• Journal of Microbiology and Biotechnology
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• 제32권6호
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• pp.749-760
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• 2022

α-Galactosidase is a debranching enzyme widely used in the food, feed, paper, and pharmaceuticals industries and plays an important role in hemicellulose degradation. Here, T26, an aerobic bacterial strain with thermostable α-galactosidase activity, was isolated from laboratory-preserved lignocellulolytic microbial consortium TMC7, and identified as Parageobacillus thermoglucosidasius. The α-galactosidase, called T26GAL and derived from the T26 culture supernatant, exhibited a maximum enzyme activity of 0.4976 IU/ml when cultured at 60℃ and 180 rpm for 2 days. Bioinformatics analysis revealed that the α-galactosidase T26GAL belongs to the GH36 family. Subsequently, the pET-26 vector was used for the heterologous expression of the T26 α-galactosidase gene in Escherichia coli BL21 (DE3). The optimum pH for α-galactosidase T26GAL was determined to be 8.0, while the optimum temperature was 60℃. In addition, T26GAL demonstrated a remarkable thermostability with more than 93% enzyme activity, even at a high temperature of 90℃. Furthermore, Ca²⁺ and Mg²⁺ promoted the activity of T26GAL while Zn²⁺ and Cu²⁺ inhibited it. The substrate specificity studies revealed that T26GAL efficiently degraded raffinose, stachyose, and guar gum, but not locust bean gum. This study thus facilitated the discovery of an effective heat-resistant α-galactosidase with potent industrial application. Meanwhile, as part of our research on lignocellulose degradation by a microbial consortium, the present work provides an important basis for encouraging further investigation into this enzyme complex.

• 한국미생물·생명공학회지
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• 제43권3호
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• pp.195-203
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• 2015

전통 발효된장으로부터 α-galactosidase를 분비 생산하는 두 종류 Bacillus licheniformis 균주로 YB-1413과 YB-1414를 분리하여 당 이용능을 비롯한 생화학적 특성 및 16S rRNA 염기서열과 중합효소 연쇄반응에 의한 임의증폭 DNA 다형성의 유전학적 특성을 비교한 결과 두 균주는 매우 유사하였지만 동일 균주는 아닌 것으로 확인되었다. 탄소원으로 wheat bran을 사용하였을 때는 두 균주의 α-galactosidase 생산성이 증가하였으나, melibiose, raffinose 또는 sucrose는 효소 생산성이 급격하게 감소하였다. 질소원으로 yeast extract가 첨가된 배지에서 효소 생산성이 높았으며, YB-1413은 1.87 U/ml, YB-1414는 1.69 U/ml의 효소 생산성을 보였다. 이들 균주가 생산하는 α-galactosidases는 pH 6.0과 45℃에서 모두 최대활성을 보였으며, 낮은 농도의 ribose와 galalctose에 의해서도 효소활성이 급격하게 저해되었다. 한편 이들 효소는 대두와 콩과식품에 존재하는 항영양인자인 raffinose와 starchyose를 완전히 가수분해 하였는데, 이로 보아 분리균 YB-1413과 YB-1414는 콩 발효식품과 대두 사료의 영양가를 개선하는데 활용할 가치가 있다고 여겨진다.

• Journal of Microbiology and Biotechnology
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• 제26권9호
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• pp.1650-1656
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• 2016

The SCO0284 gene of Streptomyces coelicolor A3(2) is predicted to encode an α-galactosidase (680 amino acids) belonging to glycoside hydrolase family 27. In this study, the SCO0284 coding region was cloned and overexpressed in Streptomyces lividans TK24. The mature form of SCO0284 (641 amino acids, 68 kDa) was purified from culture broth by gel filtration chromatography, with 83.3-fold purification and a yield of 11.2%. Purified SCO0284 showed strong activity against p-nitrophenyl-α-D-galactopyranoside, melibiose, raffinose, and stachyose, and no activity toward lactose, agar (galactan), and neoagarooligosaccharides, indicating that it is an α-galactosidase. Optimal enzyme activity was observed at 40℃ and pH 7.0. The addition of metal ions or EDTA did not affect the enzyme activity, indicating that no metal cofactor is required. The kinetic parameters V_max and K_m for p-nitrophenyl-α-D-galactopyranoside were 1.6 mg/ml (0.0053 M) and 71.4 U/mg, respectively. Thin-layer chromatography and mass spectrometry analysis of the hydrolyzed products of melibiose, raffinose, and stachyose showed perfect matches with the masses of the sodium adducts of the hydrolyzed products, galactose (M+Na, 203), melibiose (M+Na, 365), and raffinose (M+Na, 527), respectively, indicating that it specifically cleaves the α-1,6-glycosidic bond of the substrate, releasing the terminal D-galactose.

