基于基因组改组技术的角蛋白酶高产菌株选育研究

Breeding of microorganisms with high keratinase production based on genome shuffling

【目的】筛选具有高效角蛋白水解活性的微生物,为提高废弃羽毛中角蛋白资源的利用奠定基础。【方法】采用梯度稀释法,以羽毛作为惟一碳氮源对土样中的产角蛋白酶菌株进行筛选,筛选可高效降解羽毛的细菌。通过菌株的形态、生理生化特性以及16S rDNA序列分析,对菌株进行分类鉴定,并探讨其最适发酵条件。利用硫酸二乙酯(DES)和亚硝基胍(NTG)对目的菌株进行诱变,确定最适诱变条件,并进行原生质体融合,通过基因组改组选育角蛋白酶高产菌株。【结果】分离筛选到1株具有较强羽毛降解能力的菌株ZJT01,该菌落圆形凸出,革兰氏染色呈阳性,酪素水解、硝酸盐利用、柠檬酸利用等生理生化试验均呈阳性。结合菌株16S rDNA同源序列比对分析,初步鉴定其为节杆菌(Arthrobactersp.)。菌株ZJT01产角蛋白酶的最适发酵温度和时间分别为32℃和72h。其DES最适用量为9μL/mL,诱变时间为20min;NTG的最适合质量浓度是0.6mg/mL,处理时间为15min。原生质体制备的最佳条件为:溶菌酶终质量浓度10 mg/mL,酶解温度37℃,酶解0.5h。经过硫酸二乙酯(DES)和亚硝基胍(NTG)分别诱变处理,获得角蛋白酶活性提高的菌株DES-2和NTG-2;对其进行3轮基因组改组后,筛选到2株角蛋白酶活性比亲本菌株ZJT01提高了5.48倍的重组菌株,且其产酶活性可稳定遗传。【结论】通过基因组改组技术获得了2株角蛋白酶活性大幅提高的菌株F3-5和F3-6,这2株菌对羽毛的降解效果较亲本菌株ZJT01明显提高,具有较好的应用前景。

【Objective】The aim of this study was to screen microorganisms with high keratinolytic activity for improving the utilization of keratin resources in poultry.【Method】By serially dilution method,microorganisms with high keratinolytic activity were isolated from soil sample using feather as the sole source of carbon and nitrogen.Through the morphological and physiological characteristics and 16 SrDNA sequence analysis,the strain was identified and the optimum fermentation conditions were investigated.The target strain was subjected to diethyl sulfate（DES）and N-methyl-N-nitro-N-nitrosoguanidine（NTG）mu-tagenesis firstly to determine optimal mutagenesis condition,and then strains with higher keratinase production were screened by genome shuffling after protoplast fusion.【Result】A bacterial strain ZJT01 with high feather degrading ability was isolated and the colony presented circular projection.Gram stain,casein hydrolysis,utilization of nitrate,and utilization of sodium all showed positive and 16S rDNA sequence analysis designated it as Arthrobacter sp.The optimum fermentation temperature and time were 32 ℃ and 72 h,respectively.The DES mutagenic optimal condition was 9μL/mL for 20 min and the NTG condition was0.6 mg/mL for 15 min.The optimal condition for protoplast preparation was 10 mg/mL lysozyme for 30 min at 37℃.Two mutant strains named DES-2 and NTG-2 with improved keratinase activity was obtained by treatment with diethyl sulfate（DES）or N-methyl-N-nitro-N-nitrosoguanidine（NTG）.After three rounds of genome shuffling,two improved recombinants were obtained with genetic stable keratinase activity increased by 5.48 times.【Conclusion】Two strains with significantly increased keratinase activity and good application prospect（F3-5 and F3-6）were obtained.