉林

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西北农林科技大学动物科技学院, 杨凌 712100

谢骜李畅(1994-),男,重庆人,硕士研究生,研究方向为分子营养。E-mail:383566815@qq.com

收稿日期: 2019-09-03

网络出版日期: 2020-03-13

基金资助

国家重点研发计划（2018YFD0501905）；国家绒毛用羊产业技术体系（CARS-39-12）

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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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摘要

本试验旨在构建含内切葡聚糖酶基因的重组乳酸杆菌，研究其对秸秆植物性饲料的降解效果，为后期构建高效分解纤维素的工程菌奠定基础。从桑天牛（Apriona germari）体内分离出枯草芽孢杆菌（Bacillus subtilis），克隆其内切葡聚糖酶基因，通过酶切连接的方式将目的基因片段与启动子P₃₂、信号肽SP_lp0373、乳酸杆菌载体pLEM-415共同构建了内切葡聚糖酶分泌表达重组质粒pLEM-P₃₂SPNqm，并对重组乳酸菌酶学性质和其在秸秆饲料中的降解能力进行测定。结果表明：从枯草芽孢杆菌中克隆出的目的基因大小为1 490 bp，构建的重组乳酸杆菌其表达产物的蛋白大小约54 ku。进一步测定发现，重组乳酸杆菌产酶最适温度和时间分别为37℃、24 h，在最佳条件下其羧甲基纤维素酶（CMCase）活性为2.06 U/mL。酶促反应最适温度和pH分别为60℃、6.0。在50~70℃、弱酸性条件下该酶的稳定性较好。金属离子Na⁺、K⁺、Mn²⁺对酶促反应有促进作用，而Mg²⁺、Cu²⁺对酶活性抑制作用较大。重组乳酸杆菌发酵粗饲料，对玉米秸秆、苜蓿秸秆、小麦秸秆均发挥了一定的降解作用，中性洗涤纤维、酸性洗涤纤维相对降解率分别为10.42%、21.12%，4.99%、7.85%，4.57%、9.53%。综上，本试验构建的重组乳酸杆菌对常见的秸秆植物性饲料均有一定的降解能力。

关键词： 枯草芽孢杆菌; 内切葡聚糖酶; 罗伊乳酸杆菌; 基因工程; 发酵饲料

本文引用格式

谢骜李畅 , 杨雨鑫 , 王小龙 , 陈玉林 . 桑天牛肠道微生物内切葡聚糖酶基因的克隆及在乳酸杆菌中的表达[J]. 动物营养学报, 2020 , 32(3) : 1344 -1352 . DOI: 10.3969/j.issn.1006-267x.2020.03.042

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

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