• Mass Spectrometry Letters
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• 제5권2호
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• pp.52-56
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• 2014

Glycated hemoglobin (HbA1c) is used as an index of mean glycemia over prolonged periods. This study describes an optimization of enzyme digestion conditions for quantification of non-glycated hemoglobin (HbA0) and HbA1c as diagnostic markers of diabetes mellitus. Both HbA0 and HbA1c were quantitatively determined followed by enzyme digestion using isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) with synthesized N-terminal hexapeptides as standards and synthesized isotope labeled hexapeptides as internal standards. Prior to quantification, each peptide was additionally quantified by amino acid composition analysis using ID-LC-MS/MS via acid hydrolysis. Each parameter was considered strictly as a means to improve digestion efficiency and repeatability. Digestion of hemoglobin was optimized when using 100 mM ammonium acetate (pH 4.2) and a Glu-C-to-HbA1c ratio of 1:50 at $37^{\circ}C$ for 20 h. Quantification was satisfactorily reproducible with a 2.6% relative standard deviation. These conditions were recommended for a primary reference method of HbA1c quantification and for the certification of HbA1c reference material.

• 미생물학회지
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• 제31권2호
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• pp.157-165
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• 1993

A 1, 4-.betha.-D-xylanase, designated as xylanase II, was purified from the culture filtrate of Trichoderma koningii ATCC 251131 by column chromatography on Sephadex G-75, SP-Sephadex C-50, DEAE-Sephadex A-50 and Sephadex G-50 with an overall yield of 6.97%. It has a molecular weight of 21.000 and an isoelectric point of 9.4. The enzyme activity is optimal at pH 5.0 and at a temperature of 50.deg.C. Xylanase II is stable up to 50.deg.C, while 40 and 90% of its activity are lost after the incubation for 30 and 60 min at 60.deg.C. The enzyme degrades xylan with relatively high activity, as well as carboxymethylcellulose and Avicel. Its $K_{m}$ values for oat-spelt xylan, larchwood xylan and Avicel are 7.48, 1.98 and 13.33 mg/ml, respectively. The hydrolysis products of oat-spelt xylan by xylanase II are xylose, xylobiose, xylotriose and arabinoxylotriose, while the reaction products of larchwood xylan are xylose, xylobiose, xylotriose and small amount of higher oligomers. The action paterns of the enzyme demonstrate that xylanase II is endo-enzyme.

• Journal of Microbiology and Biotechnology
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• 제4권1호
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• pp.41-48
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• 1994

The xylanase from alkali-tolerant Bacillus sp. YA-14 was purified to homogeneity by CM-cellulose, Sephadex G-50, and hydroxyapatite column chromatographies. The molecular weight of the purified enzyme was estimated to be 20, 000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme slightly hydrolyzed carboxymethyl cellulose and Avicel, but did not hydrolyze soluble starch, dextran, pullulan, and ${\rho}-nitrophenyl-{\beta}$-D-xylopyranoside. The maximum degree of hydrolysis by enzyme for birchwood xylan and oat spelts xylan were 47 and 40%, respectively. The Michaelis constants for birchwood xylan and oat spelts xylan were calculated to be 3.03 mg/ml and 5.0 mg/ml, respectively. The activity of the xylanase was inhibited reversibly by $HgCl_2$, and showed competitive inhibition by N-bromosuccinimide, which probably indicates the involvement of tryptophan residue in the active center of the enzyme. The Xylanase was identified to be xylose-producing endo-type xylanase and did not show the enzymatic activities which cleave the branch point of the xylan structure.

