• 미생물학회지
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• 제37권1호
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• pp.1-8
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• 2001

거미는 육식동물로 구강의 소화라는 독특한 방법을 통하여 곤충을 비롯한 작은 동물을 먹이자원으로 이용한다. 거미의 독샘에 함유되어 있는 단백질 분해 효소 환만 아니라 소화관에 존재하는 미생물도 거미의 소화에 중요한 역할을 할 것으로 추정된다. 본 연구에서는 한국산 무당거미(Nephila clavata)의 소화관내 미생물 군집의 분포와 단백질 및 지질 분해능을 확인하고, 거미의 장내 미생물을 분리 .동정하고자 하였다. 한국산 무당거미의 소화관에 존재하는 총 개체수는 거미 18개체를 통합하여 처리하였을 때와 개체별로 처리하였을 때 모두 거미 한 마리당 $10^3-10^5$CFUs 로 매우 유사하였다. 계수된 미생물 중에서 90% 이상이 단백질 또는 지질 분해능을 나타내었다. 그리고 계수된 미생물 중에서 군종별로 1균주씩 순수 분리하였고, 분리된 미생물 중 63.3%가 각각 단백질 또는 지질 분해능을 나타내었고, 이중 50%의 균주는 단백질과 지질 분해능을 동시에 함유하는 것으로 나타났다. 형태적, 생리 .생화학적 방법을 통하여 동정한 결과, 11종류의 그람음성균(Acinetobacter calcoaceticus, A. haemolyticus, Aicaligenes faecalis, Cedecea davisae, C. neteri, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas fluorescens, Serratia marcescens, Stenotraphamonas maltophilia, Suttonella indologenes)과 11종류의 그람양성균(Bacillus cereus, B. coagulans, B. pasteurii, B. thuringiensis, Cellulomonas flavigena, Corynebacterium martruchotii, Enterococcus durans, E. faecalis, Micrococcus luteus, Staphylococcus huminis, S. sciuri)으로 분류되었다.

• BMB Reports
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• 제43권6호
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• pp.427-431
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• 2010

Cyt2Aa2 is a mosquito-larvicidal protein produced as a 29 kDa crystalline protoxin from Bacillus thuringiensis subsp. darmstadiensis. To become an active toxin, proteolytic processing is required to remove amino acids from its N- and C-termini. This study aims to investigate the functional role of amino acid residues on the N-terminal ${\beta}1$ and C-terminal ${\alpha}F$ of Cyt2Aa2 protoxin. Mutant protoxins were constructed, characterized and compared to the wild type Cyt2Aa2. Protein expression data and SDS-PAGE analysis revealed that substitution at leucine-33 (L33) of ${\beta}1$ has a critical effect on dimer formation and structural stability against proteases. In addition, amino acids N230 and I233-F237 around the C-terminus ${\alpha}F$ demonstrated a crucial role in protecting the protoxin from proteolytic digestion. These results suggested that ${\beta}1$ and ${\alpha}F$ on the Nand C-terminal ends of Cyt2Aa2 protoxin play an important role in the molecular interaction and in maintaining the structural stability of the protoxin.

• 한국균학회지
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• 제14권4호
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• pp.273-280
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• 1986

Rhizorus oryzae의 생장 및 autolysis에 따른 여러가지 autolytic enzymes의 활성도 변화와 이 autolytic enzyme system을 이용한 원형질체 생성에 관하여 조사하였다. Autolytic·phase의 culture filtrate 내에 함유된 autolytic enzyme은 Rh. oryzae 균사체로부터의 원형질체 생성에 효율적인 세포벽 분해효소로 작용하였으며 Autolytic enzyme중 원형질체 생성은 proteolytic 및 chitosanase activity와 가장 긴밀하게 관련되어 있었다. 이와같은 autolytic enzyme을 이용한 원형질체 생성은 10시간 배양한 균사체에 0.5M mannitol을 사용하였을 때 최고의 수율을 보였으며 원형질 생성의 최적온도 및 pH는 각각 $25{\sim}30^{\circ}C$ 및 $6.0{\sim}6.5$로 나타났다 한편 18시간 배양된 균사체를 osmotic stabilizer와 aut-olytic enzyme을 1 : 1이 되도록 처리하고 5시간 동안 $30^{\circ}C$에서 incubation하여 얻은 원형질체를 주사전자현미경으로 조사한 결과 효소에 의하여 가수분해되는 세포벽 주변으로부터 원형질체가 형성되는 것을 확인하였다.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2001년도 Proceedings of 2001 International Symposium
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• pp.40-45
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• 2001

