一株蛙源几丁质分解菌JD-3的分离鉴定、全基因组测序及功能分析  

Isolation,identification,whole genome sequencing,and functional analysis of a frog-derived chitin-degrading bacterium JD-3

作　　者：王蕊 林星荣 王婉婷 沈迎芳 张湑泽 WANG Rui;LIN Xingrong;WANG Wanting;SHEN Yingfang;ZHANG Xuze(College of Ecological Environment and Resources,Qinghai Minzu University,Xining,Qinghai,China;Qinghai Provincial Key Laboratory of High-value Utilization of Characteristic Economic Plants,Xining,Qinghai,China;Qinghai-Tibet Plateau Key Laboratory of Resource Chemistry and Ecological Environment Protection of the National Ethnic Affairs Commission,Xining,Qinghai,China)

机构地区：[1]青海民族大学生态环境与资源学院,青海西宁 [2]青海省特色经济植物高值化利用重点实验室,青海西宁 [3]国家民委青藏高原资源化学与生态环境保护重点实验室,青海西宁

出　　处：《微生物学报》2025年第4期1571-1586,共16页Acta Microbiologica Sinica

基　　金：国家自然科学基金(32360260);青海省自然科学基金(2020-ZJ-965Q);青海省昆仑英才项目(QHKLYCGDCXCY-2023-138);青海民族大学校级规划项目(ESDYJ24);青海民族大学产教融合研究生联合培养基地建设项目;青海民族大学校级教学成果培育项目。

摘　　要：【目的】从两栖动物肠道中分离并筛选能够有效分解几丁质的菌株,研究其发酵条件、酶学特性,并进行细菌全基因组测序及功能分析。【方法】对高原林蛙肠道内容物进行筛选,分离出1株具有产几丁质酶能力的菌株,并进行形态学和分子生物学鉴定;通过单因素试验和响应面试验对产酶条件进行优化,并研究其酶学性质;开展全基因组测序,鉴定几丁质酶基因家族。【结果】筛选到1株可有效产几丁质酶的菌株JD-3,经鉴定为麦芽香肉食杆菌(Carnobacterium maltaromaticum)。最佳产酶条件为:发酵时间2.47d,发酵温度31.4℃,初始p H4.9,接种量4%,酶活性达到12 m U/m L。酶学性质分析表明,该酶的最适反应温度为20℃,p H 3.0,在室温、酸性条件下表现出良好的稳定性。全基因组分析显示,JD-3基因组全长为4195636 bp,包含6个环状重叠群(contig)、63个t RNA基因、19个r RNA基因和3864个蛋白质编码序列(coding sequence,CDS)。在基因组中鉴定出2个几丁质酶基因,均属于GH18家族,系统发育分析将其归为2个不同的类别。【结论】从高原两栖动物肠道中分离出1株具有常温耐酸特性的几丁质分解菌,经形态学和系统发育鉴定为麦芽香肉食杆菌,这为动物消化系统微生物资源的开发利用提供了新思路。[Objective]To establish a theoretical foundation for the application and development of chitinases,this study isolated and screened chitin-degrading bacteria from the intestines of amphibians,optimized their fermentation conditions,characterized their enzymatic properties,and analyzed their whole genomes.[Methods]A strain capable of producing chitinase was isolated from the intestinal contents of Rana kukunoris and identified based on morphological characteristics and molecular biological evidence.The enzyme production conditions of the strain were optimized by single factor and response surface methodology(RSM)experiments,and the enzymatic properties were studied.Whole genome sequencing was carried out for identification of the chitinase gene family.[Results]The chitin-degrading strain JD-3 was identified as Carnobacterium maltaromaticum.This strain achieved the highest enzyme activity of 12 mU/mL after fermentation with the inoculum amount of 4%at 31.4℃and initial pH 4.9 for 2.47 d.The optimal reaction conditions of the enzyme were 20℃and pH 3.0,and the enzyme maintained good stability at room temperature and under acidic conditions.The genome of JD-3 was 4195636 bp long,containing 6 circular contigs,63 tRNA genes,19 rRNA genes,and 3864 protein coding sequences.Two chitinase genes belonging to the glycoside hydrolase family 18(GH18)were identified and phylogenetically classified into two distinct categories.[Conclusion]We isolated an acid-tolerant chitin-degrading bacterium,C.maltaromaticum JD-3,from the intestines of plateau amphibians.The findings provide new insights into the development and utilization of microbial resources in the digestive systems of animals.

关 键 词：高原林蛙 肠道微生物 几丁质分解菌 产酶条件优化 细菌全基因组 

分 类 号：Q93[生物学—微生物学]