一株产脂肪酶真菌的筛选、培养条件优化及脂肪酶结构特征

Screening and culture condition optimization of a lipase-producing fungal strain and characterization of its lipase

摘要:

脂肪酶是一类可在油水界面高效催化酯类化合物水解、醇解、酸解、酯交换及合成等反应的生物酶类，在医药、化工等多个领域发挥着重要作用。与动物、植物脂肪酶相比，微生物脂肪酶更易获取，具有更高的研究和开发利用价值。目的 从自然环境中筛选脂肪酶产生菌，优化其培养方案并研究其结构特征。方法 利用溴甲酚紫指示剂从油污土壤中分离产脂肪酶菌株，结合形态学观察和18S rRNA基因测序进行菌种鉴定；通过单因素实验及响应面试验优化菌株培养条件；对活性蛋白进行蛋白组测序，筛选潜在脂肪酶；对该脂肪酶基因进行PCR扩增和测序，并分析其多级结构。结果 从油污土壤中分离出一株产脂肪酶真菌FA3，经鉴定属于曲霉属(Aspergillus sp.)。以橄榄油为唯一碳源培养时，利用对硝基苯酚比色法测得菌株FA3胞内脂肪酶活力为263.75 U/g。优化后的培养条件为：乳化橄榄油4 mL/L，蛋白胨18 g/L，K₂HPO₄ 1 g/L，NaCl 10 g/L，MgSO₄ 0.5 mmol/L，pH 5.3，培养温度30 ℃，培养时间77.5 h。优化后酶活力达 2 120.27 U/g，提升了约8.04倍。经蛋白组分析和结构预测，该菌脂肪酶为GDSL脂肪酶，具有Gly-Asp-Ser保守序列，且存在严格保守的Ser、Gly、Asn、His残基，属于N-磺基葡萄糖胺磺基水解酶(N-sulphoglucosamine sulphohydrolase, SGNH)。结论 经过培养条件优化，菌株FA3产脂肪酶能力显著增强，具有良好的应用前景；明确了该脂肪酶的功能和结构特征，为相关蛋白的工程改造提供了依据。

Abstract:

Lipases are a group of biocatalysts that efficiently catalyze the hydrolysis, alcoholysis, acidolysis, ester exchange, and synthesis of esters at the oil-water interface. These enzymes play crucial roles in various fields, including pharmaceuticals and chemical engineering. Microbial lipases are more readily available and hold greater value for research and application than animal and plant lipases.Objective To isolate lipase-producing microorganisms from natural environments, optimize their culture conditions, and characterize the structure of the produced lipase.Methods We used bromocresol purple as an indicator to screen lipase-producing strains from oil-contaminated soil and identified the strains by morphological observation and 18S rRNA gene sequencing. We optimized the culture conditions of the strains by single-factor experiments and response surface methodology. We performed proteomic sequencing on the active proteins to identify the potential lipase. We amplified and sequenced the gene of the potential lipase by PCR and analyzed its multi-level structure.Results A lipase-producing fungal strain FA3 was isolated from oil-contaminated soil and identified as Aspergillus sp. The strain was cultured with olive oil as the sole carbon source, and the p-nitrophenol colorimetric method revealed that the intracellular enzyme activity of strain FA3 was 263.75 U/g. The optimized culture conditions were as follows: emulsified olive oil at 4 mL/L, peptone at 18 g/L, K₂HPO₄ at 1 g/L, NaCl at 10 g/L, MgSO₄ at 0.5 mmol/L, and pH 5.3. After culture at 30 ℃ for 77.5 h, the specific activity reached 2 120.27 U/g, which was about 8.04 times higher than that before optimization. The proteomic analysis and structural prediction revealed the conserved Gly-Asp-Ser motif, confirming the lipase as a typical GDSL lipase. The enzyme contained strictly conserved Ser, Gly, Asn, and His residues and it was thus classified as a member of the N-sulphoglucosamine sulphohydrolase (SGNH) hydrolase family.Conclusion After optimization of the culture conditions, strain FA3 demonstrated significantly enhanced lipase production, showing great potential for application. We clarify the functions and structural characteristics of the lipase, providing a basis for the engineering of related proteins.