• Mycobiology
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• 제37권2호
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• pp.121-127
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• 2009

Purification and characterization of intracellular cellulase produced by A. oryzae ITCC-4857.01 are reported. The enzyme was purified by ion-exchange chromatography using DEAE-cellulose followed by Gel filtration. The purification achieved was 41 fold from the crude extract with yield of 27%. The purified enzyme showed single band on poly acrylamide gel. The molecular weight as determined by SDS-PAGE and gel filtration was 38 KDa and 38.6 KDa respectively and contained only one subunit. The enzyme is glycoprotien as nature and contained 0.67% neutral sugar. The apparent Km value of the enzyme against cellulose was 0.83%. The enzyme showed the highest relative ativities on CMC followed by avicel, salicin and filter paper. The optimum pH of activity was 5.5 and very slight activity was observed at or above pH 7.5 as well as bellow pH 3.5. The optimum tempreture of the activity was $45^{\circ}C$ and the highest activity was exhibited in 35 to $45^{\circ}C$. The enzyme lost their activities almost completely (95${\sim}$100%) at $80^{\circ}C$ or above and as well as bellow $25^{\circ}C$.

• KSBB Journal
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• 제19권5호
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• pp.363-367
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• 2004

Leuconostoc mesenteroides NRRL B-1149 produces various glucoseyltransferases for the synthesis of dextran, levan and glucose-1-phosphate using sucrose as a substrate. A sucrose phosphorylase (1149SPase) was purified from L. mesenteroides NRRL B-1149 culture by using hollow fiber filtration (30 kDa cut off), Toyopearl DEAE 650 M column chromatography and following two times of DEAE-Sepharose column chromatographies. The specific activity of the purified 1149SPase was 25.7 (U/mg) with $16\%$ yield. The 1149SPase showed a molecular size of 56 kDa on denatured $10\%$ SDS-PAGE. The N-terminal amino acid sequence of the enzyme was MEIQNKAM. The optimum pH and temperature of this enzyme were 6.2~6.5 and 37^{circ}C, respectively. It had an apparent K_{m} of 6.0 mM and K_{cat} of 1.62/s for sucrose. 1149SPase crystal was formed by hanging drop diffusion technique using 20 mM calcium chloride dihydrate, 100 mM sodium acetate trihydrate pH 4.6 and $30\%$ 2-methyl-2,4-pentanediol as vaporizing and reservation solution. The 1149SPase catalyzes transferring of glucose from isomaltose or sucrose to salicin and salicyl alcohol by disproportionation reaction or acceptor reaction and synthesized two acceptor products, respectively.

• 한국식품위생안전성학회지
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• 제12권4호
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• pp.354-360
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• 1997

This study was intended that the biochemical patterns, bioserological characteristics, resistance of antibiotics, and transferable resistance patterns of 35 Dscherichia coli strains from 79 fish and shellfish samples in marine markets from August to October, 1995. The Standard plate count, coliforms and fecal coliforms were also counted in the 79 cases and analysed the correlationship each other. Geometric means of Standard plate count in seawater fish, shellfish, mollusca and crustacean were 1.4$\times$105 CFU/g, 4.0$\times$105 CFU/g, 2.4$\times$105 CFU/g, 4.7$\times$105 CFU/g, and those of coliforms were 1.3$\times$103 CFU/g, 4.8$\times$103 CFU/g, 8.9$\times$102 CFU/g, 5.8$\times$103 CFU/g. There were no fecal coliforms in the fish and mollusc. However, the geometric means of coliforms in the shellfish and crustacean (1.1$\times$101 CFU/100g, 10 CFU/100 g) were less than those of fish and mollusca. The important biochemical characteristics of E. coli distinguished from the shellfish and crustacean were motility, ornithine decarboxylase, mucate, esculin. The fermentative properties of E. coli were also sucrose, salicin, sorbitol, and raffinose. Of 35 isolates of E. coli, 13 strains (37.1%) showed the pathogenic O antisera, which were O:27 3 strains (23.1%), ):159 2 strains (15.4%) and ):148, O:119, O:142, O:158, O:136, O:18, O:128, and O:168 1 strain (7.7%),respectively.

