• Animal Bioscience
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• v.36 no.7
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• pp.1034-1043
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• 2023

Objective: Two serine protease inhibitors, peptidase inhibitor 3 (PI3) and secretory leukocyte protease inhibitor (SLPI), play important roles in protease inhibition and antimicrobial activity, but their expression, regulation, and function at the maternal-fetal interface in pigs are not fully understood. Therefore, we determined the expression and regulation of PI3 and SLPI in the endometrium throughout the estrous cycle and at the maternal-fetal interface in pigs. Methods: Endometrial tissues during the estrous cycle and pregnancy, conceptus tissues during early pregnancy, and chorioallantoic tissues during mid to late pregnancy were obtained, and the expression of PI3 and SLPI was analyzed. The effects of the steroid hormones estradiol-17β (E2) and progesterone (P4) on the expression of PI3 and SLPI were determined in endometrial explant cultures. Results: PI3 and SLPI were expressed in the endometrium during the estrous cycle and pregnancy, with higher levels during mid to late pregnancy than during the estrous cycle and early pregnancy. Early-stage conceptuses and chorioallantoic tissues during mid to late pregnancy also expressed PI3 and SLPI. PI3 protein and SLPI mRNA were primarily localized to endometrial epithelia. In endometrial explant cultures, the expression of PI3 was induced by increasing doses of P4, and the expression of SLPI was induced by increasing doses of E2 and P4. Conclusion: These results suggest that the PI3 and SLPI expressed in the endometrium and conceptus tissues play an important role in antimicrobial activity for fetal protection against potential pathogens and in blocking protease actions to allow epitheliochorial placenta formation.

• BMB Reports
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• v.40 no.4
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• pp.604-609
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• 2007

An Asp/His catalytic site of 10-formyltetrahydrofolate dehydrogenase (FDH) was suggested to have a similar catalytic topology with the Asp/His catalytic site of serine proteases. Many studies supported the hypothesis that serine protease inhibitors can bind and modulate the activity of serine proteases by binding to the catalytic site of serine proteases. To explore the possibility that soybean trypsin inhibitor (SBTI) can recognize catalytic sites of FDH and can make a stable complex, we carried out an SBTI-affinity column by using rat liver homogenate. Surprisingly, the Rat FDH molecule with two typical liver proteins, carbamoyl-phosphate synthetase 1 (CPS1) and betaine homocysteine S-methyltransferase (BHMT) were co-purified to homogeneity on SBTI-coupled Sepharose and Sephacryl S-200 followed by Superdex 200 FPLC columns. These three liver-specific proteins make a protein complex with 300 kDa molecular mass on the gel-filtration column chromatography in vitro. Immuno-precipitation experiments by using anti-FDH and anti-SBTI antibodies also supported the fact that FDH binds to SBTI in vitro and in vivo. These results demonstrate that the catalytic site of rat FDH has a similar structure with those of serine proteases. Also, the SBTI-affinity column will be useful for the purification of rat liver proteins such as FDH, CPS1 and BHMT.

• Mycobiology
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• v.31 no.4
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• pp.209-213
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• 2003

Aspergillus flavus K-03 isolated from poultry forming soil in Korea was studied for its ability to produce extracellular proteases on basal medium containing 2%(w/v) chicken feathers. The fungus was observed to be a potent producer of such enzymes. Keratinolytic enzyme secretion was the best at 15 days of incubation period at pH 9 and temperature $40^{\circ}C$. No relationship existed between the enzyme yield and increase of biomass. Enzyme production was suppressed by exogenous sugars in descending order arabinose>maltose>mannose>fructose. But glucose did not influence the enzyme activity. The keratinolytic enzyme released by the fungus demonstrated the ability to decompose keratin substrates as chicken feather when exogenous glucose was present. The keratinolytic activity was inhibited by $HgCl_2$ and serine-protease inhibitors such as phenymethylsulfonyl fluoride(100%), chymostain(88%), crystalline soybean trypsin inhibtor(80%), antipain(45%) and aprotinin(40%), and was not by cystein-protease and aspartyl-protease inhibitors. The enzyme activity is only partially inhibited by metallo-protease inhibitor. Thus, the enzyme secreted by A. flavus K-03 belongs to the alkaline serine-type protease.

