• Journal of Microbiology and Biotechnology
• /
• 제31권8호
• /
• pp.1163-1174
• /
• 2021

Casein-derived antioxidant peptides by using microbial proteases have gained increasing attention. Combination of two microbial proteases, Protin SD-NY10 and Protease A "Amano" 2SD, was employed to hydrolyze casein to obtain potential antioxidant peptides that were identified by LC-MS/MS, chemically synthesized and characterized in a oxidatively damaged HepG2 cell model. Four peptides, YQLD, FSDIPNPIGSEN, FSDIPNPIGSE, YFYP were found to possess high 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging ability. Evaluation with HepG2 cells showed that the 4 peptides at low concentrations (< 1.0 mg/ml) protected the cells against oxidative damage. The 4 peptides exhibited different levels of antioxidant activity by stimulating mRNA and protein expression of the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), as well as nuclear factor erythroid-2-related factor 2 (Nrf2), but decreasing the mRNA expression of Kelch-like ECH-associated protein 1 (Keap1). Furthermore, these peptides decreased production of reactive oxygen species (ROS) and malondialdehyde (MDA), but increased glutathione (GSH) production in HepG2 cells. Therefore, the 4 casein-derived peptides obtained by using microbial proteases exhibited different antioxidant activity by activating the Keap1-Nrf2 signaling pathway, and they could serve as potential antioxidant agents in functional foods or pharmaceutic preparation.

• 한국미생물학회:학술대회논문집
• /
• 한국미생물학회 2005년도 International Meeting of the Microbiological Society of Korea
• /
• pp.226.3-226
• /
• 2005

• 생명과학회지
• /
• 제2권1호
• /
• pp.20-25
• /
• 1992

곤충을 연구재료로 하여 생리활성을 가지는 펩타이드 및 단백질에 관한 연구는 최근에 상당히 활발히 진행되고 있는 실정이다. 그들을 그 생리활성별로 크게 분류하면 다음과 같이 3종류를 들 수 있다. (1) antibacterial and antifungal proteins. (2)neuropeptides. (3)proteases.이 외에도 곤충으로부터 성장호르몬, lectin, pheromone 및 변태에 관계되는 단백질을 분리 정제하여 그들의 생화학적 특성에 관하여 보고하고 있으나 본 총설에서는 위의 세 가지에 대해서 다루었다.

• Bulletin of the Korean Chemical Society
• /
• 제25권11호
• /
• pp.1703-1706
• /
• 2004

• 대한수의학회지
• /
• 제37권2호
• /
• pp.359-365
• /
• 1997

Staphylococcus hyicus subsp. hyicus strains isolated from pigs, chickens and cattle were examined for the production of staphylokinase after inhibition of staphylococcal proteases by two procedures with EDTA(disodium). In one, EDTA was added to the bovine fibrin-dog plasminogen agar medium in concentration of 0.07% and paper strips soaked in 2mg/ml soy bean trypsin inhibitor were then applied on the agar plates. In the other, paper strips soaked in 5% EDTA solution were applied on the bovine fibrin-dog plasminogen agar plates and the strains to be tested were then streaked at right angles with the strip. By these procedures, staphylokinase activity was detected in 8(88.9%) of 9 strains from diseased pigs and in 57(80.3%) of 71 strains from skin of healthy pigs, but not in any strains from skin of healthy chickens and milk samples of mastitic cattle. Additionally kinase activity in 9 Staphylococcus species and subspecies isolated from bovine intramammary infections was also tested by these procedures. Staphylokinase activity was detected in 74.2% of Staph aureus strains and in 25% of Staph xylosus strains.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2001년도 추계학술발표대회
• /
• pp.89-92
• /
• 2001

Aspergillus niger has been used as a host system to express many heterologous proteins. It has various advantages over other expression systems in that it is a small eukaryotic GRAS (Generally Recognized aS Safe) organism with a capacity of secreting large amount of foreign proteins. However, it has been known that the presence of an abundant protease is a limiting factor to express a heterologous protein. The proteases deficient mutants of A. niger were obtained using UV -mutagenesis. A total of 1 ${\times}$ $10^5$ spores were irradiated with 10-20% survival dose of UV, 600J/M2 at 280nm, and the resulting spores were screened on the casein -gelatin plates. Ten putative protease deficient mutants were further analyzed on the starch plates to differentiate the pro from the secretory mutant. An endogenous extracellular enzyme, glucose oxidase, was also examined to confirm that the mutant phenotype was due to the proteases deficiency rather than the mutation in the secretory pathway. The reduced proteolytic activity was measured using SDS-fibrin zymography gel, casein degradation assay, and bio-activity of a supplemented hGM -CSF (human Granulocyte-Macrophage Colony Stimulating Factor). Comparing with the wild type strain, less than 30 % of proteolytic activity was observed in the culture filtrate of the protease deficient mutant (pro -20) without any notable changes in cell growth and secretion.