Raw starch-digesting amylase (BF-2A, M.W. 93, 000 Da) from Bacillus circulans F-2 was converted to two components during digestion with subtilisin. Two components were separated and designated as BF-2A' (63, 000 Da) and BF-2B (30, 000 Da), respectively. BF-2A' exhibited the same hydrolysis curve for soluble starch as the original amylase (BF-2A). Moreover, the catalytic activities of original and modified enzymes were indistinguishable in $K_{m}$, Vmax for, and in their specific activity for soluble starch hydrolysis. However, its adsorbability and digestibility on raw starch was greatly decreased. Furthermore, the enzymatic action pattern on soluble starch was greatly different from that of the BF-2A. A smaller peptide (BF-2B) showed adsorb ability onto raw starch. By these results, it is suggested that the larger peptide (BF-2A') has a region responsible for the expression of the enzyme activity to hydrolyze soluble substrate, and the smaller peptide (BF-2B) plays a role on raw starch adsorption. A similar phenomenon is observed during limited proteinase K, thermolysin, and endopeptidase Glu-C proteolysis of the enzyme. Fragments resulting from proteolysis were characterized by immunoblotting with anti-RSDA. The proteolytic patterns resulting from proteinase K and subtilisin were the same, producing 63- and 30-kDa fragments. Similar patterns were obtained with endopeptidase Glu-C or thermolysin. All proteolytic digests contained a common, major 63-kDa fragment. Inactivation of RSDA activity results from splitting off the C-terminal domain. Hence, it seems probable that the protease sensitive locus is in a hinge region susceptible to cleavage. Extracellular enzymes immunoreactive toward anti-RSDA were detected through whole bacterial cultivation. Proteins of sizes 93-, 75-, 63-, 55-, 38-, and 31-kDa were immunologically identical to RSDA. Of these, the 75-kDa and 63-kDa proteins correspond to the major products of proteolysis with Glu-C and thermolysin. These results postulated that enzyme heterogeneity of the raw starch-hydrolysis system might arise from the endogeneous proteolytic activity of the bacterium. Truncated forms of rsda, in which the gene sequence encoding the conserved domain had been deleted, directed the synthesis of a functional amylase that did not bind to raw starch. This indicates that the conserved region of RSDA constitutes a raw starch-binding domain, which is distinct from the active centre. The possible role of this substrate-binding region is discussed.d.

• Journal of Microbiology and Biotechnology
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• 제3권2호
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• pp.129-133
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• 1993

The effect of different compositions of organic acids on the flavor profile of 10% sugar solution was investigated by the response surface methodology, and the results were used to evaluate the flavor characteristics of lactic acid fermented cereal beverages. A mixture of extruded rice flour (10%) and soymilk (7.8% dry matter) was fermented with Leuconostoc mesenteroides (Sikhae). Depending on the substrate pretreatments, for example, the malt or amylase digestion and the proteolytic enzyme hydrolysis, the sugar and organic acid composition of the product varied. The organic acid composition of the fermented beverages was in the ranges of 0.44-0.55% lactic acid, 0.05-0.09% acetic acid and 0.07-0.09% citric acid, while that of commercial apple juice was 1.59% malic acid and 0.49% acetic acid. The flavor profiles of fermented beverages added with 10% sucrose were compared to those of apple juice and a model mixture containing 0.48% citric acid, 0.39% lactic acid and 0.12% acetic acid in 10% sugar solution. The QDA diagram of fermented beverages approached to that of apple juice, when the substrate was digested by amylase but not by protease.

