一株产β-葡萄糖苷酶甘草内生菌的筛选、全基因组分析及产酶优化

Screening, whole genome analysis, and fermentation condition optimization of a β-glucosidase-producing endophyte from Glycyrrhiza uralensis Fisch

摘要:

【背景】从健康甘草须根中分离获得的一株芽孢杆菌具有高产β-葡萄糖苷酶的活性。【目的】探究分离菌株潜在的产酶遗传信息，为该菌深入研究与工业应用提供数据支撑。【方法】利用七叶苷培养基进行产β-葡萄糖苷酶的益生菌筛选，筛到一株产β-葡萄糖苷酶的芽孢杆菌，采用三代Nanopore PromethION和二代Illumina NovaSeq平台对菌株进行基因组测序与组装、并通过基因预测与功能注释等生物信息分析预测菌株潜在的β-葡萄糖苷酶基因。另外，以β-葡萄糖苷酶活性为指标，研究碳源、氮源、接种量、温度和起始pH对菌株产酶活性的影响。【结果】从甘草须根中分离得到一株具有β-葡萄糖苷酶活性的菌株，通过形态学观察、生理生化和分子生物学试验鉴定为芽孢杆菌属菌株，并命名为Bacillus rugosus A78.1。该菌株基因组大小为4 146 938 bp，G+C含量为43.86%，共编码4 255个基因。在基因组中，共注释到碳水化合物活性酶基因192个，其中β-葡萄糖苷酶基因10个，分别属于GH1和GH3家族基因。在基因本体（GO）、京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）和同源基因簇（clusters of orthologous groups of proteins，COG）数据库分别注释到2 896、4 019和3 657个基因。该菌株基因组测序结果上传至NCBI获得GenBank登录号为CP096590。菌株A78.1产β-葡萄糖苷酶的最佳碳、氮源分别为0.5%葡萄糖、1.0%酵母浸粉，最佳培养条件为温度37℃、3%接种量、pH 6.0，此条件下β-葡萄糖苷酶活力可达到（5.640±0.085） U/mL。【结论】通过全基因组测序分析及产酶优化试验确定了Bacillus rugosus A78.1优良的产β-葡萄糖苷酶能力及在碳水化合物代谢方面的潜力，为该菌株在纤维素分解、糖苷类化合物水解等生物、化工和食品领域的研究与应用提供基础。

Abstract:

[Background] Bacillus rugosus A78.1 isolated from the fibrous roots of healthy Glycyrrhiza uralensis Fisch. has high β-glucosidase activity. [Objective] To investigate the genetic information of the isolate for enzyme production and provide data support for the further research and industrial application of the strain. [Methods] A β-glucosidase-producing Bacillus strain was screened by the medium supplemented with esculin. Nanopore PromethION and Illumina NovaSeq were used for genome sequencing and assembly, and the potential β-glucosidase genes of the strain were predicted by gene prediction and functional annotation. In addition, the effects of carbon source, nitrogen source, inoculum amount, temperature, and initial pH on the enzyme-producing activity of the strain were investigated with β-glucosidase activity as an indicator. [Results] A strain capable of producing β-glucosidase was isolated from the fibrous roots of G. uralensis. It was identified as a strain of Bacillus by morphological observation, physiological and biochemical tests and named B. rugosus A78.1. The genome of this strain was 4 146 938 bp in length, with the G+C content of 43.86%, encoding 4 255 genes. In the genome, 192 carbohydrate-active enzyme genes were annotated, including 10 β-glucosidase genes belonging to the GH1 and GH3 families. A total of 2 896, 4 019, and 3 657 genes were annotated in GO (gene ontology), KEGG (Kyoto encyclopedia of genes and genomes), and COG (clusters of orthologous groups of proteins), respectively. The genome sequencing data were submitted to NCBI and obtained the GenBank accession No. CP096590. For the production of β-glucosidase, the strain should be fermented with 0.5% glucose as the carbon source, 1.0% yeast extract as the nitrogen source at 37℃ and pH 6.0 with the inoculum amount of 3%. Under these conditions, the activity of β-glucosidase produced by this strain reached (5.640±0.085) U/mL. [Conclusion] We confirmed the excellent β-glucosidase-producing ability of B. rugosus A78.1 and the potential of this strain in carbohydrate metabolism by whole genome sequencing and fermentation condition optimization for enzyme production. The findings provide a basis for the research and application of this strain in biochemical and food fields such as cellulose degradation and glycoside hydrolysis.