• Journal of Microbiology and Biotechnology
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• 제26권5호
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• pp.837-845
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• 2016

A novel endodextranase isolated from Paenibacillus sp. was found to produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides with a degree of polymerization of 7-14 from dextran. To determine the active site, the enzyme was modified with 1-ethyl-3-[3-(dimethylamino)-propyl]-carbodiimide (EDC) and α-epoxyalkyl α-glucosides (EAGs), an affinity labeling reagent. The inactivation followed pseudo first-order kinetics. Kinetic analysis and chemical modification using EDC and EAGs indicated that carboxyl groups are essential for the enzymatic activity. Three Asp and one Glu residues were identified as candidate catalytic amino acids, since these residues are completely conserved across the GH family of 66 enzymes. Replacement of Asp189, Asp340, or Glu412 completely abolished the enzyme activity, indicating that these residues are essential for catalytic activity.

• BMB Reports
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• 제42권6호
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• pp.350-355
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• 2009

Although previous studies on hexokinase (HK) II indicate both the N- and C-terminal halves are catalytically active, we show in this study the N-terminal half is significantly more catalytic than the C-terminal half in addition to having a significantly higher $K_m$ for ATP and Glu. Furthermore, truncated forms of intact HK II lacking its first N-terminal 18 amino acids ($\Delta$18) and a truncated N-terminal half lacking its first 18 amino acids ($\Delta$18N) have higher catalytic activity than other mutants tested. Similar results were obtained by PET-scan analysis using $^{18}F-FDG$. Our results collectively suggest that each domain of HK II possesses enzyme activity, unlike HK I, with the N-terminal half showing higher enzyme activity than the C-terminal half.

• BMB Reports
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• 제36권2호
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• pp.149-153
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• 2003

The dibucaine number (DN) was determined for serum cholinesterase (EC 3.1.1.8, SChE) in plasma samples. The ones with a DN of 79-82 were used, because they had the "usual" SChE variant. The enzyme was assayed colorimetrically by the reaction of 5,5'-dithiobis-[2-nitrobenzoic acid] (DTNB) with the free sulfhydryl groups of thiocholine that were produced by the enzyme reaction with butrylthiocholine (BuTch) or acetylthiocholine (AcTch) substrates, and measured at 412 nm. Dibucaine, a quaternary ammonium compound, inhibited SChE to a minimum within 2 min in a reversible manner. The inhibition was very potent. It had an $IC_{50}$ of $5.3\;{\mu}M$ with BuTch or $3.8\;{\mu}M$ with AcTch. The inhibition was competitive with respect to BuTch with a $K_i$ of $1.3\;{\mu}M$ and a linear-mixed type (competitive/noncompetitive) with respect to AcTch with inhibition constants, $K_i$ and $K_I$ of 0.66 and $2.5\;{\mu}M$, respectively. Dibucaine possesses a butoxy side chain that is similar to the butryl group of BuTch and longer by an ethylene group from AcTch. This may account for the difference in inhibition behavior. It may also suggest the existence of an additional binding site, other than the anionic binding site, and of a hydrophobic nature.

• Archives of Pharmacal Research
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• 제17권4호
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• pp.218-222
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• 1994

The natural product, spirostanol glycoside dioscin, was shown to directly inactivate human pleural fluid phospholipase $A_2{\;}(PLA_2)$ Inactivation was dose, and time dependent. The $IC_{50}$ was estimated at 18 .mu.M and virtually complete inactivation of the enzyme occurred at 50 .mu.M. Using Michaelis-Menten kinetics, dioscin inactivated the enzyme by a competitive inhibitory manner, the apparent Ki value was $6.9{\times}10_{-4}$. Reversibility was studied directly by dialysis method, the inhibition was reversible. Additioin of excess $Ca^{2+}$ concentration up to 8 mM did not antagonize the inhibitory activity of dioscin. Inactivation of several kinds of $PLA_2$ by dioscin is due to interaction with the active site of $PLA_2$ and may be a useful adjunt in the theraphy of inflammatory diseases.

• 대한의생명과학회지
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• 제8권1호
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• pp.47-52
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• 2002

To investigate an effect of cyclohexanone (CHO) treatment on the serum levels of xanthine oxidase (XO) in liver damaged animals, the rats were intraperitoneally pretreated with 50% carbon tetrachloride ($CCl_4$) in olive oil (0.1 mL/ 100 g body weight) 14 times every other day. To the $CCl_4$-pretreated rats, CHO (1.56 g/kg body weight) was injected once and then the animals were sacrificed at 4 hours after CHO treatment. The increasing rate of serum and liver XO activities to the control was higher in CHO-treated animals pretreated with $CCl_4$ than the $CCl_4$-pretreated those. Concomitantly CHO injection to the $CCl_4$-pretreated animals showed somewhat higher Vmax and lower Km value in the kinetics of liver XO enzyme. Furthermore, increasing rate of hepatic malonedialdehyde content to the control was also higher in CHO-treated animals pretreated with $CCl_4$ than $CCl_4$-pretreated those. On the other hand, the injection of CHO to the $CCl_4$-pretreated animals showed the more enhanced liver damage on the basis of liver function finding; liver weight per body weight (%), serum levels of alanine aminotransferase activity and hepatic glucose-6-phosphatase activity. In conclusion, injection of CHO to the $CCl_4$-pretreated rats led to more increased activity of serum XO and it may be caused by acceleration of hepatocyte membrane permeability and induction of enzyme protein.