• BMB Reports
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• 제30권2호
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• pp.125-131
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• 1997

This work reports studies of the catalytic and structural properties of pyridoxal kinase (ATP: pyridoxal 5' -phosphotransferase, EC. 2.7.1.35), Pyridoxal kinase catalyzes the phosphorylation of vitamin $B_6$ (pyridoxal, pyridoxamine, pyridoxine) using ATP-Zn as a phosphoryl donor. The enzyme purified from brain tissues is made up of two identical subunits of 40 kDa each. Native enzyme was inhibited by a substrate analogue, pyridoxal-oxime. Limited chymotrypsin digestion of pyridoxal kinase yields two fragments of 24 and 16 kDa with concomitant loss of catalytic activity. These fragments were isolated by DEAE ion exchange chromatography and used for binding studies with fluorescent ATP and pyridoxal analogues. The spectroscopic properties of both fluorescent pyridoxal analogue and Anthraniloyl ATP (Ant-ATP) bound to the 24 kDa fragment are indistinguishable from those of both pyridoxal analogue and Ant-ATP bound to the native pyridoxal kinase, respectively. The small 16 kDa fragment, generated by proteolytic cleavage of the kinase, does not bind any of the substrate analogues. Binding characteristics of Ant-ATP were extensively studied by measuring the changes in fluorescence spectra at various conditions. From the results presented herein, it is postulated that the structural domain associated with catalytic activity comprises approximately one-half of the molecular mass of pyridoxal kinase (24 kDa). whereas the remaining portion (16 kDa) of the enzyme contains a regulatory binding domain.

• Archives of Pharmacal Research
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• 제24권5호
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• pp.446-454
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• 2001

Peptide fragments, isolated from proteolytic cleavage of thyroglobulin at specific sites, were examined for the iodination of tyrosine residues. The 50 kDa polypeptide, which was prepared from digestion of bovine thyroglobulin and continuous preparative SDS-PAGE, was subjected to reduction with DTT and alkylation with iodoacetic acid to generate S-car-boxymethylated peptide derivative, which was further hydrohysed by endoproteinase-Asp-N. Peptide products were separated by RP-HPLC, and each fraction was analyzed by LC/ESI-MS and MALDI-MS analyses. Based on the specificity of endoproteinase-Asp-N andthe mass spectra data, a peptide fragment turned out to correspond to a peptide, DALCCVKCPEGSYFQ (1438-1452), characterized by the presence of a thioredoxin box (CVKC) and a tyrosine residue. In addition, another peptide fragment (1453-1465) containing a thioredoxin box (CIPC) and a tyrosine residue was also observed. However, any evidence of iodination of the tyrosine residue present in these peptides was not provided. Meanwhile, tyrosine residues in the peptides, DVEEALAGKYLAGRFA (1366-1381) and DYSGLLLAFQVFLL (1290-1303) were found to be iodinated; mono- or diiodinated tyrosine residues, characteristic of a hormogenic site, existed in both peptides. In addition, the tyrosine residue in the peptide (1218-1252), corresponding to a hormonogenic site was also iodinated. Thus, there was a sharp difference of the susceptibility to oxidative iodination between the tyrosine residue in a hormonogenic site and that in a thioredoxin region. From these results, it is suggested that polypeptide region adjacent to tyrosine residues may govern the susceptibility of tyrosine to oxidative iodination.

