• Mycobiology
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• 제30권3호
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• pp.170-174
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• 2002

A total of twenty-nine species and one species variety belonging to 12 genera was isolated from 30 samples of molasses on 1% glucose(10 genera, 22 species and 1 variety) and 50% sucrose(7, 21 and 1) Czapek's agar at $25^{\circ}C$ media. Aspergillus, Mucor, Mycosphaerella and Penicillium were the most common genera on the two types of media. From the above genera, the most prevalent species were: Aspergillus flavus, A. fumigatus, A. niger, Mycosphaerella tassiana, Penicillium chrysogenum, P. oxalicum and P. purpurogenum. Also, some species were only isolated on 50% sucrose such as Eurotium amstelodami, E. chevalieri, E. repens, Humicola fuscoatra, Penicillium aurantiogriseum and P. puberulum. About 65 fungal isolates isolated from 50% sucrose agar were tested for their ability to produce invertase enzyme in liquid medium and 93.8% of the isolates could produce this enzyme. From the positive isolates, 32 showed high invertase activity, 21 had moderate activity and the remaining 8 isolates were of weak activity. Sixty isolates of Aspergillus, Emericella, Eurotium, Mycosphaerella and Penicillium from the preceding study were screened for the presence of their respective mycotoxins. Larva of brine shrimp(Artema sauna L.) were used for toxicity test of the fungal crude extracts. Three isolates out of 60 tested were toxic. Using thin-layer chromatographic technique, 5 different known mycotoxin were detected aflatoxins : B1, B2, G1, G2 and citrinin.

• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.286-290
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• 2006

A genomic library of Streptococcus vestibularis ATCC49124 was constructed in an E. coli plasmid vector, and the urease-positive transformants harboring the urease gene cluster were isolated on Christensen-urea agar plates. The minimal DNA region required for urease activity was located in a 5.6 kb DNA fragment, and a DNA sequence analysis revealed the presence of a partial ureI gene and seven complete open reading frames, corresponding to ureA, B, C, E, F, G, and D, respectively. The nucleotide sequence over the entire ure gene cluster and 3'-end flanking region of S. vestibularis was up to 95% identical to that of S. salivarius, another closely related oral bacterium, and S. thermophilus, isolated from dairy products. The predicted amino acid sequences for the structural peptides were 98-100% identical to the corresponding peptides in S. salivarius and S. thermophilus, respectively, whereas those for the accessory proteins were 96-100% identical. The recombinant E. coli strain containing the S. vestibularis ure gene cluster expressed a high level of the functional urease holoenzyme when grown in a medium supplemented with 1 mM nickel chloride. The enzyme was purified over 49-fold by using DEAE-Sepharose FF, Superdex HR 200, and Mono-Q HR 5/5 column chromatography. The specific activity of the purified enzyme was 2,019 U/mg, and the Michaelis constant ($K_{m}$) of the enzyme was estimated to be 1.4 mM urea. A Superose 6HR gel filtration chromatography study demonstrated that the native molecular weight was about 196 kDa.

• Food Science and Biotechnology
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• 제18권2호
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• pp.494-499
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• 2009

To improve phenylalanine ammonia lyase (E.C.4.3.1.5-PAL) activity in recombinant Escherichia coli, Some approaches for improving phenylalanine ammonia lyase (PAL) activity in recombinant E. coli were developed following preliminary studies by means of response surface method. The results shown that permeabilization with combination of Triton X-100, cetyl trimethyl ammonium bromide (CTAB), and acetone enriched cellular recombinant PAL activity significantly, which improved over 10-fold as compared with the control (untreat cell), as high as 181.37 U/g. The optimum values for the tested variables were Triton X-100 0.108 g/L, CTAB 0.15 g/L, and acetone 45.2%(v/v). Furthermore, a second-order model equation was suggested and then validated experimentally. It was indicated that addition of surfactants and organic solvents made the cells more permeable and therefore allowed easier access of the substrate to the enzyme and excretion of the product, which increased the rate of transport of L-phenylalanine and trans-cinnamic acids. These improved methods of PAL activity enrichment could serve as a rich enzyme source, especially in the biosynthesis of L-phenylalanine.

• 생명과학회지
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• 제8권5호
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• pp.486-491
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• 1998

mouse의 임파구로부터 두 종류의 ADP-ribosyltransferase (Yac-1과 Yac-2)가 클로닝되어 특성을 규명한 바 있다. Yac-2는 ADP-ribosyltransferase 활성 뿐 아니라 높은 NAD glycohydrolase 활성도 가지고 있다. Yac-2는 두 보존된 glutamic acids 사이인 221번 위치에 arginine를 소유하고 있다. 두 효소 활성에 대한 Arg-221의 중요성을 조사하기 위해 Arg-221이 Glutamic acid (R221E)와 Alanine (R221A)으로 돌연변이 되었다. 돌연변이체인 R221E와 R221A는 두 효소에 대해 야생형과 유사한 활성을 나타내었으며 이러한 결과는 Yac-2의 Arg-221이 ADP-ribosyltransferase와 NAD glycohydrolase의 활성에 필수적인 역할을 하지 않음을 시사해준다.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2000년도 Proceedings of 2000 KSAM International Symposium and Spring Meeting
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• pp.68-75
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• 2000

