• Journal of the Korean Wood Science and Technology
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• v.34 no.3
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• pp.56-63
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• 2006

In order to evaluate the mechanism of cellulose hydrolysis, the complementary DNA encoding Glycoside Hydrolase Family (GHF)74 was cloned from Phanerochaete chrysosporium. Depending on the presence of Cellulose Binding Module (CBM), it can be classified as GHF74A or GHF74B. The GHF74A gene from P. chrysosporium (PcGHF74A) consists of 2163 bp encoding a protein of 721 amino acid residues. The PcGHF74A showed homology of 70~77% compared with the GHF74 from other filamentous fungi. The PcGHF74B, which contains CBM and is a member of family 1, was transcribed to various transcripts depending on the nature of carbon sources and their concentration. To study the possible presence of splice variants in GHF74B transcripts in P. chrysospoium, we carried out RT-PCR analysis using primers that designed based on the annotation data and sequenced data. Our result indicated that PcGHF74B was transcribed to several splicing variants in various culture conditions. Especially in the culture of 2% cellulose, three transcript products were observed. First transcript was presumed to be a full length ORF that contained 11th intron with stop codon at position 2562 bp. The second one consisted of 12 exons and 11 introns with stop codon at position 1187 bp with 7th exon. The shortest transcript consisted of 10 exons and 9 introns with stop codon at 910 bp in the 7th exon. These premature stop codon might prevent the synthesis of fully active GHF74 or contribute for the production of protein with distinct function depending on the ambient carbon sources.

• Journal of Microbiology and Biotechnology
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• v.29 no.5
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• pp.765-775
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• 2019

A new ${\alpha}$-amylase-encoding gene (amySL3) of glycoside hydrolase (GH) family 13 was identified in soda lake isolate Alkalibacterium sp. SL3. The deduced AmySL3 shares high identities (82-98%) with putative ${\alpha}$-amylases from the genus Alkalibacterium, but has low identities (<53%) with functionally characterized counterparts. amySL3 was successfully expressed in Escherichia coli, and the recombinant enzyme (rAmySL3) was purified to electrophoretic homogeneity. The optimal temperature and pH of the activity of the purified rAmySL3 were determined to be $45^{\circ}C$ and pH 7.5, respectively. rAmySL3 was found to be extremely halophilic, showing maximal enzyme activity at a nearly saturated concentration of NaCl. Its thermostability was greatly enhanced in the presence of 4 M NaCl, and it was highly stable in 5 M NaCl. Moreover, the enzyme did not require calcium ions for activity, and was strongly resistant to a range of surfactants and hydrophobic organic solvents. The major hydrolysis products of rAmySL3 from soluble starch were maltobiose and maltotriose. The high ratio of acidic amino acids and highly negative electrostatic potential surface might account for the halophilic nature of AmySL3. The extremely halophilic, calcium-independent, and surfactant-resistant properties make AmySL3 a promising candidate enzyme for both basic research and industrial applications.

• Journal of Life Science
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• v.22 no.11
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• pp.1545-1551
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• 2012

Authors report the glycoside hydrolase (GH) family 16 ${\beta}$-agarase from the strain of Agarivorans sp. JA-1, which authors previously stated as recombinant expression and characterization of GH-50 and GH-118 ${\beta}$-agarase. It comprised an open reading frame of 1,362 base pairs, which encodes a protein of 49,830 daltons consisting of 453 amino acid residues. Valuation of the total sequence showed that the enzyme has 98% nucleotide and 99% amino acid sequence similarities to those of GH-16 ${\beta}$-agarase from Pseudoalteromonas sp. CY24. The gene corresponding to a mature protein of 429 amino acids was recombinantly expressed in Escherichia coli, and the enzyme was purified to homogeneity by affinity chromatography. It showed maximal activity at $40^{\circ}C$ and pH 5.0, representing 67.6 units/mg. Thin layer chromatography revealed that mainly neoagarohexaose and neoagarotetraose were produced from agarose. The enzyme would be valuable for the industrial production of functional neoagarooligosaccharides.