• Journal of Microbiology and Biotechnology
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• 제24권7호
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• pp.936-942
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• 2014

Using racemic (R,S)-2-(4-chlorophenyl)-3-methylbutyramide, an intermediate for the chiral pyrethroid insecticide Esfenvalerate, as a sole nitrogen source in a minimal medium, several strains with high enatioselectivity (${\geq}98%$) were isolated by enrichment techniques. One of the strains, LG 31-3, was identified as Burkholderia multivorans, based on physiological and morphological tests by a standardized Biolog station for carbon source utilization. A novel amidase was purified from B. mutivorans LG 31-3 and characterized. The enzyme exhibited (S)-selective amidase activity on racemic (R,S)-2-(4-chlorophenyl)-3-methylbutyramide. Addition of the racemic amide induced the production of the enantioselective amidase. The molecular mass of the amidase on SDS-PAGE analysis was shown to be 50 kDa. The purified amidase was subjected to proteolytic digestion with a modified trypsin. The N-terminal and internal amino acid sequences of the purified amidase showed a high sequence homology with those deduced from a gene named YP_366732.1 encoding indole acetimide hydrolase from Burkholderia sp. 383.

• 한국축산식품학회지
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• 제44권4호
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• pp.951-965
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• 2024

Lactiplantibacillus plantarum is a valuable potential probiotic species with various proven health-beneficial effects. L. plantarum LM1001 strain was selected among ten strains of L. plantarum based on proteolytic activity on whey proteins. L. plantarum LM1001 produced higher concentrations of total free amino acids and branched-chain amino acids (Ile, Leu, and Val) than other L. plantarum strains. Treatment of C2C12 myotubes with whey protein culture supernatant (1%, 2% and 3%, v/v) using L. plantarum LM1001 significantly increased the expression of myogenic regulatory factors, such as Myf-5, MyoD, and myogenin, reflecting the promotion of myotubes formation (p<0.05). L. plantarum LM1001 displayed β-galactosidase activity but did not produce harmful β-glucuronidase. Thus, the intake of whey protein together with L. plantarum LM1001 has the potential to aid protein digestion and utilization.

• 한국수산과학회지
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• 제17권2호
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• pp.117-124
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• 1984

수산부산물을 효율적으로 이용하기 위한 방안의 하나로 정어리를 가공할 때 생기는 잔사를 원료로 하여금 자체에 존재하는 자가소화효소나 단백질분해효소로써 정어리간장제조를 시도하였다. 마쇄한 정어리잔사에 동량의 물을 첨가하여 가수분해시킬 때의 최적가수분해조건을 검토하고 아울러 제품의 정미성분을 분석하였다. 자가소화에 의한 경우와 bromelain이나 complex enzyme첨가구 모두 $55^{\circ}C$에서 최대활성을 나타내었고, 분해시간은 4시간이 가장 적합하였으며, 효소농도는 bromelaine의 경우 $0.4\%$, complex enzyme은 $6.0\%$가 가장 좋았다. 정어리잔사로써 만든 어간장의 단백질가수분해율 및 아미노질소는 각각 자가소화법인 경우 $82.5\%,\;5.2\%$, bromelain 첨가구는 $84.3\%,\;5.8\%$, complex enzyme 첨가구는 $92.5\%,\;5.9\%$이었다. 자가소화나 bromelain에 의해 제조된 정어리잔사로써 만든 정어리간장의 핵산관련물질은 hypoxanthine이 건물양 기준으로 각각 $17.4 {\mu}mole/g,\;16.0{\mu}mole/g$로서 가장 많았고, 유리아미노산 중 함량이 많은 것은 leucine, glutamic acid, lysine, valine 및 alanine으로서 전 유리아미노산에 대해 각각 51.3, $48.3\%$를 차지하였다. 그리고 5'-IMP와 TMAO는 엑스분질소에 대해 건물양 기준으로 각각 $0.2\%,\;0.4\%$로서 비교적 소량이었으나 총 creatinine은 엑스분질소에 대해 $9.2{\sim}10.0\%$로써 다소 높은 함량이었다. 관능검사결과 자가소화시킨 정어리잔사로써 만든 정어리간장은 효소첨가한 것이나 재래식 간장에 비해서 품질에 손색이 없었다. 정어리잔사는 단백질분해효소를 첨가하지 않은 자가소화만으로도 재래식 간장에 비해 손색없는 어간장을 제조할수 있다는 결론을 얻었다.