• 한국수산과학회지
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• 제47권5호
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• pp.516-521
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• 2014

A bacterium producing ${\alpha}$-galactosidase (${\alpha}$-$\small{D}$-galactoside galactohydrolase, EC 3.2.1.22) was isolated. The isolate, KM-1 was identified as Bacillus coagulans based on its 16S rRNA sequence, morphology, and biochemical properties. ${\alpha}$-Galactosidase activity was detected the culture supernatant of B. coagulans KM-1. The bacterium showed the maximum activity for hydrolyzing para-nitrophenyl-${\alpha}$-$\small{D}$-galactopyranoside (pNP-${\alpha}Gal$) at pH 6.0 and $50^{\circ}C$. It hydrolyzed oligomeric substrates such as melibiose, raffinose, and stachyose liberating a galactose residue, indicating that the B. coagulans KM-1 ${\alpha}$-galactosidase hydrolyzed ${\alpha}$-1,6 linkage. The results suggest that the decreased stachyose and raffinose contents in fermented soybean meal are due to the ${\alpha}$-galactosidase activity.

• 한국어병학회지
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• 제20권3호
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• pp.249-255
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• 2007

본 연구에서는 우리나라와 일본의 담수어에서 분리된 운동성 aeromonads 7균주와 American Type Culture Collection (ATCC)에서 분양받은Aeromonas hydrophila 4균주의 표현형적 특성을 API20E와 APIZYM 방법으로 평가하고, 7종류의 항생제에 대한 최소 성장 억제 농도 (minimum inhibitory concentrations; MICs) 를 측정하였다. API20E 시험 결과 시험한 모든 균주는 (n=7) 운동성 aeromonads로 동정되었다. API20E시험에서lysine decarboxylase와mannitol, rhamnose, amygdalin, arabinose를 포함한 4종류의 carbohydrates의 산 생성은 균주에 따라 다른 반응을 나타내었다. APIZYM 시험을 이용한 효소 활성능을 평가한 결과, 모든 시험된 균주가valine arylamidase, cystine arylamidase, α-chymotrypsin, α-galactosidase, β-glucuronidase, α-glucosidase, α-mannosidase, α-fucosidase반응에서 음성 반응을 나타내었으나, 비록 그 효소 활성의 강도에서 차이는 있었으나alkaline phosphatase, esterase-lipase, leucine arylamidase, β-galactosidase, N-acetyl-β-glucosaminidase 모든 균주에서 양성 반응이 나타났다. 최소 성장 억제농도를 시험한 결과, 시험된 모든 균주는 ampicillin sodium (MIC>100㎍/ml) 에 내성을 가지며 chloramphenicol (MIC≤1.6㎍/ml) 감수성을 나타내었다. 그러나 1998년 이후에 분리된 3균주 (AC9804, AC0202, GMA0361)는 tetracycline (MIC=50㎍/ml) 모두 저항성이 있었으며, AC9804는 oxolinic acid (MIC=12.5㎍/ml), GMA0361는 kanamycin sulfate (MIC>100㎍/ml)와 streptomycin sulfate (MIC>100㎍/ml)에도 저항성을 나타내었다.

• 한국식품위생안전성학회지
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• 제35권4호
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• pp.304-311
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• 2020

Galacto-oligosaccharides(GOS)는 프로바이오틱스 미생물의 성장을 증진시켜 인류 건강에 유익한 작용을 갖게 하는 프리바이오틱스이며 식품 산업에서 다양한 활용성을 갖는다. GOS는 보통 β-galactosidase에 의해 촉매 반응이 일어난 lactose로부터 생성된다. 한편, 세포 표면 발현은 살아있는 세포 표면의 펩타이드와 단백질을 세포의 기능성 성분에 융합시켜 발현시키는 것이다. 표층 발현 세포는 다양한 잠재적 이용가치를 갖는다. N 말단 부근에 위치하는 것으로 생각되는 Flo1p 응집 functional domain은 세포의 flocs로의 가역적인 응집을 유발하면서 α-mannan carbohydrates와 같은 세포벽 성분과 비공유결합을 한다. 한외여과한 유청을 농축, 분무건조한 유청막투과액(Whey Permeate, WP)을 이용하여 β-galactosidase 재조합 Pichia pastoris (P. pastoris) 로 표층 발현 처리 (surface engineering)하는 GOS의 합성법은 폐기물을 활용하는 새로운 효율적인 방법이라 할 수 있다.

