高蛋白酶活力菌株是促进豆豉成熟的重要发酵剂,除此之外,能分泌风味酶也是优良发酵剂的主要特征之一。筛选高蛋白酶活力菌株,并对菌株中风味酶相关功能基因进行研究,对开发高质量豆豉发酵剂具有重要意义。利用透明圈法初筛,蛋白酶活力测定法复筛出1株高蛋白酶活力菌株B3后对菌株进行16S rDNA分子鉴定、酶学性质研究,以及确定与风味酶相关的功能基因及其编码的蛋白酶。结果表明,优势菌株B3鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)(Genbank 序列号 MZ723401.1),蛋白酶活力达到185.6 U/mL。其酶学特性显示,最适温度为50 ℃,最适pH为7.0。对菌株B3的全基因组序列进行分析,检索到风味蛋白酶基因AprX和ampS,并预测其编码的丝氨酸蛋白酶和氨肽酶的氨基酸序列组成、理化性质、高级结构,同时进行系统发育树的建立和多序列比对研究。其中AprX基因全长为1 329 bp,共编码442个氨基酸,ampS基因全长为1 233 bp,共编码410个氨基酸。两者均编码亲水性蛋白,基因编码氨基酸序列都与Bacillus amyloliquefaciens group相似性最高。因此,B3菌株蛋白酶活力高并存在编码碱性蛋白酶和脱苦蛋白酶的特征功能基因,是发酵豆豉的潜在优良发酵剂。
The high protease activity strain is an important starter for promoting the maturation of Douchi. In addition, the ability to secrete flavor enzymes is also one of the main characteristics of a good starter. Screening strains with high protease activity and studying the functional genes related to flavor enzymes in the strains are of great significance for the development of high-quality Douchi starter. A high protease activity strain B3 was screened by the transparent circle and the protease activity assay method. Then, the strain was identified by 16S rDNA molecular, the enzymatic properties were studied, and the functional genes related to flavor enzymes and their encoded proteases were determined. The results showed that the dominant strain B3 was identified as Bacillus amyloliquefaciens (nos. MZ723401.1), and its protease activity reached 185.6 U/mL. Its enzymatic properties showed that the optimum temperature was 50 ℃, and the optimum pH was 7.0. By analyzing the whole genome sequence of strain B3, flavor protease genes AprX and ampS were retrieved. The amino acid sequences, physicochemical properties, and high-level structural properties of the encoded serine protease and aminopeptidase were predicted. At the same time, the establishment of phylogenetic tree and multiple sequence alignment were carried out. The full length of the AprX gene is 1 329 bp, encoding a total of 442 amino acids, and the full length of the ampS gene is 1 233 bp, encoding a total of 410 amino acids. Both of them encode hydrophilic proteins, and the amino acid sequences encoded by the genes have the highest similarity with the Bacillus amyloliquefaciens group. Therefore, the strain B3 has high protease activity and has characteristic functional genes encoding alkaline protease and depicroprotease, which is a potential good starter for fermented Douchi.
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