• Journal of Microbiology and Biotechnology
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• 제26권5호
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• pp.946-952
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• 2016

The eglS gene in Bacillus amyloliquefaciens encodes an endo-β-1,4-glucanase that belongs to glycosyl hydrolase family 5. In this study, a disruption mutant of gene eglS was constructed to examine its role in bacterial adaptation in plants. The mutant TB2_k, eglS gene inactivated bacterial strain, was remarkably impaired in extracellular cellulase activity. When inoculated on Brassica campestris, the TB2_k population was reduced by more than 60% compared with the wild-type strain in the root, stem, and leaf tissues. Overexpression of eglS in the wild-type strain increased the bacteria population in the plant tissues. Further studies revealed that the transcription level of eglS was correlated with bacterial population. These data demonstrate that endo-β-1,4-glucanase of B. amyloliquefaciens is required for its optimal endophytic colonization.

• 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.

• 유기물자원화
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• 제15권3호
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• pp.80-89
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• 2007

셀룰로오스 가수분해효소의 하나인 endo-${\beta}$-1,4-D-glucanase의 유전자를 지렁이 Eisenia anderi의 중장으로부터 클로닝하여 EaEG2와 EaEG3로 명명하였다. 두 유전자의 염기는 1368bp이며, 개시코돈을 포함하여 456개의 아미노산을 코딩한다. N-말단 지역의 20개 잔기들은 signal peptide이다. 두 유전자의 전체 아미노산 염기서열은 glycosyl hydrolase family 9에 속하며, 같은 종류의 셀룰로오스 가수분해효소를 분비하는 흰개미, 바퀴벌레, 가재 그리고 연체동물과 51-55%의 높은 상동성을 보였다. 지렁이의 EaEG2와 EaEG3는 가수분해활성에 중요한 세 부분이 잘 보존되어 있다. 아미노산 염기서열을 이용한 계통수 분석에서 GHF9 그룹은 절지동물, 박테리아, 식물, 환형동물 및 연체동물의 5개 그룹으로 분석되었다.

• 생명과학회지
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• 제22권2호
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• pp.142-147
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• 2012

효모 Saccharomyces diastaticus가 포자형성기에 세포질에서 생산된다고 알려진 포자형성 특이 glucoamylase (SGA)가 세포 외로 분비되는 단백질임을 증명하고자 S. dastaticus의 SGA promoter와 예상되는 분비신호서열 다음에 reporter gene으로 사용한 고초균의 CMCase 구조유전자를 융합한 재조합 플라스미드 pYSC25를 제작하고 수주세포인 S. diastaticus YIY345에 형질전환 하였다. 형질전환체를 1% CMC를 포함하는 최소한천배지에서 배양한 후 Congo red 염료로 염색하여 생성된 투명환으로부터 SGA의 분비서열에 의해 세균의 CMCase가 효모세포외로 분비되는 것을 확인하였다. 효모세포부위 별 CMCase의 활성분포를 측정하여 SGA 분비서열의 분비효율을 추정하기 위해 효모세포 배양액을 배양상등액, periplasmic 및 세포질 분획으로 나눈 다음 효소활성을 측정한 결과 CMCase 활성의 76%가 배양상등액과 periplasmic 부위에 존재하였으며 N-연결형 당쇄가 일어났으므로 SGA 분비서열은 효과적으로 작용함을 알 수 있었다. 대조균인 고초균에서 생산된 CMCase에서는 당쇄가 일어나지 않은 것을 확인하였다. 이상의 결과로부터 SGA는 아미노 말단에 존재하는, 24개의 아미노산으로 구성된 분비서열을 보유한 분비성 단백질임을 확인하였다.

• 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.

• Journal of Microbiology and Biotechnology
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• 제28권4호
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• pp.588-596
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• 2018

An endo-${\beta}$-1,4-glucanase gene, cel9K, was cloned using the shot-gun method from Paenibacillus sp. X4, which was isolated from alpine soil. The gene was 2,994 bp in length, encoding a protein of 997 amino acid residues with a predicted signal peptide composed of 32 amino acid residues. Cel9K was a multimodular enzyme, and the molecular mass and theoretical pI of the mature Cel9K were 103.5 kDa and 4.81, respectively. Cel9K contains the GGxxDAGD, PHHR, GAxxGG, YxDDI, and EVxxDYN motifs found in most glycoside hydrolase family 9 (GH9) members. The protein sequence showed the highest similarity (88%) with the cellulase of Bacillus sp. BP23 in comparison with the enzymes with reported properties. The enzyme was purified by chromatography using HiTrap Q, CHT-II, and HiTrap Butyl HP. Using SDS-PAGE/activity staining, the molecular mass of Cel9K was estimated to be 93 kDa, which is a truncated form produced by the proteolytic cleavage of its C-terminus. Cel9K was optimally active at pH 5.5 and $50^{\circ}C$ and showed a half-life of 59.2 min at $50^{\circ}C$. The CMCase activity was increased to more than 150% in the presence of 2 mM $Na^+$, $K^+$, and $Ba^{2+}$, but decreased significantly to less than 50% by $Mn^{2+}$ and $Co^{2+}$. The addition of Cel9K to a commercial enzyme set (Celluclast 1.5L + Novozym 188) increased the saccharification of the pretreated reed and rice straw powders by 30.4% and 15.9%, respectively. The results suggest that Cel9K can be used to enhance the enzymatic conversion of lignocellulosic biomass to reducing sugars as an additive.

• Journal of the Korean Wood Science and Technology
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• 제46권6호
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• pp.670-680
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• 2018

Acanthophysium sp. KMF001, a wood rotting fungus, produces a strong crude enzyme complex that efficiently produces simple sugars from wood. The transcriptomic analysis of Acanthophysium sp. KMF001 identified 14 genes for putative glycoside hydrolases. Among them, isotig01043 was expressed heterogeneously in Escherichia coli BL21(DE3), and the expressed protein exhibited an endo-${\beta}$-1,4-xylanase activity which showed the optimum reaction at pH 5.0 and $30^{\circ}C$. The enzyme kinetic values of $K_m$ and $V_{max}$ were 25.92 mg/ml and $0.628{\mu}mole/mg/ml$, respectively. The enzymatic characteristics of the expressed xylanase showed a typical fungal xylanase. However, the bioinformatics analysis suggested that the protein encoded by isotig01043 was a novel xylanase based on a low identity when it was compared with the closest protein in the NCBI database and a similar protein domain with GH16_fungal_Lam16A_glucanase, which had not been earlier suggested as a xylanase.