• 농업과학연구
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• 제18권1호
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• pp.49-73
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• 1991

발아대두 $\alpha$-galactosidase와 Aspergillus niger가 생산하는 $\alpha$-galactosidase의 효소학적 성질을 비교하기 위하여 대두 발아 중의 $\alpha$-galactosidase활성 및 소당류의 함량변화를 측정하였고, 대두 발아 과정 중 활성이 가장 강할때에 추출한 $\alpha$-galactosidase 및 Aspergillus niger를 밀기울 배양하였을 때 생성되는 $\alpha$-galactosidase를 염석, 이온교환 크로마토그래피 및 겔여과 등을 사용하여 정제한 후 정제효소의 이화학적 및 효소학적 성질을 측정, 비교하여 다음과 같은 결과를 얻었다. 1. 대두 $\alpha$-galactosidase의 활성은 대두를 $25^{\circ}C$에서 120시간 발아시켰을 때 가장 높았으며, 대두 중의 raffinose는 96시간, stachyose는 120시간 발아시켰을 때 완전히 분해되었다. 2. Aspergillus niger를 밀기울배지에서 $30^{\circ}C$, 4일간 배양했을 때 $\alpha$-galactosidase 활성이 최고에 달하였다. 3. 대두 $\alpha$-galactosidase는 황산암모늄 염석, DEAE-Cellulose 및 DEAE-Sephadex A-50 이온교환 크로마토그래피, Sephadex G-l50 겔여과 등에 의하여 6.6배까지 정제되었으며 그의 비활성이 825U/mg protein, 수율 2.5%에 달하였고, Aspergillus niger의 $\alpha$ -galactosidase는 23.7배까지 정제되었으며 그의 비활성이 1,229U/mg protein, 수율 14%에 달하였다. 4. 정제된 대두 및 Aspergillus niger의 $\alpha$-galactosidase는 HPLC, PAGE 및 SDS-PAGE에 의해서 순도가 확인되었다. 5. 정제효소의 이화학적 성질 1) Aspergillus niger의 $\alpha$-galactosidase는 periodic acid schiff 염색에 의하여 당단백질임이 확인되었다. 2) 대두 $\alpha$-galactosidase의 등전점은 pH4.8이었고, 분자량이 30,000인 monomer이었으나, Aspergillus niger의 $\alpha$-galactosidase는 등전점이 pH4.6이었고 분자량은 112,000이었으며 분자량 28,000인 monomer 4개로 구성된 tetramer이었다. 3) 대두 및 Aspergillus niger $\alpha$-galactosidase의 활성에 관여하는 아미노산은 diethyl pyrocarbonate에 의한 화학 수식에 의하여 histidine임이 확인되었다. 4) 대두 $\alpha$-galactosidase의 활성은 2-mercaptoethanol과 L-cysteine에 의하여 약간 저해되었다. 6. 정제효소의 효소학적 성질 1) 대두 $\alpha$-galactosidase의 최적 작용 pH는 pH6.0, 최적 작용온도는 $40^{\circ}C$이었고, Aspergillus niger $\alpha$-galactosidase 각각 pH6.5 및 $40^{\circ}C$이었다. 2) 대두 및 Aspergillus niger의 $\alpha$-galactosidase는 $45^{\circ}C$이하에서 비교적 안정하였으나 $60^{\circ}C$에서 10분 처리시 대두 $\alpha$-galactosidase는 25%, Aspergillus niger $\alpha$-galactosidase는 46%의 잔존활성을 나타내었다. 3) 대두 $\alpha$-galactosidase는 pH5.5~6.5, Aspergillus niger의 $\alpha$-galactosidase는 pH6.0~7.0에서 매우 안정하였다. 4) 대두 및 Aspergillus niger의 $\alpha$-galactosidase간에 기질 특이성상의 차이가 없었으며, stachyose보다 raffinose를 잘 분해하였고, gaIactose는 양효소의 활성을 저해하였다. 5) 대두 $\alpha$-galactosidase의 P-nitrophenyl-$\alpha$-gaIactopyranoside, raffinose 및 stachyose에 대한 Km값은 각각 5.3mM, 50.0mM 및 55.5mM이 었고, Aspergillus niger $\alpha$-galactosidase에 있어서는 각각 5.0mM, 37.0mM 및 55.5mM이었다. 6) 대두 $\alpha$-galactosidase의 p-nitrophenyl-$\alpha$-d-gaIactopyranoside에 대한 활성화 에너지는 13.024Kcal/mole, $Q_{10}$값은 2.0이 었으며, Aspergillus niger의 $\alpha$-galactosidase는 각각 8.515Kcal/mole 및 1.38이었다.

