海洋来源羽毛降解菌株的筛选、鉴定及酶学性质研究

微生物降解羽毛既可保护环境又能实现废物的资源化利用,具有显著的经济效益和社会效益。本研究从广西钦州港海鸭养殖淤泥筛选获得一株可高效降解羽毛的菌株Gxun-20,通过对该菌株形态特征、生理生化特性和16S rRNA基因序列进行分析,以确定菌株的分类,并对菌株产角蛋白酶的条件及酶学性质进行研究。结果表明：该菌为副短短芽孢杆菌(Brevibacillus parabrevis);菌株最适产酶条件为：温度34 ℃,初始pH 6.5,羽毛含量1.5%(质量分数),发酵48 h,角蛋白酶活性可达277.45 U/mL。菌株Gxun-20所分泌的角蛋白酶最适温度为50 ℃,且热稳定性较好,最适pH为7.5;金属离子Fe³⁺、Cu²⁺、Co²⁺、Fe²⁺和Mn²⁺对酶活性有显著抑制作用,而Ca²⁺、Na⁺对酶活性有明显促进作用;金属螯合剂乙二胺四乙酸(ethylene diamine tetraacetic acid, EDTA)可显著抑制酶的活性,蛋白酶抑制剂苯甲基磺酰氟(pheny methyl sulfonyl fluoride, PMSF)可完全抑制酶活性,表明该蛋白酶的活性中心含有金属离子和丝氨酸;异丙醇可使酶活性提高至129.34%,酶活性在十二烷基硫酸钠(sodium dodecyl sulfate, SDS)、吐温40溶液中保持稳定;酶的底物特异性结果表明,该酶对羽毛类β-角蛋白及酪蛋白降解能力较好,而对牛血清蛋白、I型胶原及人发类α 角蛋白降解能力较弱。海洋来源的副短短芽孢杆菌Gxun-20及其所产角蛋白酶在废弃羽毛降解方面具有良好的应用价值。     

深海羽毛降解菌Bacillus sp. JM7极端碱耐受角蛋白酶的酶活性质研究

Bacillus sp. JM7, a strain isolated from the deep-sea of the South China Sea, was found to efficiently degrade 79.4% native chicken feather within 30 h. Scanning electron microscopy analysis showed that JM7 strain could gradually degrade feather by modifying the microstructure of feather keratin. A total of 25 protease genes were predicted from the draft genome of JM7 strain, among which a predicted subtilisin-like serine protease(designated as Ker02562) was further characterized for its keratinolytic activity. The recombinant Ker02562 functioned at a wide range of temperatures from 30℃ to 60℃, with an optimum at 40–50℃. Ker02562 was highly active at various pHs ranging from 5.0 to 13.0, with a maximum activity observed at pH 7.0–9.0. Remarkably, recombinant Ker02562 was stable in extreme alkaline environments(pH 10–13), which was much better than most other reported keratinases. Collectively, these favorable properties could make Bacillus sp. JM7 and Ker02562 attractive to be applied in the detergent formulation and feather bioconversion.     

角蛋白酶基因工程研究

角蛋白是一种广泛存在于自然界中的硬性蛋白,主要存在于各种动物的毛发、鳞片、羽毛、蹄、角等结构中。角蛋白化学结构稳定,不易被一般的蛋白酶降解,但作为角蛋白酶的专一性底物,角蛋白可被角蛋白酶降解。角蛋白酶由微生物产生,可特异性降解羽毛等含角蛋白类的物质,在食品、医药、饲料生产和环境废物处理等方面具有潜在的应用价值。综述了微生物角蛋白酶基因分离和表达研究进展,并对角蛋白酶基因在异源表达过程中出现的问题进行了讨论。