Chromosomal Integration에 의해 제조한 Bacillus clausii C5 유래의 alkaline protease의 세제 첨가제 응용성

Abstract

Bacillus clausii I-52 which produced SDS- and H₂O₂-tolerant extracellular alkaline protease (BCAP) was isolated from heavily polluted tidal mud flat of West Sea in Incheon, Korea and stable strain (transformant C5) of B. clausii I-52 harboring another copy of BCAP gene in the chromosome was developed using the chromosome integration vector, pHPS9-fuBCAP. When investigated the production of BCAP using B. clausii transformant C5 through pilot-scale submerged fermentation (500 L) at 37℃ for 30 h with an aeration rate of 1 vvm and agitation rate of 250 rpm, protease yield of approximately 105,700 U/mL was achieved using an optimized medium (soybean meal 2%, wheat flour 1%, sodium citrate 0.5%, K₂HPO₄ 0.4%, Na₂HPO₄ 0.1%, NaCl 0.4%, MgSO₄ · 7H₂O 0.01%, FeSO₄· 7H₂O 0.05%, liquid maltose 2.5%, Na₂CO₃ 0.6%). The enzyme stability of BCAP was increased by addition of polyols (10%, v/v) and also, the stabilities of BCAP towards not only the thermal-induced inactivation at 50℃ but also the SDS and H₂O₂-induced inactivation at 50℃ were enhanced. Among the polyols examined, the best result was obtained with propylene glycol (10%, v/v). The BCAP supplemented with propylene glycol exhibited extreme stability against not only the detergent components such as α-orephin sulfonate (AOS) and zeolite but also the commercial detergent preparations. The granulized enzyme of BCAP was prepared with approximately 1,310,000 U/g of granule. Wash performance analysis using EMPA test fabrics revealed that BCAP granule exhibited high efficiency for removal of protein stains in the presence of anionic surfactants as well as bleaching agents. When compared to Savinase 6T ⓡ and Everlase 6T ⓡ manufactured by Novozymes, BCAP under this study probably showed similar or higher efficiency for the removal of protein stains. These results suggest that the alkaline protease produced from B. clausii transformant C5 showing high stability against detergents and high wash performance has significant potential and a promising candidate for use as a detergent additive.