• 한국미생물·생명공학회지
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• 제47권1호
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• pp.69-77
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• 2019

Screening microorganisms that can produce glucan hydrolases for industrial, environmental, and biomedical applications is important. Herein, we describe a novel approach to perform glucan hydrolase screening-based on analysis of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) spectra-which involves degradation of the oligo- and polysaccharides. As a proof-of-concept study, glucan hydrolases that could break down glucans made of several glucose units were used to demonstrate the MALDI-MS-based enzyme assay. First, the enzyme activities of ${\alpha}$-amylase and cellulase on a mixture of glucan oligosaccharides were successfully discriminated, where changes of the MALDI-MS profiles directly reflected the glucan hydrolase activities. Next, we validated that this MALDI-MS-based enzyme assay could be applied to glucan polysaccharides (i.e., pullulan, lichenan, and schizophyllan). Finally, the bacterial glucan hydrolase activities were screened on 96-well plate-based platforms, using cell lysates or samples of secreted enzyme. Our results demonstrated that the MALDI-MS-based enzyme assay system would be useful for investigating bacterial glucoside hydrolases in a high-throughput manner.

• Journal of Microbiology and Biotechnology
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• 제24권12호
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• pp.1699-1706
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• 2014

A lichenase gene (mt-lic) was identified for the first time through function-based screening of a soil metagenomic library. Its deduced amino acid sequence exhibited a high degree of homology with endo-${\beta}$-1,3-1,4-glucanase (having both lichenase and chitosanase activities), encoded by the bgc gene of Bacillus circulans WL-12. The recombinant lichenase overexpressed and purified from Escherichia coli was able to efficiently hydrolyze both barley ${\beta}$-glucan and lichenan. The enzyme showed maximal activity at a pH of 6.0 at $50^{\circ}C$, with Azo-barley-glucan as the substrate. The metal ions $Mn^{2+}$, $Mg^{2+}$, $Ca^{2+}$, and $Fe^{2+}$ enhanced the enzymatic activity, whereas the $Cu^{2+}$ and $Zn^{2+}$ ions inhibited the enzymatic activity. The $K_m$ and $V_{max}$ values of the purified lichenase were determined to be 0.45 mg/ml and 24.83 U/min/mg of protein, respectively.

• Journal of Microbiology and Biotechnology
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• 제21권10호
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• pp.1012-1020
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• 2011

A gene coding for an endoglucanase (EglA), of the glycosyl hydrolase family 12 and derived from Aspergillus niger VTCC-F021, was cloned and sequenced. The cDNA sequence, 717 bp, and its putative endoglucanase, a 238 aa protein with a predicted molecular mass of 26 kDa and a pI of 4.35, exhibited 98.3-98.7% and 98.3-98.6% identities, respectively, with cDNA sequences and their corresponding endoglucanases from Aspergillus niger strains from the GenBank. The cDNA was overexpressed in Pichia pastoris GS115 under the control of an AOX1 promoter with a level of 1.59 U/ml culture supernatant, after 72 h of growth in a YP medium induced with 1% (v/v) of methanol. The molecular mass of the purified EglA, determined by SDS-PAGE, was 33 kDa, with a specific activity of 100.16 and 19.91 U/mg toward 1% (w/v) of ${\beta}$-glucan and CMC, respectively. Optimal enzymatic activity was noted at a temperature of $55^{\circ}C$ and a pH of 5. The recombinant EglA (rEglA) was stable over a temperature range of $30-37^{\circ}C$ and at pH range of 3.5-4.5. Metal ions, detergents, and solvents tested indicated a slightly inhibitory effect on rEglA activity. Kinetic constants ($K_m$, $V_{max}$, $k_{cat}$, and $k_{cat}/K_m$) determined for rEglA with ${\beta}$-glucan as a substrate were 4.04 mg/ml, 102.04 U/mg, 2,040.82 $min^{-1}$, and 505.05, whereas they were 10.17 mg/ml, 28.99 U/mg, 571.71 $min^{-1}$, and 57.01 with CMC as a substrate, respectively. The results thus indicate that the rEglA obtained in this study is highly specific toward ${\beta}$-glucan. The biochemical properties of rEglA make it highly valuable for downstream biotechnological applications, including potential use as a feed enzyme.

