Effect of metallic ions and elastase inhibitors.

Ninety-one elastase-producing bacterial isolates were recovered from different localities of the Eastern Province of Saudi Arabia. Elastase from the best isolate Priestia megaterium gasm32, from luncheon samples was purified to electrophoretic homogeneity using DEAE-Sepharose CL-6B and Sephadex G-100 chromatographic techniques. The recovery was 17.7%, the purification fold was 11.7x, and the molecular mass was 30 kDa. Enzymatic activity was highly repressed by Ba2+ and almost completely lost by EDTA, but it was greatly stimulated by Cu2+ ions, suggesting a metalloprotease type. The enzyme was stable at 45°C and pH 6.0–10.0 for 2 hours. Ca2+ ions considerably enhanced the stability of the heat-treated enzyme. The Vmax and Km against the synthetic substrate elastin–Congo red were 6.03 mg/mL, and 8.82 U/mg, respectively. Interestingly, the enzyme showed potent antibacterial activity against many bacterial pathogens. Under SEM, most bacterial cells showed loss of integrity, damage, and perforation. SEM micrographs also showed a time-dependent gradual breakdown of elastin fibers exposed to elastase. After 3 hours, intact elastin fibers disappeared, leaving irregular pieces. Given these good features, this elastase may be a promising candidate for treating damaged skin fibers with the inhibition of contaminating bacteria.

从沙特阿拉伯东部省不同地点分离得到91株产弹性蛋白酶（elastase）的细菌分离株。从午餐肉样品中筛选得到的最优菌株——巨大普里斯特芽孢杆菌（Priestia megaterium）菌株gasm32所产的弹性蛋白酶，通过DEAE-琼脂糖凝胶CL-6B与葡聚糖凝胶G-100层析技术被纯化至电泳纯。该酶的回收率为17.7%，纯化倍数为11.7倍，相对分子质量为30 kDa。其酶活性可被Ba²+显著抑制，几乎可被乙二胺四乙酸（EDTA）完全灭活，但可被Cu²+极大激活，提示其属于金属蛋白酶类。该酶在45℃、pH 6.0~10.0的条件下孵育2小时仍保持稳定。Ca²+可显著提升经热处理后的该酶的稳定性。以合成底物弹性蛋白-刚果红（elastin–Congo red）为底物时，该酶的最大反应速率（Vmax）为6.03 mg/mL，米氏常数（Km）为8.82 U/mg。值得注意的是，该酶对多种细菌性病原菌具有强效抗菌活性。在扫描电子显微镜（SEM）下，多数细菌细胞出现完整性丧失、破损及穿孔现象。扫描电子显微镜图像还显示，经弹性蛋白酶处理的弹性蛋白纤维会随时间推移逐渐降解；处理3小时后，完整的弹性蛋白纤维完全消失，仅残留不规则碎片。鉴于上述优良特性，该弹性蛋白酶有望成为兼具抑制污染菌与修复受损皮肤纤维潜力的候选制剂。