• Animal Bioscience
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• 제34권5호
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• pp.867-879
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• 2021

Objective: Fibronectin 3 (FN3) and immunoglobulin like modules (Ig) are usually collocated beside modular cellulase catalytic domains. However, very few researches have investigated the role of these modules. In a previous study, we have sequenced and analyzed bacterial metagenomic DNA in Vietnamese goats' rumen and found that cellulase-producing bacteria and cellulase families were dominant. In this study, the properties of modular cellulases and the role of a FN3 in unique endoglucanase belonging to glycosyl hydorlase (GH) family 5 were determined. Methods: Based on Pfam analysis, the cellulases sequences containing FN3, Ig modules were extracted from 297 complete open reading frames (ORFs). The alkaline, thermostability, tertiary structure of deduced enzymes were predicted by AcalPred, TBI software, Phyre2 and Swiss models. Then, whole and truncated forms of a selected gene were expressed in Escherichia coli and purified by His-tag affinity column for assessment of FN3 ability to enhance enzyme activity, solubility and conformation. Results: From 297 complete ORFs coding for cellulases, 148 sequences containing FN3, Ig were identified. Mostly FN3 appeared in 90.9% beta-glucosidases belonging to glycosyl hydrolase family 3 (GH3) and situated downstream of catalytic domains. The Ig was found upstream of 100% endoglucanase GH9. Rarely FN3 was seen to be situated downstream of X domain and upstream of catalytic domain endoglucanase GH5. Whole enzyme (called XFN3GH5 based on modular structure) and truncate forms FN3, XFN3, FN3GH5, GH5 were cloned in pET22b (+) and pET22SUMO to be expressed in single and fusion forms with a small ubiquitin-related modifier partner (S). The FN3, SFN3 increased GH5 solubility in FN3GH5, SFN3GH5. The SFN3 partly served for GH5 conformation in SFN3GH5, increased modules interaction and enzyme-soluble substrate affinity to enhance SXFN3GH5, SFN3GH5 activities in mixtures. Both SFN3 and SXFN3 did not anchor enzyme on filter paper but exfoliate and separate cellulose chains on filter paper for enzyme hydrolysis. Conclusion: Based on these findings, the presence of FN3 module in certain cellulases was confirmed and it assisted for enzyme conformation and activity in both soluble and insoluble substrate.

• Korean Chemical Engineering Research
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• 제52권3호
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• pp.302-306
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• 2014

목질계 분해효소 활성 증대를 위해 밀짚을 이용한 고체발효에서 주요 발효인자의 최적화를 수행하였다. Trichoderma reesei와 Aspergillus niger를 이용한 혼합배양에서 고체발효에 주요한 영향을 미친다고 알려진 배양온도, pH, 수분함량과 고체기질 크기를 순차적 최적화를 진행하였다. 실험에 적용 된 인자 모두 목질계 분해효소 활성에 유의한 효과를 주었으며, 발효온도 $40^{\circ}C$, pH 7, 수분함량 75%와 고체기질 크기 0.25~0.5 mm가 목질계 분해효소 생산을 위한 최적 조건임을 알 수 있었다. 최적조건 하에서 밀짚을 이용한 고체발효를 수행하였을 때, 효소활성 기준 cellulase 10.3 IU, endoglucanase 100.3 IU, ${\beta}$-glucosidase 22.9 IU와 xylanase 2261.7 IU/g dry material을 배양 96시간에 확인할 수 있었다. 본 결과는 기존 효소활성 대비 각각 72.6, 48.7, 55.2와 51.9% 증가한 수치로 혼합배양과 순차적 최적화를 적용하여 효과적인 목질계 분해효소 활성 증대가 가능함을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제17권5호
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• pp.800-805
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• 2007

