微生物转谷氨酰胺酶的生产菌种诱变和发酵生产分析

Fermentation of microbial transglutaminase by new Streptomyces mutant

对本研究室从土壤分离得到的链霉菌(Streptomyces sp.)WZFF.W-12菌株的斜面孢子预培养处于初萌发状态后,以亚硝基胍(NTG)进行诱变育种试验,并根据诱变处理后菌落的某些形态变化状况与产酶能力相结合的特征。初步判断产酶性能,挑选高酶活菌株,再经过初筛和复筛,获得一性能良好的产酶突变菌株WZFF.W-12.varMN-35,转谷氨酰酶活达0.53 U/mL,比原始菌株提高了1.2倍。然后在摇瓶条件下,对其发酵过程中的主要培养基组成及各种培养条件对菌体生长和产酶的影响作用进行了研究,结果表明该菌株发酵生产转谷氨酰酶的适宜碳源为可溶性淀粉+葡萄糖,氮源是多价胨外加少量的酵母膏,优化工艺条件为种龄时间24 h、接种量10％、初始pH值6.5、温度30℃和搅拌速度200 r/min,产酶能力显著提高,用小型生化反应器可以稳定生产2.0U/mL以上的酶产品。

The precultured spores of Streptomyces sp. WZFF.W- 12 isolated from soil and preserved in our laboratory producing microbial transglutaminase (MTG) about 0. 24 U/mL, were used for genetic breeding through several N - methyl - N' - nitro - N - nitrosogunidine (NTG) - mediated genetic mutation tests. According to the resulted mutant isolation analysis, some mutants showed clear differences from the original strain including colony morphology and situations were found in direct relation to the ability to produce MTG and thereafter used to select efficient MTG - producing mutants. Through both primary and secondary screening tests, an effective mutant strain named Streptomyces sp. WZFF.W- 12. var MN - 35 was finally obtained and its MTG productivity was 0.53 U/mL, which has been increased to about 1.2 times higher than that of the initial strain. The effects of the culture conditions and the main compositions of fermentation medium on the MTG production were investigated, and the results showed that the suitable fermentation technology for the enzyme production was recommended as follows: 1.0% soluble starch with 1.5% glucose for carbon source, 2.5% multi - peptone for nitrogen source and plus with a low amount of yeast extract, initial pH 6.5, the inoculum time at 24 hours, the inoculum ratio 10% , and the shaking speed at 200 r/min. Under these conditions, the enzyme productivity was significantly enhanced and the W - 12. var MN - 35 strain could stably repeat MTG -producing ability up to 2.10 U/mL with a simple but effective and easily - controlled 2L mini - bioreactor de- signed and equipped in our laboratory.