• KSBB Journal
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• 제5권3호
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• pp.279-291
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• 1990

Chitin을 35 mesh로 분쇄하여 강산과 강알카리로 처리하여 CHITA와 CHITB를 만들고 여기에 glutaraldehyde를 작용시켜 $\beta$-glucosidase를 고정하여 CHITA-Gase 및 CHITB-Gase를 제조하였다. 이 두 종류의 고정화 효소를 기질 p-nitropheol-$\beta$-D-gliucopyranoside(PNG)과 회분식 반응기, 연속 흐름 반응기 및 플러그 흐름 반응기에서 반응시켜 최적 온도, 최적 pH, 반응 속도 상수, 물질 전달 계수, 효율 인자 및 효소 불활성화 속도 등을 구하여 반응기별 효율을 검토하였다. 반응 최적 온도는 세가지의 반응기 모두 5$0^{\circ}C$였으며, 최적 pH는 플러그 흐름 반응기에서는 Nat-Gase와 같이 pH5.0이었고 회분식 반응기 및 연속 흐름 반응기에서는 최적 pH의 이동이 일어나 pH6.0으로 이었다. 반응기의 최적 조건에서 km값은 회분식 반응기에서 CHITB-Gase$1.725$\times$10^-^5M/1$가 CHITA-Gase($1.725$\times$10^-^5M/1$)보다 작았으며, 연속 흐름 반응기 및 플러그 흐름 반응기에서는 유속 증가에 따라 Km'치가 감소하는 경향을 보였고, CHITB-Gase가 CHITB-Gase보다 더 작았다. $V^m^a^x'$값은 회분식 반응기, 연속 흐름 반응기, 플러그 흐름 반응기에서 모두 CHITA-Gase가 CHITB-Gase보다 높은 것으로 나타났다. 그리고, 물질전달계수 및 효율인자, 효소 불활성화 속도등의 값은 환경은 CHITB-Gase의 것이 나은 것으로 나타났다. 이들의 결과에서 CHITA-Gase 및 CHITB-Gase는 기질과의 반응 환경이 좋으므로 chitind은 $\beta$-glucosidase의 좋은 지지체라고 판단되어 공업적 응용이 기대된다.

• Journal of Microbiology and Biotechnology
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• 제22권12호
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• pp.1705-1713
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• 2012

A gene encoding ${\beta}$-glucosidase was cloned from Weissella cibaria 37, an isolate from human feces. Sequence analysis showed that the gene could encode a protein of 415 amino acids in length, and the translated amino acid sequence showed homology (34-31%) with glycosyl hydrolase family 1 ${\beta}$-glucosidases. The gene was overexpressed in E. coli BL21(DE3) using pET26b(+) and a 50 kDa protein was overproduced, which matched well with the calculated size of the enzyme, 49,950.87 Da. Recombinant ${\beta}$-glucosidase was purified by using a his-tag affinity column. The purified ${\beta}$-glucosidase had an optimum pH and a temperature of 5.5 and $45^{\circ}C$, respectively. Among the metal ions (5mM concentration), $Ca^{2+}$ slightly increased the activity (108.2%) whereas $Cu^{2+}$ (46.1%) and $Zn^{2+}$ (56.7%) reduced the activity. Among the enzyme inhibitors (1 mM concentration), SDS was the strongest inhibitor (16.9%), followed by pepstatin A (45.2%). The $K_m$ and $V_{max}$ values of purified enzyme were 4.04 mM and 0.92 ${\mu}mol/min$, respectively, when assayed using pNPG (p-nitrophenyl-${\beta}$-D-glucopyranoside) as the substrate. The enzyme liberated reducing sugars from carboxymethyl cellulose (CMC).

• 한국미생물·생명공학회지
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• 제37권3호
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• pp.204-212
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• 2009

대두의 발효를 통하여 생리활성을 가지고 있는 이소플라본 aglycone 함량을 높이기 위한 ${\beta}$-glucosidase와 대두에 다량 함유되어 있는 stachyose, rafinose와 같은 난소화성 oligosaccharides를 분해하기 위해 ${\alpha}$-galactosidase 효소 분비 미생물을 김치로부터 ${\alpha}$-galactosidase와 ${\beta}$-glucosidase를 생산하는 미생물을 탐색하였다. 탐색과정을 위해서 선별한 미생물을 16S rDNA sequencing 동정한 결과, Weissella cibaria 동정되어 Weissella cibaria K-M1-4로 명명하였다. Weissella cibaria K-Ml-4를 대두 액체배지에서 18시간동안 배양한 후, 생산한 효소는 배양액을 에탄을 침전, DEAE sepharose, sephacryl S-100HR column chromatography 통하여 ${\alpha}$-galactosidase의 경우, 정제도 5.3배, 수율 3.5% 그리고 ${\beta}$-glucosidase의 경우, 정제도 4.4배, 수율 2.9%로 정제되었다. ${\alpha}$-Galactosidase 효소특성은 $60^{\circ}C$에서 최대 활성을 나타내었으며, $80^{\circ}C$에서 30분 처리시 43% 잔존활성을 보였다. pH 8.0에서 최대 활성을 나타내었으며, pH 5.0-9.0에서 안정하였다. 금속이온에 대한 영향에서 $Fe^{2+}$과 $Cu^{2+}$을 첨가하였을 때 효소 활성이 증가하였다. p-Nitrophenyl-${\alpha}$-D-galacto-pyranoside (PNPG) 기질에 대한 Km은 0.98 mM이었고, Vmax는 $1.81{\mu}$mole/min 이었다. ${\beta}$-Glucosidase 효소 특성은 $50^{\circ}C$에서 최대 활성을 나타내었으며, $80^{\circ}C$에서 30분 처리시 46% 잔존활성을 보였다. pH 7.0에서 최대 활성을 나타내었으며, pH 5.0-9.0에서 안정하였다. 금속이온에 대한 영향에서 $Fe^{2+},\;Co^{2+},\;Cu^{2+}$을 첨가하였을 때 효소 활성이 증가하였다. p-Nitrophenyl-${\beta}$-D-gluco-pyranoside (PNPG)에 대한 Km값은 1.24mM이었고, Vmax는 $6.81{\mu}$mole/min 이었다.

