• Mycobiology
• /
• v.37 no.2
• /
• pp.121-127
• /
• 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
• /
• v.19 no.5
• /
• pp.363-367
• /
• 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.

• Journal of Food Hygiene and Safety
• /
• v.12 no.4
• /
• pp.354-360
• /
• 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
• /
• v.17 no.6
• /
• pp.905-912
• /
• 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.

• The Korean Journal of Pesticide Science
• /
• v.2 no.1
• /
• pp.12-17
• /
• 1998

Salicylic acid and 3-hydroxy benzoic acid are nitrated and esterified with some alcohols. Five phosphate and six carbamate compounds of alkyl-5-nitrosalicylate and alkyl-3-hydroxy-4-nitrobenzoate are synthesized by reacting alkyl-5-nitrosalicylate and alkyl-3-hydroxy-4-nitrobenzoate with diethyl-chlorophosphate and methyl isocyanate. As the bioassay results of synthesized compounds on five insects and six pathogens, the brown planthopper (BPH, Nilaparvata lugens) mortality of O-(2-carbomethoxy-4-nitrophenyl) O,O-diethylphosphate and O-(2-nitro-5-carbo methoxyphenyl) O,O-diethylphosphate was 96%, while four compounds including O-(2-carbo-(2-chloroethoxy)-4-nitrophenyl) O,O-diethylphosphate showed more than 95% of fungicidal activity on rice blast.

• Korean Journal of Veterinary Service
• /
• v.26 no.2
• /
• pp.121-128
• /
• 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.

• Microbiology and Biotechnology Letters
• /
• v.15 no.2
• /
• pp.135-139
• /
• 1987

Aspergillus sp. MK-24 was cultured at 3$0^{\circ}C$ in the medium consisting of 2% glucose, 0.2% NaNO$_3$, 0.02% $K_2$HPO$_4$, 0.02% MgSO$_4$ 7$H_2O$, 0.02% KCl, and at initial pH of 5.0. The production of the inhibitor on venom proteinase reached to the maximum in 7 days. Sodium nitrate or potassium nitrate as a nitrogen source was favorable. The production of inhibitor was not affected by the addition of most of the inorganic salt used but depressed by lead, zinc, cobalt, mercuric or silver salts.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.5
• /
• pp.901-907
• /
• 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
• /
• v.18 no.5
• /
• pp.933-941
• /
• 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.

• Microbiology and Biotechnology Letters
• /
• v.15 no.6
• /
• pp.388-395
• /
• 1987

In order to obtain a regulatory mutant strain with high cellulase activity, a newly isolated Penicillium verrculosum, strain F-3 was used as parental strain since it was proved to be an efficient cellulase producer. A number of experiments were conducted to determine the optimum conditions to in-duce mutagenesis and isolate the desirable mutant strains. Out of several restriction compounds tested, 1.5% oxgall was found to be most effective to restrict the colony size by suppressing overgrowth. Derepression of catabolites was employed as a criterion in selecting mutant strains with high cellulase productivity. Production of cellulase by Penicillium venculosum F-3 was suppressed when cultured on the media with more than 1% of glucose or glycerol. It was found that either irradiation with UV light for 19 mins or treatment with nitrosoguanidine at 200$\mu\textrm{g}$/m1 for 60 mins, induced mutagenesis at desired level, when the survival rate of the spore was 0.2% and 48%, respectively. Three mutant strains of F-3, UV-9, UV-10, and NTG-3 that had the highest cellulase productivity were finally selected, based on filter paper degradation rate, size of clearing zone on the screening plate and cellulase activity in the medium containing cellulose powder. When the mutant strains were compared with parental strain F-3, on the KC-M-W medium containing cellulose powder, the filter paper activities of UV-9, UV-10, and NTG-3 were increased by 34%, 55%, and 41%, respectively. However, the assimilation of cellobiose octaacetate by UV-9 or NTG-3 was markedly reduced. When the mutant UV-10 was grown on cellobiose octaacetate medium (CCA-4) in shaking flasks, the cellulase activities of the mutant increased by 20 to 50% compared to the parental strain. Excreation of soluble protein from the mutant also elevated up to 30%. The mutant also constitutively produced both CMCase and $\beta$-glucosidase, though at relatively low level, in the presence of glucose or cellobiose as carbon sources.