• Journal of Life Science
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• v.22 no.2
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• pp.266-280
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• 2012

Agar is a cell wall component of macro red algae that can be hydrolyzed by agarase. Agarases are classified into ${\alpha}$-agarase (E.C. 3.2.1.158) and ${\beta}$-agarase (E.C. 3.2.1.81), in accordance with their cleavage pattern, and can be grouped in the glycoside hydrolase (GH)-16, -58, -86, -96, and -118 family according to the amino acid sequences of the proteins. Many agarases and/or their genes have been detected, isolated, and recombinantly expressed from bacteria, and metagenomes have their origins in sea and terrestrial environments. Products of agarases, agarooligosaccharides and neoagarooligosaccharides, represent wide functions such as antitumor, immune stimulation, antioxidation, prebiotic, hepa-protective, antibacterial, whitening, and moisturizing effects; hence, broad applications would be possible in the food industry, cosmetics, and medical fields. In addition, agarases are also used as a tool enzyme for research. This paper reviews the sources, purifications and detection methods, and application fields of agarases. The role of agarases in agar metabolism and the function of their enzymatic products are also surveyed.

• Journal of the Korean Wood Science and Technology
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• v.35 no.6
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• pp.159-165
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• 2007

An extracellular xylanase from the brown-rot fungus Fomitopsis palustris grown on rice straw culture was purified to a single protein band. On SDS-PAGE, the molecular mass of purified enzyme was estimated to be about 43 kDa. The amino acid sequence of the proteolytic fragments showed high homology with fungal glycoside hydrolase family 10 xylanases. The $K_m$, $K_{cat}$ and $V_{max}$ for birch xylan were $31mg/m{\ell}$, $2.3{\times}10^4/min$ and 252.3 U/mg, respectively. The optimal activity of the purified xylanase from F palustris was observed at pH 4.0~5.0 and $70^{\circ}C$.

• Journal of the Korea Organic Resources Recycling Association
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• v.17 no.4
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• pp.48-58
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• 2009

Glycosyl hydrolases (GH, EC 3.2.1.-) are key enzymes which can hydrolyze the glycosidic bonds between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. The new enzyme nomenclature of glycoside hydrolases is based on their amino acid sequence similarity and structural features. Here, we examined the glycosyl hydrolase family(GHF) genes reported from earthworm species. Among overall 115 GHFs, 12 GHFs could be identified from earthworm species through CAZy database. Of 12 GHF group genes, five genes including GHF2, 5, 17, 18, 20 are thought to be potent for industrial applications. The alignment of these genes with same genes from other animal species exhibited high sequence homology and some important amino acid residues necessary for enzyme activity appeared to be conserved. These genes can be utilized as a pest control agent or applicable to the food industry, clinical therapeutics and organic wastes disposition.

• Journal of Microbiology and Biotechnology
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• v.16 no.11
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• pp.1753-1759
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• 2006

Paenibacillus sp. HY-8 isolated from the digestive tracts of the longicorn beetle, Moechotypa diphysis, produced an extracellular endoxylanase with a molecular weight of 20 kDa estimated by SDS-PAGE. The xylanase was purified to near electrophoretic homogeneity from the culture supernatant after ammonium sulfate precipitation, gel filtration, and ionexchange chromatography. The purified xylanase exhibited the highest activities at pH 6.0 and $50^{\circ}C$. The $K_m\;and\;V_{max}$ values were 7.2 mg/ml and 16.3 U/mg, respectively, for birchwood xylan as the substrate. Nucleotide sequence of the PCR-cloned gene was determined to have the open reading frame encoding a polypeptide of 212 amino acids. The N-terminal amino acid sequence and the nucleotide sequence analyses predicted that the precursor xylanase contained a signal peptide composed of 28 amino acids and a catalytically active 19.9-kDa peptide fragment. The deduced amino acid sequence shared extensive similarity with those of the glycoside hydrolase family 11 of xylanases from other bacteria. The predicted amino acid sequence contained two glutamate residues, previously identified as essential and conserved for active sites in other xylanases of the glycoside hydrolase family 11.