• 유기물자원화
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• 제16권4호
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• pp.64-73
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• 2008

glysosyl hydrolase의 하나인 CCF 유사 유전자를 지렁이 Eisenia anderi의 중장으로부터 분리하여 클로닝하였다. 이 유전자의 전체 염기서열 크기는 1,152bp로 나타났으며 개시코돈을 포함하여 384개의 아미노산을 인코딩한다. N-말단 지역의 17개 잔기들은 signal peptide이다. CCF 관련 유전자의 아미노산 서열을 분석한 결과 본 연구에서 분리한 CCF 유사 유전자는 glycosyl hydrolase family 16 (GHF16)에 속하며, 다른 종의 지렁이에서 밝혀진 CCF 및 CCF-유사 단백질과 79~99%의 높은 상동성을 보였다. 여러 지렁이 종에서 분리된 CCF 및 CCF-유사 단백질들은 가수분해활성에 중요한 polysacchride-binding motif와 glucanase motif가 100% 상동성을 나타냈다. 본 연구의 대상종과 유사종인 E. fetida에서 분리된 CCF는 이 종의 서식지가 미생물 활성이 높은 부패 유기질층이기 때문에 다른 종의 CCF에 비해 높은 기질인식 특이성을 갖고 있는 것으로 알려져 있다. 이러한 사실은 본 연구에서 분리한 CCF도 넓은 기질 특이성을 갖고 있을 가능성을 제시해 주고 있으며 이는 산업적 응용 측면에서 유용한 특징 중의 하나라고 생각된다. BLASTX를 이용한 계통수 분석 결과 GHF16 효소는 크게 후생동물군, 녹색식물군, 진정세균군 등의 세 그룹으로 나눌 수 있었으며, 후생동물군의 GHF16은 다시 촉수담륜동물형와 탈피동물형(후생동물형 포함)으로 나뉘어졌다. 본 연구에 의해 E. andrei로부터 분리된 CCF-유사 단백질의 더 많은 생물학적 특성 연구를 통해 ${\beta}$-D-글루칸의 분해 및 생산을 조절하는 산업적 활용이 가능할 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1295-1302
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• 2004

A gene encoding the mannanase of Bacillus subtilis WL-7, which had been isolated from Korean soybean paste, was cloned into Escherichia coli, and the gene product was purified from the culture filtrate of the recombinant E. coli. This mannanase gene, designated manA, consisted of 1,086 nucleotides, encoding a polypeptide of 362 amino acid residues. The deduced amino acid sequence was highly homologous to those of mannanases belonging to the glycosyl hydrolase family 26. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. The enzyme had a pH optimum at 6.0 and a temperature optimum at $55^{\circ}C$. The enzyme was active on locust bean gum, konjak, guar gum, and lichenan, while it did not exhibit activity towards yeast mannan, laminarin, carboxymethylcellulose, $\beta$­glucan, xylan, and para-nitrophenyl-$\beta$-mannopyranoside.

• 한국식품영양학회지
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• 제26권1호
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• pp.35-43
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• 2013

소화성 질환의 중요 인자인 Helicobacter pylori를 저해하는 IgY, 목이버섯, 김치와 타락 유산균을 이용하여 생육 저해 상승 효과를 분석하고자 하였다. 동결 건조된 유산균 배양 상징액을 다양한 효소 처리 결과, 지질 분해 효소를 제외하고는 활성을 나타냈다. GC 분석을 통해 유산균 동결건조액의 지방산 조성은 undecanoic acid($C_{11:0}$), palmitic acid($C_{16:0}$), steraic acid($C_{18:0}$), oleic acid($C_{18:1}$)가 L. mensenteroides LABKW5와 S. thermophilus LAB KW15에서 모두 확인되었으며, eicosadienoic acid($C_{20:2}$)는 LAB KW5에서만 나타났다. 또한 유산균 동결건조액은 다른 식중독균에서도 spot assay의 결과, 그람 음성균 중에서 특히 E. coli O157:H7, E. coli, C. sakazakii 등에서 생육 저해능이 확인되었다. 목이버섯 추출물은 열수 추출과 ethanol를 이용해 분리하였으며, HPLC를 이용하여 목이버섯 추출 다당체를 분석한 결과, glucurono-xylomannan 혹은 glucomannan이 ${\beta}$-glucan과 함께 존재하는 혼합물일 것으로 되었다. 또한, 면역란은 1차 접종 후 11일째부터 주마다 2회씩 회수하여 IgY를 분리, 정제하였다. 실험을 통해 동결 건조된 유산균 배양 상징액, IgY, 목이버섯 추출액을 혼합 배양하여 배양시간에 따른 생육 저해력의 변화를 확인한 결과, 유산균에 의해 H. pylori의 저해 효과가 있었으며, IgY와 목이버섯의 혼합 시 추가 억제 효과가 있는 것으로 나타났다. 그러므로 동결 건조된 유산균 배양 상징액, IgY, 목이버섯 추출액의 복합처리는 H. pylori를 제어하는데 효과적인 것으로 보인다.

