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To verify anti-diabetic effect of oligopeptide with His-Pro repeats (mHP peptide), the oligopeptide was first secreted and optimized using the secretion vector, pRBAS with alkaline protease gene promoter and the signal sequence in Bacillus subtilis and directly the anti-diabetic effect of the mHP peptide was investigated in insulinoma cell, RINm5F cell line. The oligopeptide gene was obtained by annealing oligonucleotides with repeated His-Pro sequence and finally was constructed as 18 dipeptides (108 bp and 4.0 kDa) coding gene, named oligopeptide with His-Pro repeats (mHP peptide) to make cyclo(His-Pro) known to be anti-diabetic effects. The region encoding the oligopeptide gene was subcloned into the pRBAS secretion vector (E.coli-Bacillus shuttle vector) after PCR amplification using the designed primers including initiation and termination codons and His tag, named pRBAS-mHP (6.56 kb). To optimize secretion of the oligopeptide, various culture conditions were investigated in Bacillus subtilis LKS. As a result, the secreted oligopeptide was maximally measured (approximately 59.6 µg/mL) in 3 L batch culture and the highest secretion was achieved at 30°C, PY medium, and carbon sources (particularly barley and glycerol). In the RINm5F cells treated with 2 mM STZ, the oligopeptide treatment (0.1 mg/mL) restored the cell viability (10%) and reduced the nitric oxide (NO) generation (35%) and DNA fragmentation (90%). And also, insulin secretion level was increased to 17% higher than in STZ-treated RINm5F cells. These results suggest that the oligopeptide with His-Pro repeats could be a candidate material for anti-diabetic agent against STZ-induced diabetes.

1) To enhance cyclo-His-Pro (CHP) content, soybean hydrolysate was obtained using the strains isolated from Chungkukjang and further purified by various purification steps. First, twenty two strains were screened from Chungkukjang containing high level of CHP. Among them, the strain No. 12, which showed higher productivity of CHP from soybean ferment and have homologous sequence with 16S rDNA of Bacillus amyloliquefaciens, was named B. amyloliquefaciens CHP-12. Through various purification processes, CHP was concentrated from soybean ferment using ultrafiltration, which showed the best efficiency of CHP production, with the yield (71.3%) and CHP content (2.14 mg/g). Moreover, when glucose tolerance test was performed in Type I Sprague-Dawley rat induced by streptozotocin using the soybean ferments [0.5 g soybean ferment/kg body weight (CHP-0.5 group)] and 1.0 g soybean ferment/kg body weight (CHP-1.0 group), there were significant differences in glucose levels between diabetes-control group (265.3 mg/dL) and soybean ferment-treated groups (CHP-0.

1) Bacillus clausii I-52 which produced SDS-and H 2 O 2 -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%, K 2 HPO 4 0.4%, Na 2 HPO 4 0.1%, NaCl 0.4%, MgSO 4 · 7H 2 O 0.01%, FeSO 4 · 7H 2 O 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.

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