The morecular cloning, gene structure, expression and enzyme activity of a serine-like proteas frome Laccotrephes Japonensis were examined. In this study, RT-PCR was used to amplify cDNA fragments for serine-like proteases from total RNA the hole body of Laccotrephes japonensis. The flanking sequences of the 5'- and 3'- end of the this gene were characterized by RACE-PCR. Sequence analysis showed that this gene contained an 963bp ORF encoding 321 amino acids. The deduced amino acid sequence of this protease showed 62% identity to the serine protease of Creontiades dilutus, 58% to Lygus loneolaris trypsin precuror LlsgP4, 54% to Triatonatoma infestans salivary trypsin. To generate Laccotrephes japonensis serine-like protease, the DNA fragment coding for serine protease is cloning into suttle vector pBACⅠ, named pBAC1-JG and infected to Spodoptera frugiperda (sf9) insect cell. The cDNA encoding JG was expressed as a 32-kDa polypeptide in baculovirus infected insect cells and the recombinant JG showed activity in the protease enzyme assay using gelatin as a substrate.

As pulp calcification occurs at least fifty percent of total teeth, the focal calcification in pulp chamber usually appears in all age groups. However, the pulp calcification is one of the important pathologic changes affecting the pulp vitality. In order to elucidate the mechanism of pulp calcification during the retrogressive degeneration of pulp tissue we performed an immunohistochemical study for proteases (MMP-3, MMP-10, and cathepsin-G), antiproteases (TIMP-1, α1- AT) and proteins involving tissue protection (TGase-2 and HSP-70). In the normal pulp tissue MMP-3 and MMP-10 were weakly expressed, but cathepsin-G and TIMP-1 were rarely expressed. Around the calcifying tissue of MMP-3, MMP- 10, and α1-AT were predominant, but TIMP-1 and cathepsin-G were sparsely expressed. On the other hands, TGase-2 and HSP-70 were condensed in the proximal fibrous tissue. These data suggest that the pulp calcification is related to retrogressive pulp degeneration, which could be resulted in the incomplete digestion of the degenerated stromal tissue by different proteases. We presume that the aberrant protease digestion of chronic pulpal pathosis, i.e., sclerotic fibrosis, chronic pulp degeneration, etc., may enhance the dystrophic calcification in dental pulp.

V. parahaemolyticus possessed an extracellular alkaline protease activity during the stationary growth phase. Various factors such as initial pH of medium, incubation temperature and shaking rate were investigated for optimizing the production of allcaline protease from V. parahaemolyticus ATCC 17802. Maximal activity of the protease was obtained when the bacteria were grown in 2% skim milk medium in 0.1 M tris/HCl buffer (pH 7.6). Maximal activity of the protease was obtained when the bacteria were grown at initial pH of 7.6, incubation temperature of 37℃ and shaking rate of 250 rpm.

V. parahaemolyticus possessed an extracellular alkaline protease activity during the stationary growth phase. Various factors such as nitrogen sources, the concentration of NaCl and metal ions were investigated for optimizing the production of alkaline protease from V. parahaemolyticus ATCC 17802. Among the nitrogen sources tested skim milk showed the distinct increase of the activity and the activity was the highest at 2% in final concentration after 60 hours incubation. The addition of NaCl and metal ions did not increase the alkaline protease activity. CoC1₂, CuC1₂, and HgCl rather highly inhibited alkaline protease production.

당화효소와 단백질분해효소의 생산능력이 강한 Aspergillus usamii mut. shirousamii S1을 접종하여 밀가루누룩을 제조할 때에 당화효소와 단백질분해효소의 생산을 위한 배양조건을 검토하였다. 밀가루를 가열처리했을 때에는 날밀가루에 비하여 단백질분해효소와 유기산의 생산은 증가되었으나 당화효소와 호정화효소의 생산은 감소되었다. 밀가루에 염산 0.5%를 함유하는 물을 급수했을 때에는 당화효소, 호정화효소 및 단백질분해효소의 생산이 감소되었다. 당화효소 생산의 최적급수비율은 32%이고, 단백질분해효소 생산의 최적급수비율은 28%이었다. 당화효소 생산의 최적온도는 36℃이고, 단백질분해효소 생산의 최적온도는 28℃이었다. 당화효소력은 120시간 배양시에 최고치에 도달하였고, 단백질분해효소력은 96시간 배양시에 최고치에 도달하였다. 당화효소의 생산은 곰팡이 접종 후 즉시 성형하는 것보다 24시간 전배양을 한 후에 성형하는 것이 좋았고, 단백질분해효소의 생산은 성형을 하지 않는 것이 좋았다.

