• Title/Summary/Keyword: Enzyme engineering

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Metagenomic Analysis of Novel Lignocellulose-Degrading Enzymes from Higher Termite Guts Inhabiting Microbes

  • Nimchua, Thidarat;Thongaram, Taksawan;Uengwetwanit, Tanaporn;Pongpattanakitshote, Somchai;Eurwilaichitr, Lily
    • Journal of Microbiology and Biotechnology
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    • v.22 no.4
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    • pp.462-469
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    • 2012
  • A metagenomic fosmid library was constructed from genomic DNA isolated from the microbial community residing in hindguts of a wood-feeding higher termite (Microcerotermes sp.) collected in Thailand. The library was screened for clones expressing lignocellulolytic activities. Fourteen independent active clones (2 cellulases and 12 xylanases) were obtained by functional screening at pH 10.0. Analysis of shotgun-cloning and pyrosequencing data revealed six ORFs, which shared less than 59% identity and 73% similarity of their amino acid sequences with known cellulases and xylanases. Conserved domain analysis of these ORFs revealed a cellulase belonging to the glycoside hydrolase family 5, whereas the other five xylanases showed significant identity to diverse families including families 8, 10, and 11. Interestingly, one fosmid clone was isolated carrying three contiguous xylanase genes that may comprise a xylanosome operon. The enzymes with the highest activities at alkaline pH from the initial activity screening were characterized biochemically. These enzymes showed a broad range of enzyme activities from pH 5.0 to 10.0, with pH optimal of 8.0 retaining more than 70% of their respective activities at pH 9.0. The optimal temperatures of these enzymes ranged from $50^{\circ}C$ to $55^{\circ}C$. This study provides evidence for the diversity and function of lignocellulose-degrading enzymes in the termite gut microbial community, which could be of potential use for industrial processes such as pulp biobleaching and denim biostoning.

Isolation and characterization of a marine bacterium Thalassomonas sp. SL-5 producing β-agarase (한천분해효소를 생산하는 해양세균 Thalassomonas sp. SL-5의 분리 및 특성)

  • Lee, Dong-Geun;Kim, Nam-Young;Jang, Min-Kyung;Lee, Ok-Hee;Lee, Sang-Hyeon
    • Journal of Life Science
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    • v.17 no.1 s.81
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    • pp.70-75
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    • 2007
  • A novel agar-degrading bacterium SL-5 was isolated from seashore of Homigot at Kyung-Buk province, and cultured in marine broth 2216 media. The bacterium SL-5 was identified as Thalassomonas genus by 16S rDNA sequencing with 96% identity. Growth rate was faster at $27^{\circ}C$ than at $37^{\circ}C$ and agarase was produced as growth-related. The optimum pH of the enzyme activity was 7.0 and the optimum temperature for the reaction was $40^{\circ}C$. Although the enzyme had no thermostability, the enzyme activity was remained over 80% at $60^{\circ}C$. The enzyme hydrolyzed neoagarohexaose to yield neoagarobiose as the main product, indicating that the enzyme is $\beta-agarase$. Thus, the enzyme would be useful for the industrial production of neoagarobiose.

Effect of $Zn^{2+}$ and Ferulic Acid on Laccase and Manganese Peroxidase Production by Funalia trogii (Funalia trogii에 의한 Laccase와 Manganese Peroxidase의 생산시 $Zn^{2+}$ 및 Ferulic Acid가 미치는 영향)

  • Park, Chul-Hwan;Han, Eun-Jung;Lee, Byung-Hwan;Lee, Jin-Won;Kim, Sang-Yong
    • KSBB Journal
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    • v.21 no.2
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    • pp.85-89
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    • 2006
  • Typical property of the white-rot fungi is their ability to degrade lignin and other aromatic compounds with non-specific extracellular enzyme. In this work, the modification of the strain(Funalia trogii ATCC 200800) and the culture condition was performed to enhance enzyme productivity. Single cell was separated by the protoplasts formation and several putative laccase and manganese peroxidase inducers were tested. By adopting the modified strain, enzyme productivity increased comparing with that of the original strain. Extracellular enzyme formation was highly stimulated by the addition of copper and various aromatic compounds in the glucose-based culture medium.