• Journal of Applied Biological Chemistry
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• 제57권3호
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• pp.197-203
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• 2014

Various parameters such as solvent selection, concentration, solid/liquid ratio, soaking time, temperature, stationary, shaking conditions, and repeated extractions were investigated in order to determine the optimum extraction conditions of ${\beta}$-glucosidase from bagasse fermented by mixed culture of Aspergillus niger NRC 7A and Aspergillus oryzae NRRL 447. Among various solvents tested, non ionic detergents gave the best results than the inorganic or organic salt solutions and distilled water. The optimum conditions for extraction of ${\beta}$-glucosidase were 30 min soaking time at $40^{\circ}C$ under shaking condition at 150 rpm, with solid/liquid ratio 1:15 (w/v), which yielded $2882.74{\pm}95.52U/g$ fermented culture (g fc) of enzyme activity. With repeated washes under the above optimum conditions, the results showed that enzyme extracted in the $1^{st}$ and $2^{nd}$ washes represents about 90% of the total activity.

• Journal of Applied Biological Chemistry
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• 제43권3호
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• pp.141-146
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• 2000

Glycosidase activities were tested from the grape berries, Vitis labruscana B. Takasumi. Among various glycosidases, $\alpha$-D-galactosidase was found to be the most active in the flesh and other glycosidases were considerably active in the order of the following: $\alpha$-D-mannosidase>$\alpha$-D-glucosidase>$\beta$-D-glucosidase>$\beta$-D-galactosidase. In the seeds, $\alpha$-D-glucosidase activity was the highest and other glycosidases such as $\alpha$-D-galactosidase, $\beta$-D-glucosidase, and $\beta$-D-galactosidase were still significantly active. The $\alpha$-D-galactosidase in the grape flesh was purified over 83-folds through salting-out with $(NH_4)_2SO_4$ and a series of chromatographies employing Sephadex G-50, Octyl-Sepharose, Q-Sepha- rose, and Biogel P-100. The enzyme was a monomer of 45 kDs as determined through SDS-PAGE and Sephacryl S-200 chromatography. The purified enzyme showed a preference of $\alpha$-D-galactose to $\beta$-D-galactose as a substrate about 5.4 times. Sulfhydryl specific reagents such as N-ethylmaleimide and iodoacetamide significantly inhibited the enzyme activity to the extents of 48 and 52% of its initial activity, respectively. The optimumpH range of $\alpha$-D-galactosidase was around 6.5-7.0. The enzyme activity increased by 46% in the presence of 1mM $Fe^{2+}$.

• 한국식품과학회지
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• 제27권4호
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• pp.598-604
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• 1995

원지, 양파 및 쑥을 추출물, 즙 또는 분말로 하여 기준식이에 5 및 10% 첨가하여 흰쥐에게 14일간 먹여 대장내용물에서 주요균의 변화와 ${\beta}-glucosidase$와 ${\beta}-glucuronidase$의 효소활성 및 인돌과 volatile basic nitrogen의 생성량을 조사하였다. 실험쥐들의 사료섭취량과 체중증가량은 쑥의 물추출물 5% 섭취구를 비롯하여 시료 첨가구들에서 대체로 높았으나, 원지물추출물 5%와 쑥의 에탄올추출물 10% 섭취구에서는 체중이 감소하였다. 쑥의 물 또는 에탄올추출물 첨가구에서 유익균인 bifidobacteria가 대조구에 비해 높았고, clostridia와 E. coli는 낮았다(p<0.05). 그외 양파즙 10% 첨가구에서도 유익균의 증가와 유해균의 감소와 같은 유리한 균총변화를 보였다. 한편 쑥분말 5% 첨가구에서 대장내용물중 ${\beta}-glucosidase$와 ${\beta}-glucuronidase$ 활성은 대조구와 비교해 통계적인 유의성이 없으나, 인돌함랑과 pH는 대조구에 비해 낮았다(p<0.05). 그러나 원지물추출물과 양파를 10% 첨가구에서는 ${\beta}-glucosidase$ 활성이, 쑥추출물들의 첨가구에서는 ${\beta}-glucuronidase$ 활성이 높았다(p<0.05). 인돌함량은 단백질 함량이 높은 원지물추출물과 쑥에탄올추출물 첨가구에서 높았다(p<0.05). 한편 휘발성 염기태 질소화합물의 함량은 처리구들에서 유의적인 차이가 없었다.