D-Tagatose is a potential bulking agent in food as a non-calorific sweetener. To produce D-tagatose from cheaper resources, plasmids harboring the L-arabinose isomerase gene (araA) from Escherichia coli was constructed because L-arabinose isomerase was previously suggested as an enzyme that mediates the bioconversion of galactose to tagatose as well as that of arabinose to ribulose. In the cultures of recombinant E.coli with pTC101, which harboring araA of E.coli, tagatose was produced from galactose in 9.9 % yield. The enzyme extract of E.coli containing pTC101 also converted galactose into tagatose in 96.4 % yield. For the economic production of D-tagatose, an L-arabinose isomerase of E.coli was immobilized using covalent binding on agarose. While the free L-arabinose isomerase produced tagatose with the rate of 0.48 mg/U$.$day, the immobilized one stably converted galactose into average 7.5 g/l$.$day of tagatose during 7 days with higher productivity of 0.87 mg/U$.$day. In the scaled up immobilized enzyme system, 99.9 g/l of tagatose was produced from galactose with 20 % equilibrium in 48 hrs. The process was stably repeated additional 2 times with tagatose production of 104.1 and 103.5 g/l.

• 대한수의학회지
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• 제35권4호
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• pp.699-712
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• 1995

The present study, to evaluate the effect of vitamin E on the oxidative stress in STZ-treated rat and BB rat, was investigated the biochemical enzyme activity in the serum, and malondialdehyde and carbonyl group in the RBC membrane, liver and microsomal fraction after vitamin E and/ or insulin treatment. Results obtained through the experiments were summarized as follows; 1. Effect of vitamin E and/or insulin treatment in STZ-treated rat 1) Lipid peroxidation level in RBC membrane, liver and microsomal fraction was significantly decreased in vi. tamin E and/or insulin treatment group, and especially more significantly decreased in vitamin E with insulin treated group. 2) Protein oxidation level in RBC membrane, liver and microsomal fraction was significantly decreased in vitamin E and/or insulin treatment group. And it was especially more significantly decreased in RBC membrane and liver of vitamin E with insulin treated group. 3) In the enzyme activity in the serum, the activity of AST and ALT was not altered in all experimental group. The increased ALP activity in STZ-treated group was significantly decreased in insulin treated group and vitamin E with insulin treated group. 4) Decreased level of albumin and creatinine after STZ treatment was significantly increased in vitamin E and/or insulin treated group. 5) Level of glucose, cholesterol and triacylglycerol in serum: Glucose level was not significantly different in vitamin E treated group compared to STZ control group. But it was significantly different in the insulin treated group and vitamin E with insulin treated group compared to STZ control group. The cholesterol content in the serum was significantly increased in STZ control group compared to normal control group. And except low dose vitamin E treatment group, it was significantly decreased in vitamin E and/or insulin treated group compared to STZ control group. The triacylglycerol content in the serum was significantly decreased in STZ control group and increased in high dose vitamin E treated group and vitamin E with insulin treated group. But it was not significantly different in low dose vitamin E treated group and insulin treated group compared to STZ control group. 2. Effect of vitamin E and/or insulin treatment in BB rat 1) Lipid peroxidation level in liver was decreased by vitamin E with insulin treatment compared to insulin treatment. But it was not different in microsomal fractions. 2) Protein oxidation level in liver and microsomal fraction was decreased by vitamin E with insulin treatment compared to insulin treatment only in microsomal fractions. These results suggest that the combination treatment of vitamin E and insulin could prevent the oxidative change of lipid and protein of the RBC membrane, liver and microsomal fraction in STZ-treated rats and BB rats.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.628-631
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• 2003

The recombinant alanine dehydrogenase (ADH) from E. coli containing Thermus caldophilus ADH was purified to homogeneity from a cell-free extract. The enzyme was purified 38-fold with a yield of 68% from the starting cell-free extract. The purified enzyme gave a single band in polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 45 kDa. The pH optimum was 8.0 for reductive amination of pyruvate and 12.0 for oxidative deamination of L-alanine. The enzyme was stable up to $70^{\circ}C$. The activity of the enzyme was inhibited by 1 mM $Zn^{2+}$, 20% hexane, and 20% $CHCl_3$. However, 10 mM $Mg^{2+}$ and 40% propanol had no effect on the enzyme activity. The Michaelis constants ($K_m$) for the substrates were $50\;\mu\textrm{M}$ for NADH, 0.2 mM for pyruvate, 39.4 mM for $NH_4+$, 2.6 mM for L-alanine, and 1.8 mM for $NAD^+$.