Two endoglucanases with processive cellulase activities, produced from Fomitopsis palustris grown on 2% microcrystalline cellulose(Avicel), were purified to homogeneity by anion-exchange and gel filtration column chromatography systems. SDS-PAGE analysis indicated that the molecular masses of the purified enzymes were 47 kDa and 35 kDa, respectively. The amino acid sequence analysis of the 47-kDa protein(EG47) showed a sequence similarity with fungal glycoside hydrolase family 5 endoglucanase from the white-rot fungus Phanerochaete chrysosporium. N-terminal and internal amino acid sequences of the 35-kDa protein(EG35), however, had no homology with any other glycosylhydrolases, although the enzyme had high specific activity against carboxymethyl cellulose, which is a typical substrate for endoglucanases. The initial rate of Avicel hydrolysis by EG35 was relatively fast for 48 h, and the amount of soluble reducing sugar released after 96 h was $100{\mu}g/ml$. Although EG47 also hydrolyzed Avicel, the hydrolysis rate was lower than that of EG35. Thin layer chromatography analysis of the hydrolysis products released from Avicel indicated that the main product was cellobiose, suggesting that the brown-rot fungus possesses processive EGs capable of degrading crystalline cellulose.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2016년도 춘계학술대회 및 임시총회
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• pp.11-11
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• 2016

Penicillium species are commonly isolated from various outdoor and indoor environments, including marine environments such as sponges, algae and sand. Penicillium is especially important because numerous bioactive compounds have been isolated. Penicillium was the most common species in intertidal zone in Korea, however the diversity and ecological roles of Penicillium in intertidal zone are not clarified. We explored diversity and ecological roles of marine-derived Penicillium from tidal flat and sea sand in Korea. The diversity of marine-derived Penicillium from Korea was investigated using both culture-dependent and culture-independent approach by ${\beta}$-tubulin sequence. In addition, we evaluated optimal temperature, halo-tolerance, and enzyme activity of Penicillium strains, such as extracellular alginase, endoglucanase, ${\beta}$-glucosidase, and protease. For culture-dependent approach, a total of 182 strains of 62 Penicillium species were isolated, with 53 species being identified. The most common species was Penicillium oxalicum, followed by P. crustosum, P. brasilianum, P. koreense, and P. griseofulvum. Species richness and composition were not significantly different by season, substrates, and seaside. For culture-independent approach using Illumina sequencing, 73 OTUSs were detected. The most frequently observed species was P. antarcticum, followed by P. koreense, P. crustosum, and P. brevicompactum. Diversity of Penicillium was higher during winter season than during summer season and in western sea than in southern sea, respectively. Community structure was significantly different by season and sea side. 52 species were detected by both methods. Unique species were isolated from each of methods - 10 from culture methods and 21 from Illumina sequencing. Furthermore, salinity adaption of the Penicillium varied depending on species. Many Penicillium species showed endoglucanase, ${\beta}$-glucosidase, and protease activity. Some species including P. paneum and P. javanicum degraded the polycyclic aromatic hydrocarbons. Thus, our results demonstrate that intertidal zone in Korea harbors diverse Penicillium community and marine-derived Penicillium play important ecological roles as decomposers of organic material in marine environments.

• 미생물학회지
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• 제51권1호
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• pp.68-74
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• 2015

Thermococcus pacificus P-4의 유전체 서열 분석을 통하여 예측되는 GH1 ${\beta}$-glucosidase를 암호화하는 유전자를 동정하였다. 그 유전자는 487 아미노산들을 암호화하는 1,464 bp 나타내었으며, 그 아미노산 서열은 Pyrococcus furiosus ${\beta}$-glucosidase와 77% 상동성을 나타내었다. 그 유전자는 Escherichia coli 시스템 내에서 복제 및 발현하였다. 재조합 된 단백질은 금속 친화크로마토그래피를 통하여 정제하고 특성을 분석하였다. 정제된 단백질(Tpa-Glu)은 pH 7.5와 $75^{\circ}C$에서 최적활성을 나타내었으며, 열안정성은 $90^{\circ}C$에서 약 6시간의 반감기를 보였다. Tpa-Glu는 pNP-${\beta}$-glucopyranoside, pNP-${\beta}$-galactopyranoside, pNP-${\beta}$-mannopyranoside, 그리고 pNP-${\beta}$-xylopyranoside 순으로 우수한 $k_{cat}/K_m$ 값을 나타내었다. 또한, Tpa-Glu는 ${\beta}$-1,3-linked polysaccharide (laminarin) 그리고 ${\beta}$-1,3-와 ${\beta}$-1,4-linked oligosaccharides에 대하여 exo-hydrolyzing 활성을 보였다. 본 연구는 초고온 고세균으로터 ${\beta}$-glucosidase가 exohydrolyzing 활성을 처음 확인한 것으로 이 효소는 laminarin의 당화공정에 ${\beta}$-1,3-endoglucanase와 함께 적용할 수 있을 것으로 기대된다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.202-205
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• 2000

