• 한국환경보건학회지
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• 제19권3호
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• pp.58-63
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• 1993

In order to elucidate the alteration of drug-metabolizing enzymes and mechanism in the animal with hepatic injury and ketosis, the regulation of P450IIE was studied in the rats with heaptic injury caused by CCl$_4$ and with ketosis caused by streptozotocin and high-fat diet. P450IIE expression in liver was examined by the combination of enzyme activities, Western immunoblot, and mRNA Northern blot analyses using specific polyclonal antibody and cDNA probe for P450IIE. Enzyme activity and amounts of immunoreactive P450IIE were rapidly decreased in a time-dependent manner after a single dose of CCl$_4$ . However, the decreases in P450IIE enzyme activity and immunoreactive protein by CCl$_4$ were not accompanied by a decline in its mRNA level. The data thus suggested a post-translational reduction of P450IIE by CCl$_4$. The enzyme activities (aniline hydroxylase) in hepatic microsomes were elevated about 2-3-fold by streptozotocin and feeding with a high fat diet. This increases in enzyme activities were also accompanied by 3-fold increases in immunoreactive P450IIE protein and its mRNA. Our data thus indicated that P450IIE induction during the ketosis appears to be due to pretranslational activation.

• 한국식품과학회지
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• 제29권1호
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• pp.145-149
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• 1997

쌀의 저장시 지방질의 가수분해나 산화에 의해 off flavor를 생성하여 쌀의 품질을 저하시키는데 있어 촉매작용을 하는 lipoxygenase 활성을 비색법으로 측정하였다. 기질농도와 효소반응속도와의 상호관계를 검토하고, 최적 pH를 조사하였으며, 도정도에 따른 현미 각 부분의 효소활성을 알아보았다. 0.2 mM의 linoleic acid/phosphate buffer (pH 7.0)와 반응시켰을 때 반응시간에 따른 효소활성은 동진이 3시간, 금오가 4시간, 간척이 5시간까지 효소활성이 증가하였다. 기질의 농도와 효소액과의 반응속도 관계를 Lineweaver-Burk plotting으로 해석한 결과 $V_{max}$는 동진, 금오 및 간척벼는 각각 57.89, 19.85, 31.38 unit/mg protein이었고, $K_m$은 동진, 금오 및 간척벼 각각 0.054, 0.045, 0.035 mM이었다. 현미의 lipoxygenase의 활성은 동진벼의 경우 pH 7.6, 간척벼는 pH 7.0, 금오벼는 pH 7.0에서 최적의 활성을 나타내었다. 도정도에 따른 현미 각 부분의 효소활성 및 비활성은 동진, 금오 및 간척벼 모두 획분 II에서 가장 높은 활성을 보였으며 획분 IV에서 가장 낮은 값의 활성을 보였다. 세 품종의 현미에 microwave로 0, 30, 60, 90초간 가열처리하였을 때 효소활성 및 효소의 비활성은 대조구에 비해 90초간 가열처리했을 때 효소활성의 경우 동진, 금오 및 간척벼는 27.4%, 52.2%, 17.1%로 감소하였으며, 효소의 비활성도 42.6%, 76.7%, 23.4% 정도로 감소하였다.

• 한국미생물·생명공학회지
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• 제19권1호
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• pp.57-63
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• 1991

토양으로부터 분리한 알칼리 내성 및 liner alkylbenzene sulfonate 내성인 lipase 생산균주를 동정하여 Pseudomonas sp. J-19로 명명하였다. 호알칼리성 lipase는 ammonium sulfate 침전, DEAE-Sephadex와 Sephadex G-100 column chromatography로 정제하였고, 정제효소의 비활성도는 35 unit/mg protein, 수율은 17이었다. 정제효소는 polyacrylamide disc gel 전기영동에서 단일 band를 나타내었고, Sephadex G-100 gel filtration과 SDS-polyacrylamide gel 전기영동에 의하여 추정된 분자량은 36,000이었다. 정제효소의 최적 pH는 10.0 최적온도는 30'C이었다. 정제 효소의 활성은 0.1 linear alkylbenzene sulfonate 첨가에 의하여 2배 증가되었고, 0.05 Tide에 의하여 2.5배 증가되었다. 정제효소는 p8.0-10.0, 4'C이하에서 안정하였다.

