一株海洋來源高效產角蛋白酶菌株的篩選、鑒定及其酶學性質研究

張妮，張紅巖，楊夢瑩，劉聰，楊立芳，申乃坤*，姜明國

1(廣西民族大學 海洋與生物技術學院，廣西多糖材料與改性重點實驗室，廣西高校微生物與植物資源利用重點實驗室，廣西 南寧， 530006)2(廣西民族大學 化學化工學院，廣西林產化學與工程重點實驗室，廣西 南寧， 530006)

Isolationandidentificationofahighlyefficientkeratinaseproducingstrainfrommarineenvironmentanditsenzymaticproperties

ZHANG Ni1,ZHANG Hongyan1,YANG Mengying1, LIU Cong2,YANG Lifang2,SHEN Naikun1*,JIANG Mingguo1

1(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)2(Guangxi Key Laboratory of Chemistry and Engineering of Forest Products, School of Chemistry and Chemical En-gineering, Guangxi University for Nationalities, Nanning 530006)

ABSTRACTThis study aimed to obtain a highly efficient keratinase producing strain, improve its enzyme-producing ability and study the properties of keratinase. A strain Gxun-30 of highly efficient degradation ability for feather keratin was isolated from the soil of feather waste in the duck breeding base in Beibu gulf by enrichment, primary screening and re-screening process．It was identified asBacillusparamycoides, according to its morphology, physiological and biochemical characteristics, and 16S rDNA sequence alignment and phylogenetic trees analysis. The keratinase producing conditions and enzymatic properties were studied．The results showed that its optimum fermentation temperature was 35°C．After fermenting at 35°C for 48 h, the feather was almost completely degraded and the keratinase activity reached 434.54 U/mL. The keratinase had maximum activity at 75°C and optimum pH at 7.5．Ca2+and Na+could greatly enhance the activity of keratinase, but its activity was strongly inhibited by Mn2+and Cu2+. And it was inhibited by phenyl methyl sulfonyl fluoride (PMSF) and ethylene diamine tetra acetic acid (EDTA), indicating that it is a metallo-serine keratinase. The enzyme could remain stable in the presence of surfactants, including sodium dodecyl sulfate (SDS), isopropyl alcohol and Tween 40. Especially, isopropyl alcohol could substantially enhance the activity to 114%. The substrate specificity results showed that this keratinase exhibited high activity towards casein, keratin and feather, but only little degradation on hair and type I collagen. The results indicated thatB.paramycoidesGxun-30 and its protease was of potential application for feather degradation, casein peptide and collagen material preparation.

Keywordsmarine bacterium; feather-degrading; isolation and identification; keratinase; characteristics of proteinases

近年來，隨著我國家禽養殖產業迅猛發展，羽毛產量也逐年遞增，超過100萬t/年，大部分卻并未得到利用，通常被填埋或焚燒[1]。不僅造成資源的巨大浪費，而且嚴重污染環境，甚至傳播雞新城疫、氣囊炎和角膜結膜炎等疾病[2]。此外，焚燒還會釋放氨、惡臭硫化氫等有害氣體，引起頭痛、惡心、腹瀉等健康問題[3]。羽毛中粗蛋白含量可達85%以上，且氨基酸組成比較齊全，特別是半胱氨酸是常見天然飼料中含量最高的[4-5]。但羽毛以角蛋白為主，是一類富含二硫鍵、結構穩定的“不溶于水的硬性蛋白”，動物消化率極低，需要處理后才能應用。目前，常采用高溫高壓、強酸強堿和擠壓膨化等物理或化學方法進行處理，不僅工藝復雜、耗能多、污染環境，而且處理過程中會破壞氨基酸，造成營養流失[6]。利用微生物降解廢棄羽毛，不僅過程溫和、效果顯著，且對氨基酸破壞較小，可高效生產稀有氨基酸、肽等產品，應用于飼料、肥料、醫藥等行業[7-9]，不僅利用廢棄蛋白資源，促進羽毛的深加工發展，對環境保護也具有積極意義。

目前已篩選出真菌、放線菌、細菌等30多種可降解羽毛的微生物[4, 10]。但大多數真菌為皮膚類真菌，具有致病性，應用價值較低[11]；……