为获得高效产角蛋白酶的菌株,提高产酶能力,研究角蛋白酶酶学性质,该文从广西北部湾海鸭养殖羽毛废弃物土壤通过富集、初筛和复筛获得1株高效降解羽毛角蛋白的菌株Gxun-30,综合形态、生理生化和16S rDNA 序列的系统进化分析结构,鉴定为拟蕈状芽孢杆菌(Bacillus paramycoides)。该菌株产酶条件及所产酶学性质研究结果为,菌株产酶最适温度35 ℃,发酵48 h,整根羽毛几乎完全降解,酶活性可达434.54 U/mL;所产角蛋白酶最适温度为75 ℃,最适pH为6.5;该酶热稳定性较好,在70 ℃以下,能保持酶活的稳定性;金属离子中Ca2+、Na+对该酶活性起到激活作用,而Mn2+、Cu2+对该酶有显著抑制作用;酶活性可被苯甲基磺酰氟(phenyl methy sulfonyl fluoride,PMSF)和乙二胺四乙酸(ethylenediamine tetraacetic acid,EDTA)抑制,表明可能为含金属离子的丝氨酸蛋白酶类;异丙醇对酶活性有明显激活作用,相对酶活性可提高至114%;且在十二烷基硫酸钠(sodium dodecyl sulfate,SDS)、吐温-40溶液中具有良好的稳定性;该酶降解羽毛、角蛋白及酪蛋白的能力较强,但对人发及胶原蛋白降解能力较弱,该特性使B. paramycoides Gxun-30菌株及所产角蛋白酶在羽毛降解、酪蛋白肽及医用胶原材料制备等方面具有潜在应用价值。
This 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 as Bacillus paramycoides, 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 that B. paramycoides Gxun-30 and its protease was of potential application for feather degradation, casein peptide and collagen material preparation.
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