• Microbiology and Biotechnology Letters
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• v.49 no.1
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• pp.65-74
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• 2021

Serine proteases are the most versatile proteolytic enzymes with tremendous applications in various industrial processes. This study was designed to investigate the biochemical properties, critical residues, and the catalytic potential of alkaline serine protease using in-silico approaches. The primary sequence was analyzed using ProtParam, SignalP, and Phyre2 tools to investigate biochemical properties, signal peptide, and secondary structure, respectively. The three-dimensional structure of the enzyme was modeled using the MODELLER program present in Discovery Studio followed by Molecular Dynamics simulation using GROMACS 5.0.7 package with CHARMM36m force field. The proteolytic potential was measured by performing docking with casein- and keratin-enriched residues, while the effect of the inhibitor was studied using phenylmethylsulfonyl fluoride, (PMSF) applying GOLDv5.2.2. Molecular weight, instability index, aliphatic index, and isoelectric point for serine protease were 39.53 kDa, 27.79, 82.20 and 8.91, respectively. The best model was selected based on the lowest MOLPDF score (1382.82) and DOPE score (-29984.07). The analysis using ProSA-web revealed a Z-score of -9.7, whereas 88.86% of the residues occupied the most favored region in the Ramachandran plot. Ser327, Asp138, Asn261, and Thr326 were found as critical residues involved in ligand binding and execution of biocatalysis. Our findings suggest that bioengineering of these critical residues may enhance the catalytic potential of this enzyme.

• The Korean Journal of Mycology
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• v.36 no.2
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• pp.178-182
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• 2008

Although the number of patients with Acanthamoeba keratitis has increased dramatically since the widespread use of contact lens, it is still very hard to cure the disease. The proteases from the Acanthamoeba were reported to play important role in the pathogenesis of keratitis. In this study, the inhibitors for extracellular serine proteases of A. castellanii were screened from the extracts of 230 mushroom samples collected from various regions of Korea. The mushrooms were extracted with methanol and water ($65^{\circ}C$). Filtered and concentrated extracts (0.3 mg/ml) were preincubated with proteases before addition of peptide substrate N-succinyl-ala-ala-pro-phe p-anilide. The selected extracts showing strong inhibitory effects were characterized. Although inhibition with single extract was not so high enough, the complete inhibition was achieved with combination of two extracts. The selected extract showed little effect on other serine proteases such as thrombin (human and bovine) and on general protease such as protease K.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.852-858
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• 2008

An extracellular protease (Mc1) was isolated from the nematode-trapping fungus Monacrosporium cystosporium by gel filtration, anion-exchange, and hydrophobic interaction chromatographies. This protease had a molecular mass of approximately 38 kDa and displayed an optimal activity at pH 7-9 and $56^{\circ}C$ (over 30 min). Its proteolytic activity was highly sensitive to the serine protease inhibitor PMSF (phenylmethylsulfonylfluoride, 0.1 mM), indicating that it belonged to the serine-type peptidase group. The Michaelis constant ($K_m$) and $V_max$ for substrate N-Suc-Ala-Ala-Pro-Phe-pNA were $1.67{\times}10^{-4}\;M$ and 0.6071 $OD_{410}$ per 30 s, respectively. This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. Moreover, the enzyme could immobilize the free-living nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, suggesting that it might playa role in infection against nematodes. The encoding gene of Mc1 was composed of one intron and two exons, coding for a polypeptide of 405 amino acid residues. The deduced amino acid sequence of Mcl showed 61.4-91.9% identity to serine proteases from other nematode-trapping fungi. Our results identified that Mcl possessed biochemical properties including optimal reaction condition and substrate preference that are different from previously identified serine proteases.