A gene coding $endo-{\beta}$,-1, 4 glucanase from Actinomyces sp. KNG40 and phytase from Hansenula polymorpha were cloned into Esherichia coli JM101 by using E. coli/Lactobacillus shuttle vector pNZ3004 and pNZ123. The plasmid p3PS(1-4) and p123(1-4) have slpA promoter and slpA signal sequence. So, I constructed expression vectors, p3PS(1-4)Endo, phy and p123(1-4)Endo, phy. These constructed vector was transformed in target host Lactobacillus gasseri and reutri. These transformed host expressed endoglucanase and phytase as extracellular fraction. In the enzyme activity of the same vector, host L, gasseri was higher activity than L. reuteri. This indicates that L. gasseri recongnize promoter and signal sequence very well.

• BMB Reports
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• 제47권5호
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• pp.256-261
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• 2014

To investigate the function of N-glycosylation of Cel5A (endoglucanase II) from Hypocrea jecorina, two N-glycosylation site deletion Cel5A mutants (rN124D and rN124H) were expressed in Saccharomyces cerevisiae. The weights of these recombinant mutants were 54 kDa, which were lower than that of rCel5A. This result was expected to be attributed to deglycosylation. The enzyme activity of rN124H was greatly reduced to 60.6% compared with rCel5A, whereas rN124D showed slightly lower activity (10%) than that of rCel5A. rN124D and rN124H showed different thermal stabilities compared with the glycosylated rCel5A, especially at lower pH value. Thermal stabilities were reduced and improved for rN124D and rN124H, respectively. Circular dichroism spectroscopy showed that the modification of secondary structure by mutation may be the reason for the change in enzymatic activity and thermal stability.

• Journal of Microbiology and Biotechnology
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• 제3권2호
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• pp.86-90
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• 1993

CMCase positive clones were screened from Cellulomonas sp. YE-5 and named pCE1, pCE2 and pCE3. Among the positive clones pCE1 was used for this study, because it has the smallest insert and the highest CMCase activity among the 3 clones, and its nucleotide sequence was determined. The CMCase gene in pCE1 was composed of 1071 bp of nucleotides coding 357 amino acids. Computer analysis showed that the pCE1 has 65% sequence homology with the endoglucanase from Cellulomonas fimi.

• Journal of Animal Science and Technology
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• 제57권7호
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• pp.23.1-23.6
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• 2015

Cellulose degrading bacteria from koala faeces were isolated using caboxymethylcellulose-Congo red agar, screened in vitro for different hydrolytic enzyme activities and phylogenetically characterized using molecular tools. Bacillus sp. and Pseudomonas sp. were the most prominent bacteria from koala faeces. The isolates demonstrated good xylanase, amylase, lipase, protease, tannase and lignin peroxidase activities apart from endoglucanase activity. Furthermore many isolates grew in the presence of phenanthrene, indicating their probable application for bioremediation. Potential isolates can be exploited further for industrial enzyme production or in bioremediation of contaminated sites.

• 한국신재생에너지학회:학술대회논문집
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• 한국신재생에너지학회 2008년도 춘계학술대회 논문집
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• pp.225-228
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• 2008

This study shows that lignocellulosic biomass saccharification work has been carried out with rice-straw by the extracellular enzyme from KMU001, and the enzymes produced in 5%(w/v) wood biomass were characterized by protein and various enzyme activity measurements. Several cellulases such as Endoglucanase(EG), $\beta$-D-1,4-Glucosidase(BGL), Cellobiohydrolase(CBH), and $\beta$-D-1,4-Xylanase (BXL) were detected. Saccharification of rice-straw by the enzyme yielded about 233mg/g of glucose after 48hrs.