• 미생물학회지
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• 제29권2호
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• pp.85-91
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• 1991

.betha.-1,4-D-Glucan glucanohydrolase(EC 3.2.1.4;F-II-IV) purified from Trichoderma koningii was identified as a glycoprotein containing 9% carbohydrate. Isoelectric point of the enzyme was estimated to be 4.9 and molecular weight was determined to be approximately 58,000. The porducts of p-nitrophenyl-cellobioside ($PNPG_{2}$) catalyzed by the enzyme were p-nitrophenol(PNP) and p-nitrophenyl-glucoside($PNPG_{1}$). The Km value for $PNPG_{2}$ was estimated to be 0.97 mM in case of the holoside lindage and 10.4 mM in case of the aglycon linkage and their kcat values were $1.8*10^{5}$$ min^{-1}$ and $7.5*10^{5}$ $min^{-1}$ respectively. The product of p-nitrophenyl cellotriose($PNPG_{3}$) was only $PNPG_{1}$. The Km value for $PNPG_{3}$ was 69.5 .$\mu$M and kcat was $1*10^{8}$ $min^{-1}$ which implicates that the enzyme have higher affinity and higher hydrolysis rate toward $PNPG_{3}$ than toward $PNPG_{2}$. The enzyme showed its optimal activity at pH 4.0-4.5 and at 60.deg.C. The effect of gluconolactone on the activity toward $PNPG_{2}$ showed competitive inhibition pattern but glucose and cellobiose did not. The enzyme contained a high content of acidic and hydroxylated amino acids in contrast to basic amino acids.

• 한국미생물·생명공학회지
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• 제25권1호
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• pp.44-50
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• 1997

$\beta $-Glucosidase (EC 3.2.1.21) was purified from Aspergillus niger SFN-416 by a sequential process of ammonium sulfate precipitation, Sepadex G-100 and DEAE-Sephacel column chromatography. Molecular weight of the enzyme was 46, 000 daltons. The K$_{m}$ and V$_{max}$ values for PNPG were 0.67 mM and 25 moles/ml $\cdot $min., respectively. The optimum pH and temperature of the enzyme activity were 3.5 and 58$\circ $C, respectively. The enzyme activity was decreased by addition of metal ions, and increased by addition of metanol, ethanol, isopropanol and 1-butanol at a concentration of 10% (v/v). Stability of the enzyme was increased by addition of isopropanol and 1-butanol at a concentration of 10% (v/v).

• Applied Biological Chemistry
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• 제34권3호
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• pp.249-257
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• 1991

대두 발아 과정 중의 ${\alpha}-galactosidase$를 추출하여 염석, 이온교환 크로마토그래피 및 겔 여과 등의 방법으로 정제한 후 정제효소의 효소학적 성질을 검토하였다. 대두 ${\alpha}-galactosidase$의 활성은 $25^{\circ}C$에서 120시간 발아시켰을 때 가장 높았으며, 대두 중의 raffinose는 96시간, stachyose는 120시간 발아시켰을 때 완전히 분해되었다. 대두 ${\alpha}-galactosidase$는 황산암모늄염석, DEAE-Cellulose 및 DEAE-Sephadex A-50 이온교환 크로마토그래피, Sephadex G-150 겔 여과 등에 의하여 비활성은 825U/mg protein으로써 6.6배까지 정제되었으며 수율은 2.5%이었고 HPLC와 PAGE에 의하여 순도를 확인하였다. 정제효소의 등전점은 pH 4.8이었고, 분자량은 30,000인 monomer이었으며 정제효소의 최적작용 PH는 6.0, 최적작용온도는 $40^{\circ}C$ 이었고, $60^{\circ}C$에서 10분 처리시 25%의 잔존 활성을 나타내었다. 정제효소는 stachyose보다 raffinose를 쉽게 분해하였으며 PNPG에 대한 Km값은 5.3 mM, 활성화 에너지는 13.02 cal/mole이었다.

