| 摘要: |
| 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。 |
| 关键词: 海洋生物学 角蛋白酶 废弃羽毛 副短短芽孢杆菌 分离鉴定 酶学性质 |
| DOI：10.3969/J.ISSN.2095-4972.2022.02.019 |
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| 基金项目:国家自然科学基金资助项目(32060020，32160017)；广西科技重大专项资助项目(AA18242026,AB21196019,AB221220020)；国家级大学生创新创业训练计划资助项目（202110608031) |
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| Isolation, identification and enzymatic property of a feather degrading strain from marine environment |
| ZHANG Hongyan,HE Jin,YANG Mengying,ZHANG Ni,WANG Fang,SHEN Naikun |
| (Guangxi Key Laboratory for Polysaccharide Materials and Modifications, Guangxi Key Laboratory of Microbial Plant Resources and Utilization, School of Marine Sciences and Biotechnology, Guangxi University for Nationalities, Nanning 530006, China) |
| Abstract: |
| Microbial degradation of feather favors the environment protection as well as waste utilization. So, it has remarkable economic and social benefits. A highly efficient feather degrading strain (designated as Gxun-20) was isolated from the sludge of duck breeding base in Qinzhou port of Guangxi. The species information was identified using combined methods of morphological observation, physiological and biochemical characteristics and 16S rRNA gene sequence analysis. The keratinase producing conditions and enzymatic properties were also studied. The strain was identified as Brevibacillus parabrevis Gxun-20 and its optimal conditions for keratinase production were determined at temperature 34 ℃, initial medium pH 6.5, feather content 1.5%. The keratinase activity could reach 277.45 U/mL with 48 h fermentation. The maximal keratinase activity was detected at 50 ℃, pH 7.5. It showed that Ca2+ and Na+ enhanced the enzyme activity significantly, while Fe3+,Cu2+,Co2+,Fe2+and Mn2+ inhibited the activity. The enzyme activity was also inhibited by ethylene diaminetetra acetic acid and phenyl methyl sulfonyl fluoride, indicating that it is a serine-metallo keratinase. The enzyme remained stable with surfactants, including isopropyl alcohol, sodium dodecyl sulfate and Tween-40, of which isopropyl alcohol significantly improved the activity up to 129.34%. The substrate specificity results showed that the keratinase exhibited high activity toward β-keratin, feather and casein, but little degradation on type I collagen, α-keratin and hair. The B. parabrevis Gxun-20 strain and its keratinases have potential application for waste feather degradation. |
| Key words: marine biology keratinase waste feather Brevibacillus parabrevis isolation and identification enzymatic property |
