• Korean Journal of Microbiology
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• v.36 no.1
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• pp.14-19
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• 2000

Algal lytic enzyme, an extracellular enzyme, was purified from the culture filtrate of Penicillium oxalicum(HCLF-34) by ultrafiltration, gel filtration chromatography, and anion exchange chromatography. The enzyme has a molecular mass of approximately 22 kDa, an it is a monomer by renaturation SDS-PAGE. The amino acid sequences of the enzyme was revealed to be NH2-Glu-Ser-Tyr-Ser-Ser-Asn-Ala-Ala-Gly-Ala-Val-Leu-Ile---, had about 84% identity with the mature light chain of aspergillopepsin II precursor and 81% identity with the mature protein of the acid proteinase EapC precursor.

• Korean Journal of Microbiology
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• v.35 no.3
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• pp.231-236
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• 1999

The fuugus(Penicil1ium oralicum; HCLF-34) secreted the cyanobacteria lytic enzyme which had a molecular weight of about 22 kDa, a optimum temperature of $20^{\circ}C$, a optimum pH of 3.5, and a temperature-stable up to $50^{\circ}C$. The chemical ions such as sodium, potassium, barium, magnesium. and mangan ions appeared positive activity. but calcium, iron, copper ions, EDTA, and PMSF displayed negative activity: this results were the same as the characterilics of other cell wall lytic enzymes. This extracellular enzyme showed lytic aclivily against SDS-insoluble peptidoglycan of Anabaenrr cylinrlrica. The cell wall lylic enzyme of Penicilliurn oxalicum(HCLF-34) seemed to be glycosidase-like enzyme in the fact that ihe concentration of rcducing sugar was increased when the peptidoglycan of Anabaena qlinrlricn md Micrococcus luteus reacted with this enzyme

• Journal of Life Science
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• v.12 no.4
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• pp.483-489
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• 2002

We investigated the biochemical properties of insect defensin expressed and secreted from Saccharomyces corevisiae. The defensin showed extremely high resistance to boiling for up to 30 min and to pH values tested from 2.0 to 12.0. The treatment of defensin with various proteases abolished antibacterial activity. However, amylases, cellulase, lipase and catalase had no effect on the activity. The defensin was purified to homogeneity through ammonium sulfate concentration of culture supernatant, SP-Sepharose column chromatography and RP-HPLC. Tricin-SDS-PAGE analysis revealed that the molecular weight of the defensin was about 4.0 kDa. The antibacterial activity of the purified defensin was verified by renaturation of stained gel and gel pouring assay using Micrococcus luteus as a test organism.

• Korean Journal Plant Pathology
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• v.12 no.1
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• pp.1-10
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• 1996

Xanthomonas campestris pv. vesicatoria의 감염으로 토마토 잎조직에 $\beta$-1, 3-Glucanases와 chitinases가 합성, 축적되었다. 그러나 접종되지 않은 건전한 잎에서는 위의 두 가지 가수분해 효소는 매우 낮은 수준으로 유지되었고, 이 두 가지 효소는 친화적 상호작용에서보다는 불친화적 상호작용에서 더욱 높은 수준으로 존재하였다. 이것은 $\beta$-1, 3-glucanases와 chitinases가 X. c. pv. vesicatoria의 생육에 대한 방어기작으로서 중요한 역할을 한다는 것을 시사해 주고 있다. Native PAGE 젤 상에서 $\beta$-1, 3-glucanases를 분리한 결과, 병징 발현이나 저항성 발현에 중요한 역할을 하는 것으로 생각되는 산성 isoform Ga 1과 염기성 isoform Gb 1의 isoform bands만 확인되었다. Isoelectric focusing을 이용하였을 때, 적어도 pI 6.4와 pI 8.6을 지닌 두 개의 $\beta$-1, 3-glucanases의 isoform을 확인할 수 있었고, 특히 불친화적 상호작용에서 더욱 뚜렷하게 유도되었다. 이것은 병 진전과정에서 X. c. pv. vesicatoria에 대해 저항성 발현에 관여한다는 것을 나타내고 있다. 산성 chitinase isoform인 Ca 1의 활성은 병원균의 감염이 진전되는 동안 감소하였다. 또한 다섯 개의 염기성 chitinase isoform이 감염된 토마토 잎 조직에서 발견되었는데, 특히 토마토의 방어기작에 관여하여 병원화적 균주 Bv5-4a에 감염된 잎에서만 유도, 축적되었다. Isoelectric focusing(IEF)을 이용한 후 적어도 2개의 산성과 4개의 염기성 chitinase isoform이 감염된 토마토 잎 추출액에서 확인되었다. Native PAGE 젤에서 isoform Cb 1에 해당되는 pI 9.5를 지닌 chitinase isoform은 오직 불친화적 상호작용에서만 확인되었다. 이온이 제거된 Triton X-100을 처리하여 renaturation 시킨 후에 SDS-PAGE 젤 상태에서 23 kDa과 26 kDa을 지닌 2개의 chitinase isoform을 확인하였다.