• BMB Reports
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• 제29권6호
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• pp.493-499
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• 1996

The biochemical properties of purified D-hydruxyisovalerate dehydrogenase from Fusarium sambucinum was elucidated. D-Hydroxyisovalerate dehydrogenase produced solely D-hydroxyisovalerate from 2-ketoisovalerate. The isoelectric point of the purified enzyme was 7.0. The enzyme was highly specific with 2-ketoisovalerate ($K_{m}=0.188$ mM, $V_{max}=8.814$ mmol/min mg) and 2-keto-3-methyl-n-valerate ($K_{m}=0.4$ mM, $V_{max}=1.851$ mmol/min mg) for the reductive reaction. This was also seen by comparing D-hydroxyisovalerate ($K_{m}=1.667$ mM, $V_{max}=0.407$ mmol/min mg) and D-hydroxy-3-methyl-n-valerate ($K_{m}=6.7$ mM, $V_{max}=0.648$ mmol/min mg) for the oxidative reaction. Thiol blocking reagents, such as iodoacetamide, N-ethylmaleimide and p-chloromecuribenzoate inhibited about 80% of enzyme activity at 0.02 mM, 50 mM and 50 mM, respectively. The enzyme activity was also inhibited by the addition of 0.1 mM of various metal ions, such as $Fe^{2+}$ (67%), $Cu^{2+}$ (88%), $Zn^{2+}$ t (76%) and $Mg^{2+}$ (9%). The enzyme was stable over three months in 50 mM potassium phosphate buffer (pH 5~7) at $-80^{\circ}C$. However the purified enzyme lost 30% of its activity in the same buffer after 24 h at $4^{\circ}C$. The studies about thermal inactivation of D-hydroxyisovalerate dehydrogenase exhibit 209.2 kJ/M of activation enthalpy and 0.35 kJ/mol K of activation entropy.

• 한국미생물·생명공학회지
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• 제4권4호
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• pp.131-137
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• 1976

산업상 이용에 적합한 분리 선정된 Naringinase 생산 균주인 Asrpergillus niger S-1은 동시에 Hesperidinase를 강력히 생산함이 확인되었으며 이 균주가 생산하는 Hesperidinase의 효소학적 성질을 요약하면 다음과 같다. 1. PH 4.0에서 30분간 반응 시켰을때 최적 온도는 6$0^{\circ}C$ 이었으며 최적 작용 pH는 5.0이었으나 pH 3.0에서는 70% 활성을 보였다. 2. pH 4.0에서 30분간 열처리 하였을 경우 6$0^{\circ}C$로 열처리한 결과 90%, 7$0^{\circ}C$일 경우 70%, 8$0^{\circ}C$일 경우 65%의 활성을 가지는 비교적 내열성 효소이었다. 그리고 pH에 대한 안정성은 4$^{\circ}C$에서 24시간 보존한 결과 pH 5.0일 경우 가장 안정 하였다. 3. $Mg^{＋＋}$ 이온은 1$\times$$10^{-2}$M 이하부터는 활성화하였으나 Cu$^{＋＋}$ Mn$^{＋＋}$ Pb$^{＋＋}$ Mo$^{＋＋}$ Ag$^{＋＋}$ Hg$^{＋＋}$등은 저go 하였다. 4. 효소를 Aceton 처리할 경우 60% Aceton 처리에서 배양 추출물의 경우 보다 11배 전제되었으며 35%가 회수 되었으며 40%의 Aceton 처리일 경우 10배 정제되었으며 5.6%가 회수 되었다. 5. 배양 추출 효소를 유안으로 염석할 경우 0.4.~0.6 분획 침전 시킬 경우 48배 정제되었으며 처음 효소의 13%가 회수되었고 0.6~0.8포화 분획에서도 19%가 회수되었고 25배 정제되었다. 6. Hesperidinase 생성은 약간량의 밀감 과피 추출물의 첨가로 현저히 증가 하였다.

• 한국축산식품학회지
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• 제29권2호
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• pp.157-163
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• 2009

This study was conducted for the isolation, purification, and characterization of a protease from Korean pear, to see its proteolytic activity on chicken actomyosin and to find the optimum pH and temperature of activity on chicken actomyosin. The protease was isolated from crude extract of Korean pear by ammonium sulfate precipitation. Further purification was done by DEAE-Sepharose ion-exchange chromatography, Mono-Q and Mini-Q column chromatography. The purified enzyme gave a single protein band on SDS polyacrylamide gel electrophoresis and the molecular weight was found to be 38 kDa. The specific activity of purified enzyme was 34,907 unit/mg with 25 fold purification and the yield was 2%. The purified enzyme incubated with chicken actomyosin showed high activity. The optimum pH and temperature for enzyme activity on chicken actomyosin were 6.5 and $70^{\circ}C$, respectively. A protease was purified from Korean pear for the first time and characterized. It was found to be promising for meat tenderization.

