马尾松毛虫肠道细菌的分离鉴定与产蛋白酶细菌的筛选

doi:10.3969/j.issn.1004-1524.2020.08.15

浙江农业学报 ›› 2020, Vol. 32 ›› Issue (8): 1446-1456.DOI: 10.3969/j.issn.1004-1524.2020.08.15

马尾松毛虫肠道细菌的分离鉴定与产蛋白酶细菌的筛选

江宇航^1,2, 李宏伟^1,2, 蔡赛波^1,2, 林连兵^1,2, 张棋麟^1,2,*

1.昆明理工大学生命科学与技术学院,云南昆明 650500;
2.云南省高校饲用抗生素替代技术工程研究中心,云南昆明 650500

收稿日期:2020-03-09 出版日期:2020-08-25 发布日期:2020-08-28
通讯作者: *,张棋麟,E-mail: zhangql@kust.edu.cn
作者简介:江宇航(1996—),男,四川遂宁人,硕士研究生,研究方向为昆虫肠道微生物的开发与利用。E-mail: 1584727524@qq.com
基金资助:
国家自然科学基金(31960286); 云南省教育厅科学研究基金(2019J0050)

Isolation and identification of intestinal culturable bacteria of Dendrolimus punctatus and screening of protease-producing bacteria

JIANG Yuhang^1,2, LI Hongwei^1,2, CAI Saibo^1,2, LIN Lianbing^1,2, ZHANG Qilin^1,2,*

1. Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China;
2. Engineering Research Center for Replacement Technology of Feed Antibiotics of Yunnan College, Kunming 650500, China

Received:2020-03-09 Online:2020-08-25 Published:2020-08-28

摘要/Abstract

摘要： 为分离鉴定马尾松毛虫幼虫肠道细菌,从中挖掘产蛋白酶的肠道细菌资源。以马尾松毛虫(Dendrolimus punctatus)幼虫为材料,通过常规细菌培养方法体外获得部分肠道细菌,利用16S rDNA鉴定细菌种类,采用酪蛋白选择培养基筛选具有产蛋白酶能力的菌株,依据GB/T 23527—2009《蛋白酶制剂》的检测方法,测定蛋白酶活性。结果表明:从马尾松毛虫肠道分离出18个不同种类的非厌氧细菌,隶属于厚壁菌门(Firmicutes)的芽孢杆菌属(Bacillus),变形菌门(Proteobacteria)的亚硫酸杆菌属(Sulfurobacillus)、葡萄球菌属(Staphylococcus)、嗜冷杆菌属(Psychrobacter)、泛菌属(Pantoea)、假单胞菌属(Pseudomonas)、不动杆菌属(Acinetobacter)和柠檬酸杆菌属(Citrobacter)。经酪蛋白选择培养基筛选后共获得6株产蛋白酶细菌,均为芽孢杆菌。酶活性测定结果显示:这些产蛋白酶细菌均具有较高的产蛋白酶能力,尤其是菌株MW-39地衣芽孢杆菌(Bacillus licheniformis),酶活性最高可达255.36 U·mL^-1;其次为MW-50解淀粉芽孢杆菌(Bacillus amyloliquefaciens)和MW-4枯草芽孢杆菌(Bacillus subtilis),酶活性最高可分别达193.85 U·mL^-1和165.81 U·mL^-1。分析3个蛋白酶活性最高菌种的蛋白酶酶学特性,发现它们均具有一定的热稳定性和酸碱耐受性。单因素方差分析结果显示,在pH 5.0~9.0和30~50 ℃各菌株产生的蛋白酶活性相对较高,MW-39、MW-4和MW-50的蛋白酶最适pH分别为8.0、7.5、8.0,最适温度分别为45、40、40 ℃。本研究揭示了马尾松毛虫幼虫肠道多个细菌种类,丰富了产蛋白酶细菌资源,为进一步开发利用昆虫肠道产蛋白酶细菌资源提供了参考。

关键词: 马尾松毛虫, 肠道细菌, 芽孢杆菌, 蛋白酶, 酶学特性

Abstract: To isolate and identify intestinal bacteria from Dendrolimus punctatus larvae and to tap resources of protease-producing bacteria. D. punctatus larvae was used as experimental material in this paper, several intestinal bacteria were obtained by conventional bacterial culture method in vitro. 16S rDNA was used to identify these bacteria, and protease-producing strains were screened by casein protein selection medium. Protease activity was determined according to GB/T 23527—2009 “protease preparation” test method. The results showed that 18 different species of non-anaerobic bacteria were obtained, including Bacillus genera belonging to Firmicutes, and Sulfurobacillus, Staphylococcus, Psychrobacter, Pantoea, Pseudomonas, Acinetobacter and Citrobacter belonging to Proteobacteria. Six strains of the protease-producing bacteria all belonging to Bacillus genus were screened by casein selective medium. The results of enzyme activity showed that all of these protease-producing bacteria had high protease-producing ability, especially strain MW-39 (Bacillus licheniformis), the highest enzyme activity reached to 255.36 U·mL^-1, followed by MW-50 (Bacillus amyloliquefaciens) and MW-4 (Bacillus subtilis), their highest enzyme activity reached to 193.85 U·mL^-1 and 165.81 U·mL^-1, respectively. Enzymatic characteristics of protease produced by three bacterial species with the highest enzymatic activity were further analyzed, and their proteases showed a certain thermal stability and acid-base tolerance. One-way ANOVA analysis showed that protease activity of investigated bacteria were high at pH 5.0-9.0 and 30-50 ℃, and the optimum pH of protease produced by MW-39, MW-4 and MW-50 were 8.0, 7.5 and 8.0, respectively, and the optimum temperatures were 45 ℃, 40 ℃ and 40 ℃. This study revealed various species of intestinal bacteria in larvae of D. punctatus, enriched resources of protease-producing bacteria, and could provide reference for further development and utilization of protease-producing bacteria in insect intestine.

Key words: Dendrolimus punctatus, intestinal bacteria, Bacillus, protease, enzymological characteristics

中图分类号:

S816.7

江宇航, 李宏伟, 蔡赛波, 林连兵, 张棋麟. 马尾松毛虫肠道细菌的分离鉴定与产蛋白酶细菌的筛选[J]. 浙江农业学报, 2020, 32(8): 1446-1456.

JIANG Yuhang, LI Hongwei, CAI Saibo, LIN Lianbing, ZHANG Qilin. Isolation and identification of intestinal culturable bacteria of Dendrolimus punctatus and screening of protease-producing bacteria[J]. , 2020, 32(8): 1446-1456.

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马尾松毛虫肠道细菌的分离鉴定与产蛋白酶细菌的筛选

Isolation and identification of intestinal culturable bacteria of Dendrolimus punctatus and screening of protease-producing bacteria

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