土壤中高产蛋白酶菌株产酶条件及酶学性质

【背景】微生物蛋白酶已经成为工业用蛋白酶的主要来源,筛选具有特殊环境适应性的微生物成为生物酶资源的开发热点。【目的】通过对青藏高原土壤微生物产蛋白酶菌株的筛选、优化及相关特性研究,寻找新的蛋白酶资源,为高原菌种资源利用提供科学依据。【方法】采用形态学和分子生物学对筛选菌株进行菌种鉴定,利用单因素试验和正交试验对菌株进行发酵条件优化及酶学性质的探究。【结果】筛选出一株高产蛋白酶菌株XC2,经鉴定菌株XC2为枯草芽孢杆菌(Bacillus subtilis)。XC2最优产酶条件:可溶性淀粉4.0%,牛肉膏1.0%,K~+0.6%,培养温度34°C、初始pH 7.0、接种量2.0%的条件下200 r/min振荡培养13 h,所产蛋白酶活力最高为638.5 U/mL。XC2所产蛋白酶最适反应温度60°C,最适pH9.0;40-50°C、pH8.0-10.0条件下酶活稳定性较高;Mn~(2+)对酶活力有明显激活作用,而Zn~(2+)、Cu~(2+)、Fe~(2+)、Fe3+对酶活力有明显抑制作用。【结论】枯草芽孢杆菌XC2有较强的产碱性蛋白酶的能力,具有较好的应用前景。

Identification and characterization of a high protease-producing strain from soil

Background] Microbial protease has become the main source of industrial enzyme preparation. Screening microorganisms with special environmental adaptability has become a research hotspot. Objective] To isolate and screen the strain with high protease activity from the alpine meadow soil on the Tibetan Plateau, and optimize the enzyme production conditions and explore enzymatic properties. Methods] Strains were identified by morphology and molecular biological identification. The fermentation condition and enzymatic properties were studied by single factor and/or orthogonal test. Results] Strain XC2 with high protease activity was identified as Bacillus subtilis. The optimum fermentation conditions were: soluble starch 4.0%, beef extract 1.0%, K+ 0.6%, 34 °C, pH 7.0, inoculum amount 2.0%, rotation speed 200 r/min after 13 h. The highest protease activity of XC2 reached 638.5 U/mL. The optimum reaction temperature and pH of the protease was 60 °C and 9.0 respectively. The enzyme activity is relative stable between 40 and 50 °C or pH 8.0?10.0. The protease activity of XC2 was significantly enhanced by Mn2+, but significantly reduced by Zn2+, Cu2+, Fe2+ and Fe3+. Conclusion] Bacillus subtilis XC2 can efficiently produce alkaline protease, with good application prospect.