• BMB Reports
• /
• 제44권10호
• /
• pp.665-668
• /
• 2011

In order to clarify the impact of Ca-binding sites (Ca1 and 2) on the conformational stability of neutral proteases (NPs), we have analyzed the thermal, pH and organic solvent stability of a NP variant, V189P/A195E/G203D/A268E (Q-mutant), from Salinovibrio proteolyticus. This mutant has shown to bind calcium more tightly than the wild-type (WT) at Ca1 and to possess Ca2. Q-mutant was resisted against autolysis, thermoinactivation and pH denaturation in a Ca-dependent manner and exhibited better activity in organic solvents compared to the WT enzyme. These results imply that Ca1 and Ca2 are important for the conformational stability of NPs.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제13권1호
• /
• pp.37-43
• /
• 2006

Enterococcus faecalis was isolated from the body fluid of dead Galleria mellonella larvae. Upon injection of E. faecalis into the hemocoel of G. mellonella, the bacteria destroyed parts of humoral defense systems in the hemolymph. In a test for the proteolytic activity of E. faecalis CS, it was confirmed that the enzyme degraded three well-known a-helical antimicrobial peptides, cecropin A, melittin and halocidin, and abolished their activities. We also determined putative cleavage sites on the primary sequences of three peptides through purification and mass analysis of peptide fragments digested by E. faecalis CS. Furthermore it was found that apolipophorin-III, recently known as a critical recognition protein for invading microbes in the hemolymph of G. mellonella, was also degraded by E. faecalis CS. Taken together, the present work shows that the protease in secretions from E. faecalis destroyed two critical humoral immune factors in the hemolymph of G. mellonella larvae. In addition, this paper demonstrates that the relationship between the host insect and the pathogenic bacteria might provide a valuable model system to study the enterococcal virulence mechanism, which may be relevant to mammalian pathogenesis.

• Journal of Plant Biotechnology
• /
• 제37권3호
• /
• pp.327-336
• /
• 2010

Cysteine proteases have been known as a critical factor in plant defense mechanisms in pineapple, papaya, or wild fig. Papain or ficin is one kind of cysteine proteases that shows toxic effects to herbivorous insects and pathogenic bacteria. However, resistance to bacterial soft rot of plants genetically engineered with cysteine protease has been little examined thus far. We cloned a cysteine protease cDNA from Ananas comosus and introduced the gene into Chinese cabbage (Brassica rapa) under the control of the cauliflower mosaic virus 35S promoter. The transgene was stably integrated and actively transcribed in transgenic plants. In comparisons with wild-type plants, the $T_2$ and $T_3$ transgenic plants exhibited a significant increase in endo-protease activity in leaves and enhanced resistance to bacterial soft rot. A cDNA microarray analysis revealed that several genes were more abundantly transcribed in the transgenic than in the wild type. These genes encode a glyoxal oxidase, PR-1 protein, PDF1, protein kinase, LTP protein, UBA protein and protease inhibitor. These results suggest an important role for cysteine protease as a signaling regulator in biotic stress signaling pathways, leading to the build-up of defense mechanism to pathogenic bacteria in plants.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제25권2호
• /
• pp.187-193
• /
• 2012

Serine proteases are major insect enzymes involved in the digestion of dietary proteins and in the process of blood meal digestion. In this study, cDNA was constructed using the whole body of Laccotrephes japonensis. The flanking sequences of the 5- and 3- end of this gene were characterized by RACE-PCR. Sequence analysis showed that this gene contained a 963-bp ORF encoding 320 amino acids. The deduced amino acid sequence showed 62% identity with the Creontiades dilutus serine protease, 58% with the Lygus lineolaris trypsin precursor, and 54% with the Triatoma infestans salivary trypsin. To assess the expression of the L. japonensis serine protease (JGsp), the JGsp gene was cloned into a baculovirus transfer vector, pBac-1, and expressed in Sf9 cells (Spodoptera frugiperda). SDS-PAGE and western blot analysis have shown that the JGsp recombinant protein was a monomer with a molecular weight of about 32 kDa. Recombinant JGsp has shown activity in the protease enzyme assay using gelatin as a substrate.