• Journal of Microbiology and Biotechnology
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• v.26 no.9
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• pp.1650-1656
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• 2016

The SCO0284 gene of Streptomyces coelicolor A3(2) is predicted to encode an α-galactosidase (680 amino acids) belonging to glycoside hydrolase family 27. In this study, the SCO0284 coding region was cloned and overexpressed in Streptomyces lividans TK24. The mature form of SCO0284 (641 amino acids, 68 kDa) was purified from culture broth by gel filtration chromatography, with 83.3-fold purification and a yield of 11.2%. Purified SCO0284 showed strong activity against p-nitrophenyl-α-D-galactopyranoside, melibiose, raffinose, and stachyose, and no activity toward lactose, agar (galactan), and neoagarooligosaccharides, indicating that it is an α-galactosidase. Optimal enzyme activity was observed at 40℃ and pH 7.0. The addition of metal ions or EDTA did not affect the enzyme activity, indicating that no metal cofactor is required. The kinetic parameters V_max and K_m for p-nitrophenyl-α-D-galactopyranoside were 1.6 mg/ml (0.0053 M) and 71.4 U/mg, respectively. Thin-layer chromatography and mass spectrometry analysis of the hydrolyzed products of melibiose, raffinose, and stachyose showed perfect matches with the masses of the sodium adducts of the hydrolyzed products, galactose (M+Na, 203), melibiose (M+Na, 365), and raffinose (M+Na, 527), respectively, indicating that it specifically cleaves the α-1,6-glycosidic bond of the substrate, releasing the terminal D-galactose.

• Journal of Microbiology and Biotechnology
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• v.32 no.8
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• pp.1064-1071
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• 2022

Arabinogalactans have diverse biological properties and can be used as pharmaceutical agents. Most arabinogalactans are composed of β-(1→3)-galactan, so it is particularly important to identify β-1,3-galactanases that can selectively degrade them. In this study, a novel exo-β-1,3-galactanase, named PoGal3, was screened from Penicillium oxalicum sp. 68, and hetero-expressed in P. pastoris GS115 as a soluble protein. PoGal3 belongs to glycoside hydrolase family 43 (GH43) and has a 1,356-bp gene length that encodes 451 amino acids residues. To study the enzymatic properties and substrate selectivity of PoGal3, β-1,3-galactan (AG-P-I) from larch wood arabinogalactan (LWAG) was prepared and characterized by HPLC and NMR. Using AG-P-I as substrate, purified PoGal3 exhibited an optimal pH of 5.0 and temperature of 40℃. We also discovered that Zn²⁺ had the strongest promoting effect on enzyme activity, increasing it by 28.6%. Substrate specificity suggests that PoGal3 functions as an exo-β-1,3-galactanase, with its greatest catalytic activity observed on AG-P-I. Hydrolytic products of AG-P-I are mainly composed of galactose and β-1,6-galactobiose. In addition, PoGal3 can catalyze hydrolysis of LWAG to produce galacto-oligomers. PoGal3 is the first enzyme identified as an exo-β-1,3-galactanase that can be used in building glycan blocks of crucial glycoconjugates to assess their biological functions.

• Journal of Marine Bioscience and Biotechnology
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• v.8 no.1
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• pp.1-9
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• 2016

The hydrolysis of biomass to fermentable sugar (saccharification) and to oligosaccharide is an essential process in biotechnology including biorefinery and biofood. Various macroalgae are commercially cultivated in several Asian countries as a useful resource for food and agar production. Agar is a major component of the cell walls of red algae that can be hydrolyzed by agarase. Agarases are classified into ${\alpha}$-agarase (E.C. 3.2.1.158) and ${\beta}$-agarase (E.C. 3.2.1.81) according to the cleavage pattern and grouped in the glycoside hydrolase (GH) family (GH-16, GH-58, GH-86, GH-96, and GH-118) based on the amino acid sequences of the proteins. Agarases have been isolated from various bacteria found in seawater and marine sediments. To increase productivity of the enzyme, a research on recombinant enzymes has been done. The application of recombinant agarase can be possible in the various filed such as energy, food, cosmetics, medical and so on. This paper reviews the source, biochemical characteristics and production system of recombinant agarases for further study.