Characterization of Carboxymethylcellulase(CMCase) Produced by Recombinant E. coli Containing CMCase Gene for Cellulomonas sp. YE-5

  • Park, Sung-Won;Her, Nam-Yun;Kim, Dong-Seob;Park, Sun-Jin;Lee, Han-Seung;Park, Hak-Jong;Yu, Ju-Hyun
    • Preventive Nutrition and Food Science
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    • v.2 no.2
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    • pp.174-179
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    • 1997
  • CMCase produced by recombinant E. coli JM109 (pCEH#4) containing CMCase gene from Cellulomonas sp. YE-5 was purified to 24.3 fold and 2.6% yield by ammoniumsulfate precipitation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-100. The optimum pH and temperature for CMCase activity were pH 7.0 and 5$0^{\circ}C$. The enzyme was stable between pH 5.0 and 10.0, and up to 6$0^{\circ}C$. The molecular weight of he enzyme was estimated to be approximately 40,000 daltons by SDS-PAGE. Analysis of the amino acid composition showed that the enzyme contained many glycines and acidic amino acids. The enzyme was an endo-type CMCase and the final enzyme reaction product from hydrolysis of Cm-cellulose by the enzyme was cellobiose. {TEX}$K_{M}${/TEX} value determined with CM-cellulose was 1.28mM.

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Purification and Characterization of Arginase from Schizosaccharomyces pombe

  • Kang, Jung-Hoon
    • BMB Reports
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    • v.28 no.3
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    • pp.232-237
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    • 1995
  • Arginase was purified to homogeneity from Schizosaccharomyces pombe. The purified enzyme is a tetramer with a subunit molecular weight of 42,000. Activity is optimal at pH 10.0 and at $60^{\circ}C$ The enzyme migrated during isoelectric focusing showing a pl=5.4. The enzyme exhibited hyperbolic kinetics at pH 10.0 with an apparent $K_m$ for L-arginine of 18 mM. Arginase activity was strongly inhibited by L-glutamate.

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Changes in Availability of Toxic Trace Elements (TTEs) and Its Effects on Soil Enzyme Activities with Amendment Addition

  • Lee, Sang-Hwan;Park, Misun;Kim, Min-Suk
    • Ecology and Resilient Infrastructure
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    • v.7 no.2
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    • pp.134-144
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    • 2020
  • In-situ stabilization is a remediation method using amendments to reduce contaminant availability in contaminated soil. We tested the effects of two amendments (furnace slag and red mud) on the availability of toxic trace elements (TTEs) and soil enzyme activities (dehydrogenase, phosphatase, and urease). The application of amendments significantly decreased the availability of TTEs in soil (p < 0.05). The decreased availability of TTE content in soils was accompanied by increased soil enzyme activities. We found significant negative relationships between the TTE content assessed using Ca(NO3)2-, TCLP, and PBET extraction methods and soil enzyme activities (p < 0.01). Soil enzyme activities responded sensitively to changes in the soil environment (pH, EC, and availability of TTEs). It could be concluded that soil enzyme activities could be used as bioindicators or ecological indicators for soil quality and health in environmental soil monitoring owing to their high sensitivity to changes in soil.

Operation Modes Can Affect the Activity of Immobilized Enzyme onto Silk Fibroin Nanofibrous Membrane

  • Oh, Hanjin;Lee, Ki Hoon
    • International Journal of Industrial Entomology and Biomaterials
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    • v.27 no.2
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    • pp.322-325
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    • 2013
  • In the present study, we report that the selection of operation mode is important to take the full advantage of nanofibrous membrane in enzyme immobilization. Silk fibroin nanofibrous membrane has been prepared by electrospinning, and a-chymotrypsin was immobilized as a model enzyme. When the immobilized enzyme was operated in the membrane reactor mode, the Michaelis constant, Km, was lower and the Vmax was higher compared to the batch reactor mode. No concentration gradient was observed in the membrane reactor mode and the immobilized enzyme was stable even after 7 times of re-use. Our results suggests that the enzyme immobilized nanofibrous membrane should be operated in the membrane reactor mode rather than in the bath reactor mode.