• Journal of Microbiology and Biotechnology
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• 제17권6호
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• pp.905-912
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• 2007

A novel ${\beta}-glucosidase$ gene, bglA, was isolated from uncultured soil bacteria and characterized. Using genomic libraries constructed from soil DNA, a gene encoding a protein that hydrolyzes a fluorogenic analog of cellulose, 4-methylumbelliferyl ${\beta}-D-cellobioside$ (MUC), was isolated using a microtiter plate assay. The gene, bglA, was sequenced using a shotgun approach, and expressed in E. coli. The deduced 55-kDa amino acid sequence for bglA showed a 56% identity with the family 1 glycosyl hydrolase Chloroflexus aurantiacus. BglA included two conserved family 1 glycosyl hydrolase regions. When using $p-nitrophenyl-{\beta}-D-glucoside$ (pNPG) as the substrate, the maximum activity of the purified ${\beta}-glucosidase$ exhibited at pH 6.5 and $55^{\circ}C$, and was enhanced in the presence of $Mn^{2+}$. The $K_m\;and\;V_{max}$ values for the purified enzyme with pNPG were 0.16 mM and $19.10{\mu}mol/min$, respectively. The purified BglA enzyme hydrolyzed both pNPG and $p-nitrophenyl-{\beta}-D-fucoside$. The enzyme also exhibited substantial glycosyl hydrolase activities with natural glycosyl substrates, such as sophorose, cellobiose, cellotriose, cellotetraose, and cellopentaose, yet low hydrolytic activities with gentiobiose, salicin, and arbutin. Moreover, BglA was able to convert the major ginsenoside $Rb_1$ into the pharmaceutically active minor ginsenoside Rd within 24 h.

• 농약과학회지
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• 제2권1호
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• pp.12-17
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• 1998

버드나무 껍질중의 배당체인 salicin으로부터 유래된 salicylic acid와 그 유사화합물인 3-hydroxy benzoic acid의 질산화된 중간체를 출발물질로 하여 몇몇의 알콜과 에스테르화반응을 거쳐 유기인계 및 카바메이트계 화합물의 전구체를 합성하였다. 합성된 전구체의 수산기에 diethylchlorophosphate와 methyl isocyanate를 반응시켜 유기인계 및 카바메이트계 화합물 11종을 합성하였다. 합성된 화합물은 살충, 살균활성실험을 실시하였다. 유기인계 화합물의 벼멸구에 대한 활성은 500 ppm의 농도에서 O-(2-carbomethoxy-4-nitro phenyl) O,O-diethylphosphate 화합물이 96%의 살충력을 보였다. 반면 carbamate 화합물의 경우는 500ppm농도에서 살충력이 전혀 나타나지 않았다. 살균효과는 유기인계 화합물인 경우 도열병에 대해서 ester에 관계없이 250 ppm농도에서 95% 이상의 방제가를 나타냈으며, 그 이외의 병원균에서는 낮은 방제가를 보였다.

• 한국동물위생학회지
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• 제26권2호
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• pp.121-128
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• 2003

This study was carried out to identify and investigate antimicrobial susceptibility for Mannheimuia haemolytical which is responsible for shipping fever. Samples were collected from nasal and lung of 100 adult healthy cattle which are slaughtered in Samsung meat corporation located in Incheon metropolitan city. lung lesion index have been investigated within 0-5 range according to Shewen and Willkie(Can J Vet Res 52:30-36, 1988). Eighty-seven of 100 cattle were under normal condition with 0-1 ranges. A total of 129 strains were collected from blood and tryptic soy agar. Among these strains, 100 strains were identified with Staphylococcus, Streptococcus and enterobacteria containing E coli. Biochemical and fermentation assay of arabinose, trehalose, xylose, mannose, mannitol, lactose and salicin were tested to identify with Mannheimia sp. for 7 strains shown haemolytic activity on blood agar. Five strains were identified with Mannheimia haemolytica and 2 strains were untyped. In seasonal survey, Mannheimia sp recovered from fall to winter(5 of 7) have been highly isolated rather than those from spring to summer(2 of 7). Mannheimiz haemolytica were susceptible to antibacterials tested in this study but more resistant to oxytetracycline and streptomycin.

• 한국미생물·생명공학회지
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• 제15권2호
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• pp.135-139
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• 1987

Aspergillus 속 균주 MK-24로부터 venom proteinase inhibitor의 생산조건을 검토한 결과 질소원으로서는 유기능질소가 균생육에 있어서는 매우 좋았으나 물질생성에 있어서는 무기능질소보다 못하였다. 무기질소원으로서는 sodium nitrate가 가장 좋은 효과를 보였다. 단소원으로서는 glucose가 대부분 당류와 비슷한 결과를 나타내었고 특히 arabitol, xylitol, salicin 등은 본 균주가 단소원으로 사용치 못하는 것으로 추정되었으며, vitamin 류는 물질생성에 무관하였으며 금속염류로서는 효과적인 것은 없으나, Ag$^+$, Co$^{++}$, $Zn^{++}$ 등은 억제하였다. 배양온도와 pH는 각각 3$0^{\circ}C$와 pH 5가 균의 생육과 저해물질생성에 제일 적당하였고, 단소원과 질소원으로서 glucose 2%, NaNo$_3$ 0.3%을 가한 액체배지에서 7일간 정치배양 하였을 때 저해물질생육이 가장 좋았다.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.901-907
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• 2008

