• Animal Bioscience
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• v.34 no.5
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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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• v.52 no.3
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• pp.302-306
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• 2014

The present study was carried out to optimize fermentation parameters for the production of cellulolytic enzymes through solid substrate fermentation of Trichoderma reesei and Aspergillus niger grown on wheat straw. A sequential optimization based on one-factor-at-a-time method was applied to optimize fermentation parameters including temperature, pH, moisture content and particle size. The results of optimization indicated that $40^{\circ}C$, pH 7, moisture content 75% and particle size between 0.25~0.5 mm were found to be the optimum condition at 96 hr fermentation. Under the optimal condition, co-culture of T. reesei and A. niger produced cellulase activities of 10.3 IU, endoglucanase activity of 100.3 IU, ${\beta}$-glucosidase activity of 22.9 IU and xylanase activity of 2261.7 IU/g dry material were obtained. Cellulolytic enzyme production with optimization showed about 72.6, 48.8, 55.2 and 51.9% increase compared to those obtained from control experiment, respectively.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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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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• 2016.05a
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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.

• Korean Journal of Microbiology
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• v.51 no.1
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• pp.68-74
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• 2015

Based on the genomic analysis of Thermococcus pacificus P-4, we identified a putative GH1 ${\beta}$-glucosidase-encoding gene (Tpa-glu). The gene revealed a 1,464 bp encoding 487 amino acid residues, and the deduced amino acid residues exhibited 77% identity with Pyrococcus furiosus ${\beta}$-glucosidase (accession no. NP_577802). The gene was cloned and expressed in Escherichia coli system. The recombinant protein was purified by metal affinity chromatography and characterized. Tpa-Glu showed optimum activity at pH 7.5 and $75^{\circ}C$, and thermostability with a half life of 6 h at $90^{\circ}C$. Tpa-Glu exhibited hydrolyzing activity against various pNP-glycopyranosides, with kcat/Km values in the order of pNP-${\beta}$-glucopyranoside, pNP-${\beta}$-galactopyranoside, pNP-${\beta}$-mannopyranoside, and pNP-${\beta}$-xylopyranoside. In addition, the enzyme exhibited exo-hydrolyzing activity toward ${\beta}$-1,3-linked polysaccharide (laminarin) and ${\beta}$-1,3- and ${\beta}$-1,4-linked oligosaccharides. This is the first description of an enzyme from hyperthermophilic archaea that displays exo-hydrolyzing activity toward ${\beta}$-1,3-linked polysaccharides and could be applied in combination with ${\beta}$-1,3-endoglucanase for saccharification of laminarin.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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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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• v.47 no.5
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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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• v.3 no.2
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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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• v.57 no.7
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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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• 2008.05a
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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.