• Journal of Microbiology and Biotechnology
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• 제15권5호
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• pp.1067-1072
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• 2005

A metagenomic library was constructed using a fosmid vector, and total genomic DNA was extracted directly from soil at Cisolok (hot spring area, Indonesia). This library was composed of 10,214 clones and screened for lipolytic enzyme on tributyrin agar plates. An esterase gene (estMa) was subcloned and sequenced from a positive lipolytic active clone. Esterase EstMa was encoded by a 954-bp open reading frame and showed low ($11-33\%$) amino acid similarity to known esterases. The amino acid sequence analysis demonstrated that the enzyme is a new member of lipolytic enzyme family VI. The estMa gene encodes a preprotein of 317 amino acids with a predicted molecular mass of 34,799 Da. The purified enzyme exhibited optimal activity at $50^{\circ}C$ and pH 6.5. The $K_m,\;and\;V_{max}$ values of EstMa for the hydrolysis of p-nitrophenyl valerate were $45.3\;{\mu}M$ and 4.45 U/mg, respectively.

• Asian-Australasian Journal of Animal Sciences
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• 제16권1호
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• pp.83-87
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• 2003

The study evaluates the enzymatic dry matter (DM) degradability and water holding capacity of leaf and stem fractions of orchardgrass (Dactylis glomerata L.) at different growth stages with or without the presence of surfactant Tween 80. While Tween 80 significantly (p<0.05) increased water and enzyme holding capacities in the leaf blades fraction, less was observed in the fraction of leaf sheath and stem of orchardgrass. The enzyme holding capacity in the leaves was also altered more than that for water holding capacity. This resulted in the increased rate and extent of enzymatic hydrolysis of the leaf blade fractions at two growth stages, whereas little was with leaf sheath and stem fractions. It was also observed that at 0.005% concentrations of Tween 80 the enzymatic DM degradability of young leaf blades was higher (p<0.05) by 20-30% compared to that of the control, as well as for water and enzyme holding capacity. For matured leaf blades the DM degradability were increased with over 0.01% concentrations of the surfactant, but the increase was less than leaf blades of young orchardgrass. This result suggests the possibility of using the surfactant Tween 80 to improve forage digestibility in the rumen.

• Natural Product Sciences
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• 제9권4호
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• pp.235-240
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• 2003

Tectorigenin has an apoptosis-inducing ability and immunosuppressive activity. We investigated the effect of tectorigenin on Phase I and II enzyme activities to elucidate the pharmacological action of the immunosuppressive tectorigenin in the diabetic rat. This compound was obtained from the hydrolysis of tetoridin isolated from the flower of Pueraria thumbergiana (Leguminosae). This crude drug (Puerariae Flos) has been used as a therapeutic for diabetes mellitus in traditional Korean medicine. Tecotrigenin inhibited the formation of malondialdehyde (MDA) and hydroxy radicals in serum and liver but promoted superoxide dismutase (SOD) activity. Low MDA contents and low xanthine oxidase and aldehyde oxidase activities were observed in the tectorigenin-treated rats, suggesting that such Phase I enzyme activities are the major source of lipid peroxidation. However, tectorigenin increased Phase II enzyme activities such as SOD, glutathione peroxidase and catalase, suggesting the activation of free radical-scavenging enzymes. The activities of tectorigenin were comparable to those of glibenclamide, which was employed as a positive control. These results suggest that tectorigenin may share some biological properties with glibenclamide in insulin-dependent-diabetes mellitus (IDDM).

• KSBB Journal
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• 제9권5호
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• pp.499-505
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• 1994

Bacillus macerans cyclodextrin glucanotrans­f ferase(CGTase)를 전분흡착법과 DEAE--cellulose 칼럼 크로마토그래피로 정제하였다. 효소의 분자량 은 67,000이였고 monomer였다. 정제된 효소는 전 분을${\alpha}$-, ${\beta}$-, ${\gamma}$-CD 로 전환시켰으며, CD생성비율은 각각 1 : 1.68: 0.32였다. ${\alpha}$-CD와 D-glucose의 coupling반응 초기에는 maltohexose가 주로 생성되었고, 그 후에 다른 oligosaccharide들이 생성되었다 .. a­C CD의 가수분해반응 초기에는 주로 maltotetrose가 생성되였고 그 이후에는 소량의 다른 이Igosa­C ccharide들이 생성되었다. 정제된 효소의 좋은 기질인 maltotriose 로부터 maltosyl 이나 D-glucopy ranosyl group이 전이될 수 있는데, 본 연구에서는 D정lucosyl transfer가 우세하였다.