당화효소와 단백질분해효소의 생산능력이 높고 향도 좋은 Rhizopus japonicus T2와 밀가루로써 누룩을 제조할 때에 당화효소와 단백질분해효소의 생산을 위한 배양조건을 검토하였다. 날밀가루로 누룩을 만들었을 때에는 가열처리 한 밀가루로 만들었을 때보다 당화효소의 생산은 현저하게 높아졌으나 산성 protease의 생산은 감소하였다. 밀가루에 0.5%의 염산을 함유하는 물을 급수했을 때에는 당화효소와 중성 protease의 생산이 감소하였다 당화효소와 단백질분해효소의 생산에 가장 적합한 급수비율은 밀가루에 대하여 28%이었다. 곰팡이를 접종한 후 즉시 성형을 하는 것보다 10∼20시간 전배양을 한 후에 성형을 하는 것이 당화효소의 생산에 좋았고 단백질분해효소의 생산은 성형을 하지 않은 것이 좋았다. 당화효소 생산의 최적온도도 28℃이었고 단백질분해효소 생산의 최적온도도 28℃이었다. 28℃에서 배양할 때에 당화효소 생산을 위하 최적배양시간은 36∼72시간이었고 단백질분해효소 생산을 위 한 최적배양시간은 36시간이었다.

담배거세미나방(Spodoptera litura: SI), 누에(Bombyx mori: Bm) 및 흰불나방(Hyphantria cunea : Hc)으로부터 분리된 핵다각체병 바이러스(nuclear polyhedrosis virus : NPV)의 다각체의 단백질의 특성과 그에 대한 alkaline protease의 영향을 SDS-PAGE 및 혈정학적 방법으로 분석했다. Alkaline protease를 부활화시킨 후 다가체 단백질을 SDS-PAGE한 결과, BmNPV의 경우 30kD, SINPV와 HcNPV는 31kD의 단일 major band 및 이것들의 종합체(polymer)인 57kD와 66kD의 minor band들이 관찰되었다. Alkaline protease의 부활화를 성략한 SI NPV 다각체를 알칼리 용액으로 시간 차이를 두고 처리한 후 SDS-PAGE한 결고, 알칼리 처리시간이 경과함에 따라서 alkaline protease활성에 의해 다각체 단백질이 일정한 pattern으로 저분자화됨이 뚜렷하였다. SINPV 와 BmNPV 다각체 단백질의 항체를 제조하여 SINPV, BmNPV및 HcNPV 다각체 단백질간의 혈정학적 상동성을 이중형역광산법과 Western blot으로 비교한 결과는 3종 모두에서 공통 antigenic determinants의 존재가 인정되었으며 major 다각체 단백질의 종합체 형성과 alkaline protease에 의한 분해를 확인했다.

The spermatogenesis is the process by which spermatozoa are generated in the testes. The spermatozoa travel male reproductive tract during which they meet many substances secreted from reproductive organs. One of the substances is epididymal protease inhibitor (EPPIN) that is involved in the post-testicular maturation including capability of fertilizing the eggs. The expression of EPPIN gene was investigated in various tissues of sexually mature and regressed male Syrian hamsters by reverse transcription polymerase chain reaction (RT-PCR). The EPPIN gene was identified in the testis and epididymis of the male Syrian hamsters and compared to the genes reported previously. There was no expression of EPPIN gene in reproductively and completely regressed testes of Syrian hamster. These results suggest that the expressions of the EPPIN gene are associated with the reproductive capability in the Syrian hamsters.