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

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2.47 d，发酵温度31.4 ℃，初始pH 4.9，接种量4%，酶活性达到12 mU/mL。酶学性质分析表明，该酶的最适反应温度为20 ℃，pH 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4 195 636 bp，包含6个环状重叠群(contig)、63个tRNA基因、19个rRNA基因和3 864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。

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

Abstract:

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 4 195 636 bp long, containing 6 circular contigs, 63 tRNA genes, 19 rRNA genes, and 3 864 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.

Key words:Rana kukunoris;gut microbiota;chitin-degrading bacterium;optimization of enzyme production conditions;bacterial whole genome sequence