• 한국미생물·생명공학회지
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• 제28권5호
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• pp.251-257
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• 2000

Serratia marcescens purine nucleoside phosphorylase (PNP) was purfied to homogeneity by streptomycin sulfate treatment, Sephacry HR S-200 gel filtration chromatography and AMP-agarose affinity chromatography. The specific activity of the enzyme was increased 49-fold during purification with an overall yield of 7.0%. The molecular weight was 168kD as estimated by Sephadex G-150 gel filtration chromatography. The S. marcescens enzyme was composed of six identical subunits with subunit molecular weight of 28kD, as estimated by SDS-PAGE. The Km values of S. marcescens enzyme for inosine and deoxyinsoine were 0.38 and 1.20 mM, respectively. The ph optimum was near 8.0, and the enzyme was relatively heat-stable protein. The enzyme was inactivated com-pletely by 0.5 mM of $Cu^{ 2+}$.

• 한국미생물·생명공학회지
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• 제16권6호
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• pp.438-442
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• 1988

The enzyme produced by Rhizopus japonicus was able to convert selectively ginsenoside-Rb$_1$which is the most abundant ginseng saponin, into ginsenoside-Rd which was known to be superior to ginsenoside-Rb$_1$pharmaceutically. The convertible enzyme was purified homogeneous from wheat bran culture of Rhizopus japonicus by ammonium sulfate fractionation and column chromatography of TEAE-cellulose, DEAE-Sephadex A-50, Sephadex G-150, Sepharose 2B. Specific activity of the purified enzyme was increased to a bent 96 folds and yield was appeared to be 11% of culture extract. Evidence for homogenity was obtained from polyacrylamide and SDS-polyacrylamide gel electrophoresis. Molecular weight of the enzyme was estimated about 88, 000 daltons by Sephadex G-l50 gel filtration and SDS-polyacrylamide gel electrophoresis, and it did not consist of any subunit.

• Archives of Pharmacal Research
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• 제20권2호
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• pp.115-121
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• 1997

Ile-269 in horse liver alcohol dehydrogenase isoenzyme S(HLADH-S) was mutated to serine by phosphorothioate-based site-directed mutagenesis in order to study the role of the residue in coenzyme binding. The specific activity of the mutant(1269S) enzyme to ethanol was increased 49-fold. All turnover numbers of 1269S enzyme toward 9 primary alcohols were increased. The mutant enzyme showed 3.6, 4.6, 11.6-fold higher catalytic efficiency for $5{\beta}$-androstane-3, 17-dione, $5{\beta}$-cholanic acid-3-one and retinal than wild-type, respectively. The reaction mechanism of 1269S enzyme was ordered bi bi as wild-type's. These results indicate that the hydrophobic interaction of Ile-269 residue with coenzyme plays an important role in dissociation of coenzyme from enzyme-coenzyme complex, which has been known as the rate limiting step of ADH reaction.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1995년도 한국생물과학협회 학술발표대회
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• pp.82-82
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• 1995

Regulation of enzyme synthesis by transcriptional and translational control systems provides rather stable adaptation to change of amino acid level in the growth medium, while manipulation of enzyme activity through endproduct feedback inhibition represents rather short-term and reversible ways of adjusting metabolic fluctuation of amino acid level. Various control mechanisms interplay to regulate genes encoding enzymes for amino acid biosynthesis in the yeast, Sacchromyces cerevisiae. When amino acids are in short supply, genes under a cross-pathway regulatory mechanism Or general amino acid control (general control) increase their action, in which Gcn4p is the major positive regulator of gene expression. When cells are cultured in minimal medium, basal level expression is also regulated by supplementary control elements, where inorganic phosphate level is additionally involved. Most of amino acid biosynthetic genes are also regulated by the level of endproduct of the pathway. This pathway-specific regulatory mechanism is called specific amino acid control (specific controD, under which gene expression is reduced when endproduct is present in the medium. Derepression of a gene through general control can be usually overridden by repression through specific control, where the endproduct level of that particular pathway is high and not limiting. In this presentation, regulatory factors for basal level expression and general control of yeast amino acid biosynthesis will be discussed, m addition to pathway-specific repression patterns and interaction between CrOSS- and specific-control mechanisms. Preliminary results are also presented from the investigation of the cloned genes in the threonine biosynthetic pathway of the yeast. yeast.