一株可高效降解羽毛的铜绿假单胞菌的分离、鉴定、产酶条件优化及其酶活研究

Isolation, identification, optimization of enzyme-producing conditions and enzymatic activity of a feather-degradable Pseudomonas aeruginosa strain

摘要:

【目的】筛选海洋环境产角蛋白酶菌株，研究其发酵条件及酶学性质，为后续开发和利用海洋微生物降解废弃羽毛提供菌种资源和理论依据。【方法】采集广西北部湾某海鸭养殖场淤泥，用酪蛋白平板初筛和角蛋白酶活复筛获得羽毛降解效果好的菌株，并进行形态学和分子生物学鉴定；利用单因素和正交试验对菌株产酶条件进行优化，最后对酶学性质及羽毛降解产物的游离氨基酸组成进行研究。【结果】筛选到1株可高效降解羽毛的菌株，经鉴定为铜绿假单胞菌（Pseudomonas aeruginosa Gxun-7）。最佳产酶条件为：羽毛25 g/L，Zn²⁺ 0.10 g/L、初始pH 8.0、发酵温度32.5℃、发酵时间48 h，酶活力达124.03 U/mL，较优化前提高了2.3倍；酶学性质分析表明，该角蛋白酶最适作用温度和pH分别为70℃和8.0，化学试剂巯基乙醇可使酶活提高6.16倍，而苯甲基磺酰氟（PMSF）使相对酶活降至15.00%，该酶耐盐性较好（20% NaCl中相对酶活为74.29%）；羽毛降解产物中检测到16种氨基酸，7种为必需氨基酸，总的游离氨基酸含量高达2 329.80 mg/L，其中缬氨酸含量最高为575.89 mg/L。【结论】海洋来源的P. aeruginosa Gxun-7具有高效降解羽毛角蛋白能力，所产碱性角蛋白酶具有耐温、耐盐等特性，具有潜在的应用价值。

关键词:羽毛降解;角蛋白酶;铜绿假单胞菌;酶活

Abstract:

[Objective] The aim of this study was to screen keratinase-producing strains from marine environment, investigate fermentation conditions and enzymatic properties, so as to provide strain resources and theoretical basis for the subsequent development and utilization of marine microbes to degrade feather waste.[Methods] The sludge from a marine duck farm in Beibu Gulf, Guangxi was used for bacterial isolation, and the strain with efficiently feather degradation ability was obtained by casein plate preliminary screening and keratinase activity re-screening. The strain was identified by morphology and molecular biology, and the enzyme-producing conditions were optimized by single-factor and orthogonal experiment. Finally, amino acid composition of feather degradation products and enzymatic properties were also studied.[Results] A strain of efficiently degrading feather was selected and identified as Pseudomonas aeruginosa Gxun-7. The optimum enzyme-producing conditions:feather 25 g/L, Zn²⁺ 0.10 g/L, initial pH 8.0, fermentation temperature 32.5℃, fermentation time 48 h, and the keratinase activity was 124.03 U/mL which was 2.3 times as high as before optimization. The analysis of enzyme properties showed that the optimum temperature was 70℃ and the optimum pH was 8.0. In the chemical reagent, mercaptoethanol could enhance enzymatic activity by 6.16 times, while phenylmethylsulfonyl fluoride (PMSF) decreased its relative activity to 15.00%. And it had excellent salt resistance (the relative activity in 20% NaCl retained 74.29%). There were 16 kinds of amino acids in the feather degradation products, including 7 kinds of essential amino acids. The total free amino acids content was as high as 2 329.80 mg/L, and the highest valine content was 575.89 mg/L.[Conclusion] P. aeruginosa Gxun-7 from marine environment had the ability to degrade feather keratin efficiently. The alkaline keratinase had excellent temperature and salt resistances, which had potential application value.

Key words:feather degradation;keratinase;Pseudomonas aeruginosa;enzymatic activity