• BMB Reports
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• 제44권12호
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• pp.805-810
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• 2011

Methionine sulfoxide reductase A (MSRA) is a ubiquitous enzyme that has been demonstrated to reduce the S enantiomer of methionine sulfoxide (MetSO) to methionine (Met) and can protect cells against oxidative damage. In this study, we isolated a novel MSRA (SlMSRA2) from Micro-Tom (Solanum lycopersicum L. cv. Micro-Tom) and characterized it by subcloning the coding sequence into a pET expression system. Purified recombinant protein was assayed by HPLC after expression and refolding. This analysis revealed the absolute specificity for methionine-S-sulfoxide and the enzyme was able to convert both free and protein-bound MetSO to Met in the presence of DTT. In addition, the optimal pH, appropriate temperature, and $K_m$ and $K_{cat}$ values for MSRA2 were observed as 8.5, $25^{\circ}C$, $352{\pm}25\;{\mu}M$, and $0.066{\pm}0.009\;S^{-1}$, respectively. Disk inhibition and growth rate assays indicated that SlMSRA2 may play an essential function in protecting E. coli against oxidative damage.

• Journal of Applied Biological Chemistry
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• 제58권3호
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• pp.209-218
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• 2015

Fructooligosaccharides have been mainly produced by microbial fructosyltransferases (FTase) enzymes. The present work focuses on the optimization of medium composition and cultivation parameters affecting FTase produced by Penicillium aurantiogriseum AUMC 5605 in shake flask cultivation. FTase production was optimized in two steps using DeMeo's fractional factorial design. A 1.46-fold increase in FTase production (105.4 U/mL) was achieved using the optimized culture medium consisting of (g/L): sucrose, 600; yeast extract, 10; $K_2HPO_4$, 5; $MgSO_4{\cdot}7H_2O$, 0.5; $(NH_4)_2SO_4$, 1.0 and KCl, 0.5. The obtained results showed that the maximum FTase enzyme activity was produced at initial cultivation pH values ranging from 6.0-6.5, at agitation speed of 200 rpm and using vegetative fungal cells as inoculum. Moreover, results showed that optimization of medium composition and some cultivation parameters resulted in an increase of about 93.7% in the enzyme activity than the nonoptimized cultivation conditions after 96 h of cultivation. Additionally, maximum production and specific production rates recorded 2340 U/L/h and 102 U/L/h/g cells, respectively.

• Food Science and Biotechnology
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• 제15권3호
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• pp.453-457
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• 2006

Activity and stability of kiwifruit actinidine was determined in various conditions of pH, salt, and temperature using N-${\alpha}$-CBZ-lysine P-nitrophenyl ester as the substrate. Actinidine activity was low below pH 6, and undetectable below pH 3. The enzyme was stable in a pH range of 6.0-8.5. At $4^{\circ}C$ the enzyme was inactive in the presence of greater than 36% vinegar and in 2 M NaCl. Actinidine at $25^{\circ}C$ was unstable in 24% vinegar but stable in up to 3 M NaCl. With regard to freeze-thaw stability, actinidine retained 85% residual activity after being frozen at $-20^{\circ}C$ for 3 days. Based on Arrenius and Lineweaver-Burk plots, actinidine became unstable at greater than $45^{\circ}C$ with only 30% residual activity remaining after 6 min. The Km, kcat, and kcat/Km values of actinidine were $56\;{\mu}M$, 67/sec, and $1.2\;{\mu}M/sec$, respectively.

• Archives of Pharmacal Research
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• 제22권3호
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• pp.232-236
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• 1999

In this study, we present evidence that cotreatment of aloesin and arbutin inhibits tyrosinase activity in a synergistic manner by acting through a different action mechanism. Aloesin or arbutin similarly inhibited enzyme activity of human- and mushroom-tyrosinases with an IC50 value of 0.1 or 0.04 mM, respectively. Lineweaver-Burk plots of the enzyme kinetics data showed that aloesin inhibited tyrosinase activity noncompetitively with a Ki value of 5.3 mM, whereas arbutin did it competitively (Maeda, 1996). We then examined whether cotreatment of these agents inhibits the tyrosinase activity in a synergistic manner. The results showed that 0.01 mM aloesin in the presence of 0.03 mM arbutin inhibited activity of mushroom by 80% of the control value and the reverse was also true. The inhibitory effects were calculated to be synergistic according to the B rgi method. Taken together, we suggest that aloesin along with arbutin inhibits in synergy melanin production by combined mechanisms of noncompetitive and competitive inhibitions of tyrosinase activity.

• 한국미생물·생명공학회지
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• 제20권3호
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• pp.295-301
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• 1992

토양으로부터 alkaline protease 생성능이 강한 Streptomyces griseus HC-1141을 분리하였으며, 정제 효소의 최적작용 pH 와 온도는 8.0, 60'C 였으며, pH 7.0-9.0의 범위와 $60^{\circ}C$이하에서 안정하였다. 금속 이온중 $Mn^{2+}$, $Ca^{2+}$, 등에 의해 활성이 증대되었으나 $Hg^{2+}$, $Cu^{2+}$, $Zn^{2+}$, $Ba^{2+}$, $Fe^{2+}$ 등에 의해 효소 활성이 저해되었고, 효소활성 저해제 중 Epsilon-aminocaproic acid, 2,4-dinitrophenol, iodine 등에 의해서는 현전한 효소활성저해가 관찰되지 않았으나 ethylenediaminetetraacetic acid와 p-chloromercuribenzoic acid에 의해 활성저해가 관찰되어 효소분자 중 SH기가 활성에 어느정도 관여하는 metallo enzyme으로 추정되었다. 정제효소의 $K_m$, $V_{max}$ 및 활성화에너지는 $2.229{\times}10^{-4}$M, $46.08 {\mu}$g/min, 3.643 kcal/mol 이었으며 hemoglobin과 egg albumin보다 casein을 더 잘 가수분해하였다. 또한 여러 가지 detergent에 대하여 강한 저항성을 가지고 있었다.