• The Korean Journal of Pharmacology
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• v.32 no.3
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• pp.433-444
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• 1996

We describe a novel approach to evaluate quantitatively the amounts of denatured proteins in cells upon heat exposure. A thiol compound, diamide [azodicarboxylic acid bis (dimethylamide)] causes protein cross-linking with exposed sulfyhydryl residues of denatured proteins. Since denatured proteins expose normally well-hidden sulfhydryl groups, these will be preferentially cross-linked by diamide. Thus diamide acts to 'trap' denatured proteins. We observed that protein aggregates (high molecular weight protein aggregates, HMA) appeared on SDS-polyacrylamide gels run under non-reducing conditions and that the amount of HMA can be quantified by scanning the gels using a gas flow counter. Heating cells followed by a fixed dose of diamide exposure resulted in HMA increases in a heat-dose dependent manner, demonstrating that the quantitation of HMA could serve as a measure of heat-denatured proteins. We compared thermotolerant and nontolerant cells and found decreased HMA in tolerant cells upon heat treatment. As an attempt to examine the kinetics of protein renaturation (or 'repair'), we measured the amounts of aggregates formed by the addition of diamide at various times after heat shock. Such experiments demonstrate an equally rapid disappearance of HMA in previously unheated and in thermotolerant cells. Levels of HMA in tolerant cells increased significantly after electroporation of HSP70 specific mAbs, suggesting an involvement of HSP70 in reducing HMA levels in thermotolerant cells upon heat exposure. Immunoprecipitation studies using anti-HSP70 antibody indicated an association of HSP70 with heat-denatured proteins. Our results suggest that heat induces protein denaturation, and that elevated level of HSP70 present in thermotolerant cells protects them by reducing the level of protein denaturation rather than by facilitating the 'repair' (or degradation) process.

• Journal of Microbiology and Biotechnology
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• v.30 no.11
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• pp.1720-1728
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• 2020

We have previously characterized AprESJ4, the major fibrinolytic enzyme from Bacillus subtilis SJ4 (Yao et al., 2019). During that study, we observed a 68 kDa protein with fibrinolytic activity. In this study, we cloned the gene (vprSJ4) encoding the 68 kDa protein, a mature Vpr and minor protease secreted by Bacillus species. vprSJ4 encodes a preproenzyme consisting of 810 amino acids (aa) including signal sequence (28 aa) and prosequence (132 aa). The mature enzyme (650 aa) has a predicted molecular weight of 68,467.35. Unlike Vprs from other B. subtilis strains, VprSJ4 has 4 additional amino acids (DEFA) at the C-terminus. vprSJ4 was overexpressed in Escherichia coli. PreproVprSJ4 was localized in inclusion bodies, and subjected to in vitro renaturation and purification by an affinity column. SDS-PAGE and western blot showed that autoprocessing of preproVprSJ4 occurred and 68 kDa and smaller proteins were produced. The optimum pH and temperature of the recombinant VprSJ4 were pH 7.0 and 40℃, respectively. Kinetic parameters of recombinant VprSJ4 were measured by using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. Coexpression of vprSJ4 and aprESJ4 using pHY300PLK increased the fibrinolytic activity a further 117% when compared with aprESJ4 single expression using the same vector in B. subtilis WB600.