• Journal of Microbiology and Biotechnology
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• 제18권9호
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• pp.1555-1563
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• 2008

A novel $\alpha$-amylase ($\alpha$-1,4-$\alpha$-D-glucan glucanohydrolase, E.C. 3.2.1.1), ScAmy43, was found in the culture medium of the phytopathogenic fungus Sclerotinia sclerotiorum grown on oats flour. Purified to homogeneity, ScAmy43 appeared as a 43 kDa monomeric enzyme, as estimated by SDS-PAGE and Superdex 75 gel filtration. The MALDI peptide mass fingerprint of ScAmy43 tryptic digest as well as internal sequence analyses indicate that the enzyme has an original primary structure when compared with other fungal a-amylases. However, the sequence of the 12 N-terminal residues is homologous with those of Aspergillus awamori and Aspergillus kawachii amylases, suggesting that the new enzyme belongs to the same GH13 glycosyl hydrolase family. Assayed with soluble starch as substrate, this enzyme displayed optimal activity at pH 4 and $55^{\circ}C$ with an apparent $K_m$ value of 1.66 mg/ml and $V_{max}$ of 0.1${\mu}mol$glucose $min^{-1}$ $ml^{-1}$. ScAmy43 activity was strongly inhibited by $Cu^{2+}$, $Mn^{2+}$, and $Ba^{2+}$, moderately by $Fe^{2+}$, and was only weakly affected by $Ca^{2+}$ addition. However, since EDTA and EGTA did not inhibit ScAmy43 activity, this enzyme is probably not a metalloprotein. DTT and $\beta$-mercaptoethanol strongly increased the enzyme activity. Starting with soluble starch as substrate, the end products were mainly maltotriose, suggesting for this enzyme an endo action.

• Journal of Microbiology and Biotechnology
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• 제31권10호
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• pp.1446-1454
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• 2021

Herein, we cloned and expressed an endo-β-1,4-glucanase gene (celA1805) from Bacillus subtilis B111 in Escherichia coli. The recombinant celA1805 contains a glycosyl hydrolase (GH) family 8 domain and shared 76.8% identity with endo-1,4-β-glucanase from Bacillus sp. KSM-330. Results showed that the optimal pH and temperature of celA1805 were 6.0 and 50℃, respectively, and it was stable at pH 3-9 and temperature ≤50℃. Metal ions slightly affected enzyme activity, but chemical agents generally inhibited enzyme activity. Moreover, celA1805 showed a wide substrate specificity to CMC, barley β-glucan, lichenin, chitosan, PASC and avicel. The K_m and V_max values of celA1805 were 1.78 mg/ml and 50.09 µmol/min/mg. When incubated with cellooligosaccharides ranging from cellotriose to cellopentose, celA1805 mainly hydrolyzed cellotetrose (G4) and cellopentose (G5) to cellose (G2) and cellotriose (G3), but hardly hydrolyzed cellotriose. The concentrations of reducing sugars saccharified by celA1805 from wheat straw, rape straw, rice straw, peanut straw, and corn straw were increased by 0.21, 0.51, 0.26, 0.36, and 0.66 mg/ml, respectively. The results obtained in this study suggest potential applications of celA1805 in biomass saccharification.

• Journal of Microbiology and Biotechnology
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• 제28권12호
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• pp.2036-2045
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• 2018

An endo-${\beta}$-1,4-glucanase gene, cel5L, was cloned using the shot-gun method from Bacillus sp.. The gene, which contained a predicted signal peptide, encoded a protein of 496 amino acid residues, and the molecular mass of the mature Cel5L was estimated to be 51.8 kDa. Cel5L contained a catalytic domain of glycoside hydrolase (GH) family 5 and a carbohydrate-binding module family 3 (CBM_3). Chromatography using HiTrap Q and CHT-II resulted in the isolation of two truncated forms corresponding to 50 (Cel5L-p50) and 35 kDa (Cel5L-p35, CBM_3-deleted form). Both enzymes were optimally active at pH 4.5 and $55^{\circ}C$, but had different half-lives of 4.0 and 22.8 min, respectively, at $70^{\circ}C$. The relative activities of Cel5L-p50 and Cel5L-p35 for barley ${\beta}$-glucan were 377.0 and 246.7%, respectively, compared to those for carboxymethyl-cellulose. The affinity and hydrolysis rate of pNPC by Cel5L-p35 were 1.7 and 3.3 times higher, respectively, than those by Cel5L-p50. Additions of each to a commercial enzyme set increased saccharification of pretreated rice straw powder by 17.5 and 21.0%, respectively. These results suggest CBM_3 is significantly contributing to thermostability, and to affinity and substrate specificity for small substrates, and that these two enzymes could be used as additives to enhance enzymatic saccharification.