• 한국미생물·생명공학회지
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• 제11권1호
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• pp.15-21
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• 1983

전보에서 분리 동정한 Y-33 균주의 $\beta$-galactosise 효소생산조건을 검토하고 효소를 정제하여 정제효소의 성질을 조사한 결과는 다음과 같다. 1. 효소생산을 위한 최적초발 pH는 7.0이었고 최적온도는 $65^{\circ}C$이었다. 2. 효소는 lactose와 galactose에 의하여 유도되어졌으며 세포내효소이었다. 3. 조효소액을 1차 DEAE-cellulose, 2차 DEAE-cellulose column chromatography 및 Sephadex G-150로 gel filtration하여 정제도가 28.3배, 수율이 15.2%의 정제효소를 얻었다. 4. 정제효소는 polyacrylamide gel 전기 영동에 의하여 순도가 검정되었다. 5. 정제효소의 유당가수분해를 위한 최적작용 온도는 $65^{\circ}C$. pH는 6.5이었다.

• 한국미생물·생명공학회지
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• 제37권2호
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• pp.118-124
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• 2009

에스터라제 EM2L8 유전자를 E. coli 균에서 발현하고 에스터라제 활성을 분석한 결과, $40-45^{\circ}C$에서 최적의 효소활성을 보였다. $15^{\circ}C$에서 최대활성의 45% 활성을 보였고 $15-45^{\circ}C$ 사이의 활성화에너지는 4.9 kcal/mol로 계산됨으로써 전형적인 저온 적응효소인 것으로 밝혀졌다. 또한, $4^{\circ}C$에서 장기보관해도 효소활성이 전혀 줄어들지 않음을 통해서 저온에서 안정한 효소임을 알게 되었다. 반응액에 에탄올, 메탄올, 아세톤을 15% 농도까지 첨가해도 효소활성이 줄어들지 않았으며 DMSO의 경우, 40% 농도까지 첨가해도 효소활성이 유지되는 것으로 나타났다. 이 효소 40 U을 Tris-HCl 용액(1.2 mL, pH 9.0)에 넣고 $30^{\circ}C$에서 (R,S)-ECHB(0.5%, 38 mM)의 분해반응을 수행한 결과, 기질이 가수분해되어 CHBacid가 생성되며 기질의 분해속도는 $6.8\;{\mu}mole/h$로 계산되었다. (R)-ECHB 보다 (S)-ECHB 기질을 빠르게 분해하였으며 전환수율이 80%일 때, e.e.s 값이 40%로 측정되었다. 반응액에 DMSO를 10% (v/v) 농도로 각각 첨가한 결과, 기질의 분해 속도는 $10.4\;{\mu}mole/h$로 증가되었다. 하지만 DMSO의 유무와 상관없이 전환수율에 따른 e.e.s 값은 유사하게 나타났다. 결론적으로 이 효소는 저온과 각종 유기용매 하에서도 높은 안정성과 활성을 갖고 있기 때문에 각종 의약품의 유기합성공정에서 효소촉매로 활용될 수 있을 것으로 기대된다.

• Journal of Animal Science and Technology
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• 제60권10호
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• pp.24.1-24.8
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• 2018

Background: Following the ban on the importation of import-dependent fed ingredients in most developing countries, the need to look inward for local content is now compelling. Thus, leaf meals that have phytogenic additive potentials are envisaged will be a viable feed ingredient in rabbit diets. Methods: The effect of dietary inclusion of gliricidia leaf meal (GLM) with or without multi-enzyme (E) supplementation in rabbits was investigated using ninety-six 35-day old rabbits of crossbreed (Newzealand and Chinchilla). One basal diet that met the requirements of growing rabbit was formulated (Diet 1). Thereafter, another two diets were formulated to contain 15% GLM and 15% GLM plus multi-enzyme at 1 g/kg and designated as diets 2 and 3 respectively. The rabbits were randomly distributed into the 3 diets (32 rabbits/treatment; 4 rabbits/replicate) and fed their respective experimental diets for 8 weeks. Results: The body weight and daily weight gain of the rabbits fed on GLM free diet and those on GLM-based diets (diets 1 and 2) were similar at finishing period of 63-91 day but have lower (P < 0.01) values than those rabbits fed GLM + E based diet (diet 3) at finishing period (63-91 days) and whole fattening period (35-91 days). The apparent dry matter and crude protein digestibility of rabbits fed control diet and those fed 15% GLM based diet were lower (P < 0.05) than those fed 15% GLM + E-based diet. Triglycerides concentration of rabbits fed 15% GLM-based diet without enzyme addition were lower (P < 0.05) than those observed for rabbits on the rest test diets. Cholesterol and Low-Density Lipoprotein levels of rabbits fed 15% GLM and 15% GLM + E-based diets were lower (P < 0.05) than those fed the GLM free diet. The superoxide dismutase and glutathione peroxidase of rabbits fed the GLM free diet (diet 1) were significantly (P < 0.05) lower than those fed the 15%GLM and 15% GLM + E-based diets. Conclusion: Dietary inclusion of GLM at 15% of the diet did not have a negative effect on the rabbits postweaning period (35-63 days) but will require multi-enzyme supplementation to enhance growth indices at finishing period (63-91 day) without precipitating negative effect on the rabbits' health status.