Chemical Modification of Porcine Brain myo-Inositol Monophosphate Phosphatase by N-bromosuccinimide

  • Lee, Byung-Ryong;Bahn, Jae-Hoon;Jeon, Seong-Gyu;Ahn, Yoon-Kyung;Yoon, Byung-Hak;Kwon, Hyeok-Yil;Kwon, Oh-Shin;Choi, Soo-Young
    • BMB Reports
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    • v.32 no.3
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    • pp.294-298
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    • 1999
  • Myo-inositol monophosphate phosphatase is a key enzyme in the phosphoinositide cell-signaling system. Incubation of myo-inositol monophosphate phosphatase from porcine brain with N-bromosuccinimide (NBS) resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first-order kinetics with the second-order rate constant of $3.8{\times}10^3\;M^{-1}min^{-1}$. The time course of the reaction was significantly affected by the substrate myo-inositol-1-phosphate, which afforded complete protection against the loss of catalytic activity. Spectrophotometric studies indicated that about one oxindole group per molecule of enzyme was formed following complete loss of enzymatic activity. It is suggested that the catalytic function of myo-inositol monophosphate phosphatase is modulated by the binding of NBS to a specific tryptophan residue at or near the substrate binding site of the enzyme.

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Geostatistical Analysis of Soil Enzyme Activities in Mud Flat of Korea

  • Jung, Soohyun;Lee, Seunghoon;Park, Joonhong;Seo, Juyoung;Kang, Hojeong
    • Ecology and Resilient Infrastructure
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    • v.4 no.2
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    • pp.93-96
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    • 2017
  • Spatial variations of physicochemical and microbiological variables were examined to understand spatial heterogeneity of those variables in intertidal flat. Variograms were constructed for understanding spatial autocorrelations of variables by a geostatistical analysis and spatial correlations between two variables were evaluated by applications of a Cross-Mantel test with a Monte Carlo procedure (with 999 permutations). Water content, organic matter content, pH, nitrate, sulfate, chloride, dissolved organic carbon (DOC), four extracellular enzyme activities (${\beta}-glucosidase$, N-acetyl-glucosaminidase, phosphatase, arylsulfatase), and bacterial diversity in soil were measured along a transect perpendicular to shore line. Most variables showed strong spatial autocorrelation or no spatial structure except for DOC. It was suggested that complex interactions between physicochemical and microbiological properties in sediment might controls DOC. Intertidal flat sediment appeared to be spatially heterogeneous. Bacterial diversity was found to be spatially correlated with enzyme activities. Chloride and sulfate were spatially correlated with microbial properties indicating that salinity in coastal environment would influence spatial distributions of decomposition capacities mediated by microorganisms. Overall, it was suggested that considerations on the spatial distributions of physicochemical and microbiological properties in intertidal flat sediment should be included when sampling scheme is designed for decomposition processes in intertidal flat sediment.

AllEC: An Implementation of Application for EC Numbers Prediction based on AEC Algorithm

  • Park, Juyeon;Park, Mingyu;Han, Sora;Kim, Jeongdong;Oh, Taejin;Lee, Hyun
    • International Journal of Advanced Culture Technology
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    • v.10 no.2
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    • pp.201-212
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    • 2022
  • With the development of sequencing technology, there is a need for technology to predict the function of the protein sequence. Enzyme Commission (EC) numbers are becoming markers that distinguish the function of the sequence. In particular, many researchers are researching various methods of predicting the EC numbers of protein sequences based on deep learning. However, as studies using various methods exist, a problem arises, in which the exact prediction result of the sequence is unknown. To solve this problem, this paper proposes an All Enzyme Commission (AEC) algorithm. The proposed AEC is an algorithm that executes various prediction methods and integrates the results when predicting sequences. This algorithm uses duplicates to give more weights when duplicate values are obtained from multiple methods. The largest value, among the final prediction result values for each method to which the weight is applied, is the final prediction result. Moreover, for the convenience of researchers, the proposed algorithm is provided through the AllEC web services. They can use the algorithms regardless of the operating systems, installation, or operating environment.