该研究以短小芽孢杆菌(Bacillus pumilus)为对象,系统探究了其胞外蛋白酶的分离纯化工艺、酶学特性及水解能力,旨在挖掘具有新型肽键选择性的蛋白酶资源。通过超滤浓缩、离子交换层析及凝胶层析,从短小芽孢杆菌发酵液中成功分离到2种丝氨酸蛋白酶组分P0和P1,其比酶活力分别为5 768 U/mg和6 873 U/mg。通过SDS-PAGE初步确定P0和P1的分子质量分别为20.3 kDa和34 kDa。酶学性质研究表明,P0和P1的最适作用温度为50~55 ℃,最适pH分别为8.5和9.6;两者在pH 7~10及≤45 ℃较稳定;P0组分的活性不受金属离子影响,对SDS有较好耐受性(1% SDS下可保留86%活力);EDTA、Zn2+、Mn2+、Fe2+可显著抑制P1组分的活性,而Co2+、Ca2+、Cu2+可显著促进其酶活力。肽键选择性的结果显示,2种蛋白酶在胰岛素β链上的酶切位点相似,但与胰蛋白酶和木瓜蛋白酶有显著差异。蛋白酶P0和P1对大豆蛋白有高效的水解能力:酶解6 h后,水解液中80%以上产物为分子质量<5 kDa的肽段,其中60%是分子质量<1 kDa的小肽。结果表明,短小芽孢杆菌蛋白酶具有较宽泛的肽键选择性,可以实现底物蛋白的深度水解。
To discover protease resources with novel peptide bond selectivity,the purification procedures, enzymatic properties and hydrolytic capacity of extracellular proteases from Bacillus pumilus were systematically investigated.Two serine protease fractions, designated P0 and P1, were successfully isolated from the B.pumilus fermentation broth through ultrafiltration, ion-exchange and gel filtration chromatography.Their specific activities reached 5 768 U/mg and 6 873 U/mg, respectively.The molecular weights of P0 and P1 were estimated to be 20.3 kDa and 34 kDa based on SDS-PAGE.Both proteases exhibited maximum activity at 50-55 ℃, with optima pH at 8.5 (P0) and 9.6 (P1).Stability profiles showed both proteases retained activity within pH 7-10 and at temperatures ≤45 ℃.P0 activity showed no sensitivity to tested metal ions and demonstrated higher tolerance to SDS (retaining 86% activity in 1% SDS).Conversely, EDTA, Zn2+, Mn2+, and Fe2+ significantly inhibited P1 activity, while Co2+, Ca2+, and Cu2+ significantly enhanced it.Cleavage specificity analysis revealed similar cleavage sites on the insulin β-chain for P0 and P1, differing significantly from trypsin and papain.Both proteases could efficiently hydrolyze soybean protein.Hydrolysis for 6 hours produced a hydrolysate where peptides with molecular weights <5 kDa constituted over 80% of the products, and small peptides (molecular weight <1 kDa) accounted for 60% of these.These results indicate that proteases P0 and P1 from B.pumilus possess broad cleavage specificity and can efficiently hydrolyze substrate proteins.
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