• 한국연초학회지
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• 제25권1호
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• pp.70-79
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• 2003

$\beta$ -Glucosidase-catalysed synthesis of glucosides with aromatic alcohols and monoterpene alcohols as accepters and cellobiose as a donor in the presence of various commercial $\beta$ -glucosidases were described. $\beta$ -Glucosidases from Aspergillus niger spp,. Trichoderma spp., Penicillium sup. and bitter almond have been shown to catalyze synthesis of $\beta$ -glucosides of benzyl alcohol, 2-hydroxybenzyl alcohol, 4-hydroxybenzyl alcohol, 2-phenylethyl alcohol, geraniol and citronellol in the presence of cellobiose as sugar donor. Among enzyme preparations tested, each $\beta$ -glucosides prepared from Aspergillus niger were isolated in the pure state by Diaion HP-20 and silica gel column chromatography. The products were identified as $\beta$ -glucosyl products of benzyl alcohol, 2-hydroxyhenzyl alcohol, 4-hydroxybenzyl alcohol, 2-phenyl ethyl alcohol, geraniol and citronellol by spectrometry (UV, IR, $^1$H-NMR, $^{13}$ C-NMR) and enzymatic hydrolysis with $\beta$ - glucosidase. Monoterpene alcohols with a sterically hindered hydroxyl group, such as linalool, $\ell$-menthol and $\alpha$-terpineol were not used as acceptors in transglycosylation reaction.

• 한국미생물·생명공학회지
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• 제21권5호
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• pp.440-445
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• 1993

The fungi SFN 416 strain which produced a stable beta-glucosidase was isolated from nature and identified to Aspergillus niger. Optimal conditions of enzyme reaction were temperature 36C, pH-5.0, reaction time-40 minutes. The enzyme was stable below 60C and in the range of pH 4.5-6.5. The enzyme was greatly inhibitied by Ag+ and slightly activated by Ca2+ (0.5mM) and Cu2+ (5 mM).

• Korean Membrane Journal
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• 제8권1호
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• pp.58-66
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• 2006

The ${\beta}-glucosidase$ from olive fruit is of particular interest compared to the ones from other sources because it has shown to have high specifity to convert the oleuropein into dialdehydes, which have antibacterial activity and are of high interest for their application in the food and pharmaceutical fields. The enzyme is not yet commercially available and advanced clean and safe technologies for its purification able to maintain the functional stability are foreseen. The purification of this protein from fruit extracts has been already tempted by electrophoresis but either enzyme deactivation or high background with unclear profiles occurred. In this work, fruit extracts obtained from the ripening stage that showed the highest enzyme activity have been processed by diafiltration and ultrafiltration. Asymmetric membranes made of polyamide or polysulphone having 50 and 30 kDa molecular weight cut-off, respectively, were tested for the diafiltration process. Ultrafiltration membranes made of polyethersulfone with 4 kDa molecular weight cut-off were used to concentrate the dia-filtered permeate solutions. The efficiency of the separation processes was evaluated byenzyme activity tests using the hydrolysis of p-D-nitrophenyl-${\beta}$-D-glucopyranoside (pNPGlc) as reaction model. Qualitative and quantitative electrophoresis were applied to analyze the composition of protein solution before and after the membrane separation; in addition dot blot and western blot analyses were applied to verify the presence of ${\beta}-glucosidase$ in the processed fractions. The overall results showed that the ${\beta}-glucosidase$ functional stability was preserved during the membrane operations and the removal of 20 kDa proteins allowed to increase the specific activity of the enzyme of about 52% compared to the one present in the initial fruit extract.

• 약학회지
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• 제53권3호
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• pp.114-118
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• 2009

Bioemulsifiers are those chemicals which are produced from microorganisms but which have both hydrophilic and hydrophobic groups. $\alpha$-Glucosidase inhibitor ($\alpha$-GI) produced from Bacillus lentimorbus B-6 (B-6) showed bioemulsifying activity. But $\beta$-glucosidase inhibitor produced from B-6 didn't show emulsifying activity. $\alpha$-GI was purified from supernatant of B-6 grown in minimal culture medium containing glucose and sodium glutamate by Sephadex G-100 column chromatography and isolated from $\beta$-GI by dialysis against water. Toluene was determined as the best substrate for emulsifying activity of $\alpha$-GI. $\alpha$-GI showed thermostability at $100^{\circ}C$ for 15 min, high salt tolerance up to 32% NaCl and wide range of pH-stability at pH $4\sim10$. Emulsifying character of $\alpha$-GI can be useful for the liposome formation for the treatment of diabetes mellitus.