• 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 $Fe^{2+}$.

• Asian-Australasian Journal of Animal Sciences
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• 제27권5호
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• pp.700-705
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• 2014

A 12-week feeding trial was conducted with 87 g rainbow trout to evaluate the effects on growth performances, feed efficiency and nutrient digestibility of adding ${\beta}$-mannanase and ${\alpha}$-galactosidase enzymes, solely or in combination. Seven diets were prepared by adding ${\beta}$-mannanase, ${\alpha}$-galactosidase and mixed enzyme at two different levels (1 g/kg and 2 g/kg) to control diet (without enzyme) including soybean meal. Mixed enzymes (1 g/kg, 2 g/kg) were prepared by adding ${\beta}$-mannanase and ${\alpha}$-galactosidase at the same doses (0.5+0.5 g/kg and 1+1 g/kg). At the end of the experiment, addition of ${\beta}$-mannanase, ${\alpha}$-galactosidase and mixed enzyme to diet containing 44% soybean meal had no significant effects on growth performance and gain:feed (p>0.05). In addition, adding ${\beta}$-mannanase, ${\alpha}$-galactosidase and mixed enzyme in different rations to trout diets had no affect on nutrient digestibility and body composition (p>0.05).

• Journal of Microbiology and Biotechnology
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• 제30권11호
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• pp.1659-1669
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• 2020

1,3-α-3,6-anhydro-L-galactosidase (α-neoagarooligosaccharide hydrolase) catalyzes the last step of agar degradation by hydrolyzing neoagarobiose into monomers, D-galactose, and 3,6-anhydro-L-galactose, which is important for the bioindustrial application of algal biomass. Ahg943, from the agarolytic marine bacterium Gayadomonas joobiniege G7, is composed of 423 amino acids (47.96 kDa), including a 22-amino acid signal peptide. It was found to have 67% identity with the α-neoagarooligosaccharide hydrolase ZgAhgA, from Zobellia galactanivorans, but low identity (< 40%) with the other α-neoagarooligosaccharide hydrolases reported. The recombinant Ahg943 (rAhg943, 47.89 kDa), purified from Escherichia coli, was estimated to be a monomer upon gel filtration chromatography, making it quite distinct from other α-neoagarooligosaccharide hydrolases. The rAhg943 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into D-galactose, neoagarotriose, and neoagaropentaose, respectively, with a common product, 3,6-anhydro-L-galactose, indicating that it is an exo-acting α-neoagarooligosaccharide hydrolase that releases 3,6-anhydro-L-galactose by hydrolyzing α-1,3 glycosidic bonds from the nonreducing ends of neoagarooligosaccharides. The optimum pH and temperature of Ahg943 activity were 6.0 and 20℃, respectively. In particular, rAhg943 could maintain enzyme activity at 10℃ (71% of the maximum). Complete inhibition of rAhg943 activity by 0.5 mM EDTA was restored and even, remarkably, enhanced by Ca²⁺ ions. rAhg943 activity was at maximum at 0.5 M NaCl and maintained above 73% of the maximum at 3M NaCl. K_m and V_max of rAhg943 toward neoagarobiose were 9.7 mg/ml and 250 μM/min (3 U/mg), respectively. Therefore, Ahg943 is a unique α-neoagarooligosaccharide hydrolase that has cold- and high-salt-adapted features, and possibly exists as a monomer.