产碱性蛋白酶地衣芽孢杆菌合成培养基筛选与优化

Screening and optimization of chemically defined medium for alkalineprotease production by Bacillus licheniformis

该研究对于产碱性蛋白酶地衣芽孢杆菌(Bacillus licheniformis)的合成培养基进行了系统的筛选与优化。基于豆粕成分分析及文献报道构建初始化学合成培养基CDM_(0),并通过单一组分移除实验,初步确定对菌体生长有显著影响(生长降低20%以上)的组分,包括3种碱基、4种维生素、11种氨基酸以及一些金属离子和无机盐。在此基础上进行了组分添加实验,分别添加单一组分移除实验从CDM_(0)中剔除的组分,进而筛选出对菌体生长具有20%以上促进作用的5种关键营养物质,并用全排列方法得到了26种组合添加方式,经实验确定了添加的最佳组合为(NH_(4))_(2)SO_(4)、腺嘌呤和天冬酰胺。此外,还研究了甘油、5种单糖和3种双糖对菌体生长及碱性蛋白酶合成的影响,最终以半乳糖为碳源确定了一种优化后的化学全合成培养基CDM_(3)。实验结果表明,与CDM_(0)相比,CDM_(3)显著提高了菌体的生长速率和碱性蛋白酶的产量,其中酶活力提升至10300.7 U/mL,提高了约2.5倍。该实验结果为今后研究该菌株的代谢特性奠定了基础。

In this study,a systematic screening and optimization approach was employed to identify and characterize the optimal chemically defined medium for cultivating Bacillus licheniformis to produce alkaline protease.The initial chemically defined medium CDM_(0)was constructed based on the analysis of the composition of soybean meal and relevant literature reviews.The components that significantly affected the growth of the bacterium(growth reduced by more than 20%)were preliminarily identified through single component removal experiments,including three bases,four vitamins,11 amino acids,as well as various metal ions and inorganic salts.Based on this,the components that were taken out of CDM_(0)were added respectively and then five key nutrients with a more than 20%enhancement in cell growth were screened out.Then the optimal combination of(NH_(4))_(2)SO_(4),adenine,and asparagine was obtained from a total of 26 combinations of the five key nutrients.Furthermore,the effects of glycerol,five monosaccharides,and three disaccharides on bacterial growth and alkaline protease chemically defined were also investigated.Ultimately,an optimized chemical complete synthesis medium,CDM 3,was developed with galactose as the carbon source.The experimental results demonstrated that the cell growth and alkaline protease production under CDM 3 were found to be significantly increased compared to the initial medium CDM_(0),where the enzyme viability was elevated by 2.5-fold to 10300.7 U/mL.The findings of this experiment provide a foundation for future research on the metabolic characteristics of this strain.