| 王宁，李小波，刘文彬，唐彬，汪洁，朱家勇，金小宝,2022,美洲大蠊肠道耐热碱性蛋白酶产生菌筛选及其酶特性[J].环境昆虫学报,44(1):212-220 |
| 美洲大蠊肠道耐热碱性蛋白酶产生菌筛选及其酶特性 |
| Screening and identification of thermostable alkaline protease-producing bacteria from the gut of Periplaneta americana and its enzyme characteristics |
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| DOI： |
| 中文关键词: 美洲大蠊 昆虫肠道菌 嗜碱性芽孢杆菌 碱性蛋白酶 耐热酶 |
| 英文关键词:Periplaneta americana insect gut microbes alkaliphilic Bacillus alkaline protease thermostable enzyme |
| 基金项目:广东省公益研究与能力建设项目（2017A020211008）；广东省普通高校基础研究与应用基础项目（2018KZDXM041）；广东省自然科学基金（2020A1515011097） |
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| 中文摘要: |
| 昆虫肠道菌是重要的微生物资源。为拓展微生物酶资源菌种来源，本研究以含Na2CO3和脱脂奶粉的培养基从美洲大蠊Periplaneta americana (L.)肠道中筛选产碱性蛋白酶的嗜碱细菌，进行形态学和16S rDNA序列鉴定，并分析温度、pH和Na2CO3对菌株生长的影响；采用福林-酚法和SDS-PAGE酶谱法测定温度、pH对菌株所产蛋白酶活性和稳定性的影响。结果显示，从美洲大蠊肠道分离到一株嗜碱细菌PaGA523-1，革兰氏阳性，具芽孢，经鉴定为列城芽孢杆菌Bacillus lehensis，其最适生长pH范围为9~11，最适生长温度为37℃，可在含0.03~0.09 mol/L Na2CO3的肉汤蛋白胨液体培养基中生长。在含Na2CO3的玉米粉-黄豆饼粉发酵培养基中，菌株PaGA523-1可产生蛋白酶。该蛋白酶的最适反应温度为62℃，最适反应pH范围为9~11。蛋白酶在不同pH缓冲液（7~11）和热处理（40~60℃）后仍具有一定活性，在60℃处理1 h后，仍保留其初始活性的62.68%。SDS-PAGE酶谱法检测结果也定性说明菌株可产生具有一定程度热稳定性的碱性蛋白酶。本研究获得了一株嗜碱性的昆虫肠道芽孢杆菌，该菌可分泌热稳定碱性蛋白酶，在蛋白质水解方面具有潜在应用价值。 |
| 英文摘要: |
| Insect gut microbes are important microbial resources. In order to expand the enzyme-producing microbial resources, in this study, the medium containing Na2CO3 and skimmed milk was used to screen alkaline protease-producing alkaliphilic bacteria from the gut of American cockroach, Periplaneta americana. Then the isolated strain was identified based on colony morphology and 16S rDNA sequence analysis, and the effects of temperature, pH and Na2CO3 on the growth of the strain were analyzed, too. The effects of temperature and pH on the activity and stability of alkaline protease which produced by the strain was detected by Folin-phenol method and SDS polyacrylamide gel electrophoresis (SDS-PAGE)zymography analysis. As results, the alkaliphilic bacteria strain PaGA523-1, isolated from the American cockroach gut, was found short rod shaped, gram-positive, formed spores, and identified as Bacillus lehensis. Its optimal growth pH range and temperature were 9~11 and 37℃, respectively. The strain could grow well in liquid broth peptone medium which containing 0.03~0.09 mol/L Na2CO3.The strain PaGA523-1 could produce thermostable alkaline protease in the corn flour-soybean meal flour fermentation medium. The optimum reaction temperature of the protease was 62℃, and the optimum reaction pH range was 9~11. The protease still remained a certain extent activity after treated with different pH buffers (7~11)and heat (40~60℃), and that it even remained 62.68% of its initial activity after treated for 1 h at 60℃. Results of the SDS-PAGE zymography test also showed qualitatively that the strain could produce alkaline protease which had a certain degree of thermal stability. In this study, an alkaliphilic Bacillus strain which could secrete thermostable alkaline protease was obtained from insect gut. The alkaline protease showed great potential for application in protein hydrolysis. |
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