Embaby Amira M, Saeed Hesham, Hussein Ahmed
Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Egypt.
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas, USA.
J Basic Microbiol. 2016 Dec;56(12):1317-1330. doi: 10.1002/jobm.201600073. Epub 2016 Jun 23.
Present study underlines an unusual non-cumbersome-powerful strategy for purification of SHG10 keratinolytic alkaline protease from Bacillus licheniformis SHG10 DSM 28096 with robust stability properties. The enzyme was impressively purified to homogeneity with specific activity, purification fold, and yield of 613.82 U mg , 58.91 and 99%, respectively, via a sequential two-step purification strategy: precipitation with 65% (NH ) SO and flow through fractions of DEAE-cellulose DE 53 column. SDS-PAGE conferred a monomeric enzyme with a molecular mass of 30.4 kDa. The enzyme demonstrated optimal activity at pH (10.0-11.0) and at 65 °C. It exhibited full stability at pH (6.0-11.0) over 38 h at 4 °C and at 65 °C for 15 min. Remarkable enhanced enzyme activity (130.15 and 126.37%) was retained in presence of commercial laundry detergents Oxi and Ariel after 1 h, respectively. Organic solvent stability of the enzyme was verified in butanol, ether, acetonitrile, isopropanol, and chloroform. Imposingly, full storage stability (100%) of the enzyme along 1 year in -20 °C was confirmed. K -V was 0.00174 mM-534.2 mM Sub · min · mg protein and 1.266 mg-28.89 mg Sub · h · mg protein on N-Suc-Ala-Ala-Pro-Phe-pNA and keratin azure, respectively. Robust stability properties of SHG10 keratinolytic alkaline protease along with rapid-efficient purification underpin its potential commercialization for industrial exploitation.
目前的研究强调了一种从地衣芽孢杆菌SHG10 DSM 28096中纯化SHG10角蛋白分解碱性蛋白酶的独特、简便且高效的策略，该酶具有强大的稳定性。通过连续两步纯化策略，即先用65%硫酸铵沉淀，再通过DEAE-纤维素DE 53柱的流通部分，该酶令人印象深刻地纯化至均一，比活性、纯化倍数和产率分别为613.82 U mg、58.91和99%。SDS-PAGE显示该酶为单体，分子量为30.4 kDa。该酶在pH值为10.0 - 11.0和65°C时表现出最佳活性。在4°C下38小时以及在65°C下15分钟内，它在pH值为6.0 - 11.0时表现出完全稳定性。在分别与市售洗涤剂Oxi和Ariel接触1小时后，该酶仍保留了显著增强的酶活性（分别为130.15%和126.37%）。该酶在丁醇、乙醚、乙腈、异丙醇和氯仿中验证了有机溶剂稳定性。令人印象深刻的是，证实该酶在-20°C下保存1年具有完全稳定性（100%）。在N - Suc - Ala - Ala - Pro - Phe - pNA和角蛋白天青上，Km - Vmax分别为0.00174 mM - 534.2 mM Sub·min·mg蛋白质和1.266 mg - 28.89 mg Sub·h·mg蛋白质。SHG10角蛋白分解碱性蛋白酶强大的稳定性以及快速高效的纯化，为其工业开发的潜在商业化奠定了基础。