• Bulletin of the Korean Chemical Society
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• 제25권10호
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• pp.1499-1502
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• 2004

An amperometric biosensor based on carbon paste electrodes (CPEs) for the determination of urea was constructed by enzyme (urease/GL-DH)-modified method. Urea was hydrolyzed to ${NH_4}^+$ by catalyzing urease onto the enzyme-modified electrode surface in sample solution. In the presence of ${\alpha}$-ketoglutarate and reduced nicotinamide adenine dinucleotide(NADH), a liberated ${NH_4}^+$ produce to L-glutamate and $NAD^+$ by Lglutamate dehydrogenase (GL-DH). After the chemical reaction was proceeded, the electrochemical reaction was occurred that an excess of the NADH was oxidized to $NAD^+$. The oxidation current of NADH was monitored at +1.10 volt vs. Ag/AgCl. An optimum conditions of biosensor were investigated: The optimum pH range for catalyzed hydrolysis reaction of urea was pH 7.0-7.4. The linear response range and detection limit were $2.0\;{\times}\;10^{-5}{\sim}2.0\;{\times}\;10^{-4}M\;and\;5.0\;{\times}\;10^{-6}M$, respectively. Another physiological species did not interfere, except L-ascorbic acid.

• Journal of Microbiology and Biotechnology
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• 제17권5호
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• pp.733-738
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• 2007

An extracellular exoinulinase($2,1-\beta-D$ fructan fructanohydrolase, EC 3.2.1.7), which catalyzes the hydrolysis of inulin into fructose and glucose, was purified 23.5-fold by ethanol precipitation, followed by Sephadex G-100 gel permeation from a cell-free extract of Kluyveromyces marxianus YS-1. The partially purified enzyme exhibited considerable activity between pH 5 to 6, with an optimum pH of 5.5, while it remained stable(100%) for 3 h at the optimum temperature of $50^{\circ}C$. $Mn^{2+}\;and\;Ca^{2+}$ produced a 2A-fold and 1.2-fold enhancement in enzyme activity, whereas $Hg^{2+}\;and\;Ag^{2+}$ completely inhibited the inulinase. A preparation of the partially purified enzyme effectively hydrolyzed inulin, sucrose, and raffinose, yet no activity was found with starch, lactose, and maltose. The enzyme preparation was then successfully used to hydrolyze pure inulin and raw inulin from Asparagus racemosus for the preparation of a high-fructose syrup. In a batch system, the exoinulinase hydrolyzed 84.8% of the pure inulin and 86.7% of the raw Asparagus racemosus inulin, where fructose represented 43.6mg/ml and 41.3mg/ml, respectively.

• Archives of Pharmacal Research
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• 제8권3호
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• pp.149-157
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• 1985

The HCL hydrolyzate of the non-histone protein fractionated from the rat liver nuclei which have been incubated inthe presence of S-adenosyl-L-[methyl-$^{14}C$ ]-methionine shows at least four unidentified radioactive peaks on a basic amino acid analysis chromatogram. One of these unknown compounds (designated as compound 3) is also formed by the rat liver homogenated with the exogenous addition of an appropriate protein substrate. Since boiled rat liver homogenate or fresh homogenate in the absence of an exogenous protein substrate failed to form compound 3, its formation can be considered to be enzyme-catalyzed. The enzyme which yields compound 3 shows a preference of protein substrate in the order of reductively methylated hemoglobin > native > histone type II-A. The rat enzyme is nuclear in location associated with chromatin, and exhibits the highest activity in the liver among various rat organs. A compound 3-forming enzyme is also present in Neurospora crassa, since endogenous formation of the compound 3 can be demonstrated with the crude extract of this mold. The chemical identity of compound 3 is not yet known. However, it resisted to the following treatments; 6 N HCL and 0.1 N Na NaOH hydrolysis at $110^{\circ}C$, OR L-amino acid oxidase.