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2003년도 제39회 학술심포지움
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• pp.23-37
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• 2003

A novel inhibitor against maltose-producing a-amylase was prepared via stepwise degradation of a high molecular weight acarbose (HMWA) using Thermus maltogenic amylase (ThMA). The structure of the purified inhibitor was determined to be ${\alpha}$-D-glucopyranosyl-${\alpha}$-acarviosinyl-D-glucopyranose (GlcAcvGlc). Progress curves of p-nitrophenyl-${\alpha}$-D-maltoside (PNPG2) hydrolysis by various amylolytic enzymes, including maltogenase (MGase), ThMA, and cyclodextrinase(CDase) I-5, in the presence of acarbose or GlcAcvGlc indicated a slow-binding mode of inhibition. The inhibition potency of GlcAcvGlc for MGase, ThMA, and CDase I-5 was 3 orders of magnitude higher than that of acarbose.

• 한국수산과학회지
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• 제31권5호
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• pp.637-644
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• 1998

우리나라 근해에 많이 분포하고 있는 말똥성게 (sea urchin, Hemicentrotus pulcherrimus)의 내장으로부터 Triton X-100을 이용하여 $\beta$-galactosidase를 추출하고, $40\~80\%$ (w/v) $(NH_4)_2SO_4$, DEAE-Sephadex A-25 및 CM-Cellulose 이온교환 크로마토그래피, Con A-Sepha-rose 4B 친화성 크로마토그래피를 사용하여 분리, 정제하여 그 생화학적 특성을 조사한 결과는 다음과 같다. 정제된 $\beta$-galactosidase는 단일의 단백질로 이루어진 효소로 판명되었고, 효소의 정제도는 조효소에 비해 384.6배 증가하였고, 수율은 $1.26\%$이었다. 정제효소의 최적 pH와 온도는 각각 3.0 및 $50^{\circ}C$ 이었다. 효소의 활성은 $Ba^{2+}$와 같은 금속이온에 의해 촉진되었고, $Hg^{2+},\;Sn^{2+}$ 및 DFP에 의해 현저하게 저하되었으며, 당인 galactose 와 lactose에 의해 저하되어 기질 저해 효과가 나타남을 알 수 있었다. 효소의 분자량은 SDS-PAG 전기이동과 Sephadex G-150 겔여과를 실시한 결과 97 kDa로 나타났다. 합성기질인 PNPG를 사용하여 효소의 속도론적 상수를 측정한 결과 $K_m$은 15.0mM, $V_{max}$는 $\mu$mole/min$\cdot$mg$\cdot$protein으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제13권1호
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• pp.134-138
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• 2003

The secondary and tertiary structures of ${\beta}$-galactosidase from L. lactis ssp. lactis 7962 were designed using Nnpredict and Sybyl version 6.3. By using site-directed mutagenesis, the mutated enzymes, Tyr-475-phe and Glu-506-Asp, were generated based on the structural modeling of L. lactis ssp. lactis 7962. The enzymes Tyr.-475-Phe and Glu-506-Asp had <$1\%$ of the activity of the native enzyme with ONPG as substrate. The $V_{max}$ values of the mutated enzymes were greatly reduced (1,800~40,000-1314) compared with the value for the native ${\beta}$-galactosidase. However, the $K_m$ values of Tyr-475-Phe and Glu-506-Asp with ONPG, PNPG, PNPF, and PNPA were not significantly different from those of the native enzyme. The results obtained support the suggestion that Tyr-475 and Glu-506 constitute very important parts of the catalytic machinery of the ${\beta}$-galactosidase.

• Journal of Microbiology
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• 제44권3호
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• pp.276-283
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• 2006

The thermophilic fungus Thermoascus aurantiacus 179-5 produced large quantities of a glucosidase which preferentially hydrolyzed maltose over starch. Enzyme production was high in submerged fermentation, with a maximal activity of 30 U/ml after 336 h of fermentation. In solid-state fermentation, the activity of the enzyme was 22 U/ml at 144 h in medium containing wheat bran and 5.8 U/ml at 48 h when cassava pulp was used as the culture medium. The enzyme was specific for maltose, very slowly hydrolyzed starch, dextrins (2-7G) and the synthetic substrate (${\alpha}$-PNPG), and did not hydrolyze sucrose. These properties suggest that the enzyme is a type II ${\alpha}$-glucosidase. The optimum temperature of the enzyme was $70^{\circ}C$. In addition, the enzyme was highly thermostable (100% stability for 10 h at $60^{\circ}C$ and a half-life of 15 min at $80^{\circ}C$), and stable within a wide pH range.