• Journal of Microbiology
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• v.41 no.1
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• pp.58-62
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• 2003

An extracellular pretense of Bacillus amyloliquefaciens S94 was purified to apparent homogeneity. The enzyme activity was strongly inhibited by general inhibitor for serine protease, PMSF, suggesting that the enzyme is a serine pretense. The purified enzyme activity was inhibited by leucine peptidase inhibitor, bestatin, suggesting that the enzyme is a leucine endopeptidase. The maximum proteolytic activity against different protein substrates occurred at pH 10, 45$^{\circ}C$ (protein substrate) and pH 8, 45$^{\circ}C$ (synthetic substrate). The purified enzyme was specific in that it readily hydrolyBed substrates with Leu or Lys residues at P$_1$ site. The pretense had characteristics of a cold-adapted protein, which was more active for the hydrolysis of synthetic substrate in the range of 15$^{\circ}C$ to 45$^{\circ}C$, specially at low temperature.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.165-165
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• 1996

균의 감염에 의해 유도되는 패혈성 shock는 균이 분비하는 elastase가 관여하며, 외부에서 serine pretense inhibitor의 biopolymer의 투여로 균에 의해 유도된 패혈성 shock를 억제시킬 수 있다고 보고되고 있다. 이에 본 연구진은 패혈성 치료제의 개발의 목적으로, 국내에서 민간 약으로 많이 이용되고 있는 백편두로부터 새로운 elastase inhibitor를 분리, 정제하여 부분 아미노산 서열 및 특성을 조사하였기에 발표하고자 한다. 백편두 추출액을 여러 차례에 걸쳐 column chromatography을 수행하면서 얻어지는 각 fraction에 대하여 elastase MCA-기질 및 trypsin MCA-기질을 이용하여 활성 측정 후 elastase 기질 및 trypsin 기질에 대하여 활성을 억제하는 fraction들을 모아 $C_{18}$ open column chromatography 및 $C_{18}$ HPLC 과정을 수행하여 2종류의 trypsin 활성 억제 물질과 1종류의 elastase inhibitor를 분리, 정제하여 각각을 Ti1, Ti2 및 Ti3로 명명하였다. 전기영 동 상에서 단일 hand를 확인한 후 각각의 inhibitor들의 부분 아미노산 서열을 결정하였다.

• Journal of agriculture & life science
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• v.45 no.6
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• pp.201-212
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• 2011

The alkaline protease gene was cloned from a halo-tolerant alkalophilic Bacillus clausii I-52 isolated from the heavily polluted tidal mud flat of West Sea in Inchon Korea, which produced a strong extracellular alkaline protease (BCAP). Based on the full genome sequence of Bacillus subtilis, PCR primers were designed to allow for the amplification and cloning of the intact pro-BCAP gene including promoter region. The full-length gene consists of 1,143 bp and encodes 381 amino acids, which includes 29 residues of a putative signal peptide and an additional 77-amino-acid propeptide at its N-terminus. The mature BCAP deduced from the nucleotide sequence consists of 275 amino acids with a N-terminal amino acid of Ala, and a relative molecular weight and pI value was 27698.7 Da and 6.3, respectively. The amino acid sequence shares the highest similarity (99%) to the nattokinase precursor from B. subtilis and subtilisin E precursor from B. subtilis BSn5. The substrate specificity indicated that the recombinant BCAP could hydrolyze efficiently the synthetic substrate, N-Suc-Ala-Ala-Pro-Phe-pNA,and did not hydrolyze the substrates with basic amino acids at the P1 site. The recombinant BCAP was strongly inhibited by typical serine protease inhibitor, PMSF, indicating that BCAP is a member of the serine proteases.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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• pp.181-186
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• 2000

MAPI (microbial alkaline protease inhibitor) was isolated from cultrue broth of Streptomyces chromofuscus SMF28. The Ki values of MAPI for the representative serine proteases such as chymotrypsin and proteinase K were 0.28 and $0.63{\;}\mu\textrm{M}$, respectively, and for the cysteine proteases cathepsin B and papain were 0.66 and $0.28{\;}\mu\textrm{M}$, respectively. These data indicate that MAPI is not a potent selective inhibitor of serine or cysteine proteases. Progress curves for the inhibition of three proteases by MAPI exhibithe characteristic patterns; MAPI exhibited slow-binding inhibition of cathepsin B. It was rapidly associated with chymotrypsin before the addition of substrate and then reactivation of MAPI-inhibited enzyme was investigated in the presence of substrate. On the other hand, MAPI-proteinase K interaction was typical for those classical inhibitors. When MAPI was incubated with trypsin, there was an extensive reduction in the ingibitory activities of MAPI corresponding to 66.5% inactivation of MAPI, indicating that trypsin-like protease may play a role in the decrease of the inhibitory activity during cultivation.