Thermotoga neapolitana $\beta$-glucosidase (BglA) was subjected to site-directed mutagenesis in an effort to increase its ability to synthesize arbutin derivatives by transglycosylation. The transglycosylation reaction of the wild-type enzyme displays major ${\beta}(1,6)$ and minor ${\beta}(1,3)$ or ${\beta}(1,4)$ regioselectivity. The three mutants, N291T, F412S, and N291T/F412S, increased the ratio of transglycosylation/hydrolysis compared with the wild-type enzyme when pNPG and arbutin were used as a substrate and an acceptor, respectively. N291T and N219T/F412S had transglycosylation/hydrolysis ratios about 3- and 8-fold higher, respectively, than that of the wild-type enzyme. This is due to the decreased hydrolytic activity of the mutant rather than increased transglycosylation activity. Interestingly, N291T showed altered regioselectivity, as well as increased transglycosylation products. TLC analysis of the transglycosylation products indicated that N291T retained its ${\beta}(1,3)$ regioselectivity, but lost its ${\beta}(1,4)$ and ${\beta}(1,6)$ regioselectivity. The altered regioselectivity of N291T using two other acceptors, esculin and salicin, was also confirmed by TLC. The major transglycosylation products of the wild type and N291T mutant were clearly different. This result suggests that Asn-291 is highly involved in the catalytic mechanism by controlling the transglycosylation reaction.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.933-941
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• 2008

An extracellular $\beta$-glucosidase produced by Monascus purpureus NRRL1992 in submerged cultivation was purified by acetone precipitation, gel filtration, and hydrophobic interaction chromatography, resulting in a purification factor of 92-fold. A $2^2$ central-composite design (CCD) was performed to find the best temperature and pH conditions for enzyme activity. Maximum activity was observed in a wide range of temperature and pH values, with optimal conditions set at $50^{\circ}C$ and pH 5.5. The $\beta$-glucosidase showed moderate thermostability, was inhibited by $HgCl_2$, $K_2Cr_O_4$, and $K_2Cr_2O_7$, whereas other reagents including $\beta$-mercaptoethanol, SDS, and EDTA showed no effect. Activity was slightly stimulated by low concentrations of ethanol and methanol. Hydrolysis of p-nitrophenyl-$\beta$-D-glucopyranoside (pNPG), cellobiose, salicin, n-octyl-$\beta$-D-glucopyranoside, and maltose indicates that the $\beta$-glucosidase has broad substrate specificity. Apparently, glucosyl residues were removed from the nonreducing end of p-nitrophenyl-$\beta$-D-cellobiose. $\beta$-Glucosidase affinity and hydrolytic efficiency were higher for pNPG, followed by maltose and cellobiose. Glucose and cellobiose competitively inhibited pNPG hydrolysis.

• 한국미생물·생명공학회지
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• 제15권6호
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• pp.388-395
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• 1987

천연섬유소 분해활성이 우수하고 그 효소 유도기구도 Trichoderma reesei와는 다른 Penicillium verruculosum F-3을 모균주로 사용하여 돌연변이 처리에 의한 유전적 개량을 시도함으로써 cellulase 생산성이 증진된 조절변이주를 얻고자 변이주 유도조건 변이주에 의한 cellulase 생성조건을 검토하였다. 한천평판상에서의 변이주의 선택분리 효율을 향상시키기 위하여 각종 colony 소형화제의 영향을 검토한 바 Oxgall을 배지에 1.5% 첨가하였을 때가 가장 좋았다. Cellulase 고생산성 변이주 선정의 한 지표로서 대사산물 억제의 해제를 선택했다. P. verruculosum F-3은 glucose 또는 glycerol 농도 1% 이상에서 본 효소생성이 억제되었다. 변이주 유도조건으로서 UV조사의 경우는 19분 처리로 약 0.2%, NTG 처리의 경우는 200$\mu\textrm{g}$/$m\ell$ 농도로 1시간처리로 48%의 생존율을 나타냈다. 변이처리 한 균주를 여지붕괴도, cellulose agar plate에서의 clear zone의 크기, cellulose powder 배지로 배양한 조효소액의 여지분해활 성을 조사하여 우수균주로서 UV-9, UV-10 및 NTG-3을 최종 선발했다. 각종 탄소원을 함유하는 배지에서의 cellulase 생산성을 조사한 바 KC-M-W 배지로 배양한 UV-9, UV-10 및 NTG-3의 여지분해 활성은 친주보다 각각 34%, 55%, 41% 증가되었으나, UV-9 및 NTG-3은 COA 자화능이 현저히 저하되었다. 변이주 UV-10은 COA-4 배지로 배양했을 때 친주에 비해 단백질량 30%, Avicel 분해활성 30%, 여지분해활성 20%, salicin 분해활성 50% 증가가 인정되었고, 비록 역가는 낮았지만 glucose 및 cellobiose를 함유하는 배지에서 CMC 및 salicin 분해활성을 구성적으로 생산하였다.