• 보존과학연구
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• 통권19호
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• pp.3-22
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• 1998

The effect of pH on degradation of paper by some fungi, which able to degrade cellulose, was investigated. Trichoderma koningii, Aspergillus nigerand Penicillium nigulosum were cultured at $28^{\circ}C$ for 16 days in the selective medium (PH3, PH4, PH5, PH6, PH7, PH8, PH9, PH10, PHC) containing paper as substrate. Each paper was pretreated with each pH buffer (pH 3∼pH 10, D.W.)prior to addition to the selective medium. Enzyme activities in the each culture medium were measured spectroph to metrically using C.M.C., Avicel, PNPG as the substrates for endoglucanase, exoglucanase and $\beta$-glucosidase, respectively. In all experimental fungi, the enzyme activities of PH3 and PH9 medium were usually much higher than those of other experimental groups. However in the PH6medium, enzyme activity was lower than other groups. To analyze the concentration and pattern of protein in the each culture medium, the medium was concentrated by lyophilization. The protein concentration of PH3 and PH9 medium were relatively high (T.koningii; 6.31mg, 6,19mg, A.niger; 1.62mg, 1.96mg, P.nigulosum;2.50mg, 2.73mg, respectively), but that of PH6 was relatively low. The protein pattern of each medium was analyzed by using SDS-PAGE and VDS Image Master Analysis Program. The concentrations of bands in the each lane were usually high at lane2 (PH3) and lane8 (PH9) and low at lane5 (PH6). Therefore, the incresed cellulolytic activity of fungus against acidified paper could be result of structural change and deterioration of paper caused by being acidified.

• 한국식품영양학회지
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• 제3권1호
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• pp.57-68
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• 1990

In order to obtain the fundamental informations on cellulase of Trichoderma viride QM 9414 for its production and utilization, some physico-chemical properties of the enzyme were reviewed. When T. viride QM 9414 was cultured on wheat bran medium, filter paper-disintegrating and carboxymethyl cellulose-saccharifying activity were increased with the cell growth, and thereafter CMC-saccharifying activity was kept on almost the same leved while filter-paper disintegrating activity was decreased sharply. And B-glucosidase was formed maximally on the late stationary phase of growth. The crude cellulase of cell-free extracts was purified by (NH4)2SO4 fractionation, Sephadex-G 200 column chromatography and DEAE Sephadex A-50 column chromatography. Filter paper-disintegrating, CMC-saccharifying and B-glucosidase activity were purified 10-fold, 47-fold and 38-fold, respectively. The crude enzyme was proved to be a complex of three different enzyme proteins which were showing filter paper-disintegrating, CMC-saccharifying and B-glucosidase activity. The optimal pH of the three enzyme components was alike pH 4.0, and the optimal temperature for CMC-saccharifying, filter paper-disintegrating and B-glucosidase activity were 4$0^{\circ}C$, 45$^{\circ}C$ and 5$0^{\circ}C$ respectively. The Km and Vmax values of CMC saccharifying activity for CMC were 0.485% and 3.10, and the Km and Vmax vallues of B-glucosidase for PNPG were 0.944$\times$10-3M and 0.097, respectively. The Km and Vmax values of filter paper-disintegrating activity for Avicel were determined to be 0.09% and 0.178, respectively. B-Glucosidase activity was competitively inhibited by glucose, and the Ki value for this enzyme was 3.54$\times$10-3M, CMC saccharifying activity was found to be greatly inhibited by cellobiose.

• Applied Biological Chemistry
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• 제36권3호
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• pp.191-196
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• 1993

본 연구는 합성기질인 $X-{\alpha}-Gal$를 이용하여 발효유 및 유제품내의 Bifidobacteria 생균수를 측정할 목적으로 하였다. 젖산균과 Bifidobacteria의 ${\alpha}-galactosidase$ specific activity를 측정한 결과 Bifidobacteria 균주에서는 높은 ${\alpha}-galactosidase$ activity를 가지고 있었으며, 그 중 Bif. longum KCTC 3215의 specific activity는 8.57 unit/mg protein으로 가장 높게 나타났다. Lactobacillus, Streptococcus, Pediococcus와 Leuconostoc 균주에서는 활성이 미약하거나 없었다. 합성기질인 $X-{\alpha}-Gal$을 MRS agar 배지에 $100{\;}{\mu}M$ 첨가한 결과 Bifidobacteria는 blue colony로, Lac. bulgaricus, Lac. casei와 Leu. mescenteroides 균주는 light blue colony로, 그 외 젖산균에서는 white colony로 나타났다.