• Journal of Life Science
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• v.18 no.11
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• pp.1617-1624
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• 2008

Chitin and chitosan, which is deacetylated form of chitin, are one of the most abundant biomass on the earth. They showed various biological activities including antimicrobial activity, heavy metal chelating, immune system activation, and have very diverse applications in food, pharmaceutical, medicinal, and environmental industry. There have been reported many chitin/chitosan-hydrolyzing enzymes, their structures and genes from three domains, archaea, bacteria, and eukarya. Carbohydrate hydrolyzing enzymes are classified in CAZy (Carbohydrate Active Enzymes) database according to their amino acid sequence similarity. Interestingly, chitinases and chitosanases are classified in various glycosyl hydrolase(GH) families, GH2, GH5, GH7, GH8, GH18, GH19, GH20, GH46, GH48, GH73, GH75, GH80, GH84, and GH85. Here, we review characteristics and structures of chitin/chitosan hydrolyzing enzymes according to glycosyl hydrolase families in order to provide information about gene mining.

• 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.

• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1933-1942
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• 2016

A novel ${\alpha}-glucoside$ hydrolase (named PspAG97A) from glycoside hydrolase family 97 (GH97) was cloned from the deep-sea bacterium Pseudoalteromonas sp. K8, which was screened from the sediment of Kongsfjorden. Sequence analysis showed that PspAG97A belonged to GH97, and shared 41% sequence identity with the characterized ${\alpha}-glucoside$ BtGH97a. PspAG97A possessed three key catalytically related glutamate residues. Mutation of the glutamate residues indicated that PspAG97A belonged to the inverting subfamily of GH97. PspAG97A showed significant reversibility against changes in salt concentration. It exhibited halophilic ability and improved thermostability in NaCl solution, with maximal activity at 1.0 M NaCl/KCl, and retained more than 80% activity at NaCl concentrations ranging from 0.8 to 2.0 M for over 50 h. Furthermore, PspAG97A hydrolyzed not only ${\alpha}-1,4-glucosidic$ linkage, but also ${\alpha}-1,6-$ and ${\alpha}-1,2-glucosidic$ linkages. Interestingly, PspAG97A possessed high catalytic efficiency for long-chain substrates with ${\alpha}-1,6-linkage$. These characteristics are clearly different from other known ${\alpha}-glucoside$ hydrolases in GH97, implying that PspAG97A is a unique ${\alpha}-glucoside$ hydrolase of GH97.

• 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.

• 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.

• Journal of Microbiology and Biotechnology
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• v.21 no.4
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• pp.347-358
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• 2011

Fomitopsis palustris, a brown-rot basidiomycete, causes the most destructive type of decay in wooden structures. In spite of its great economic importance, very little information is available at the molecular level regarding its complex decay process. To address this, we generated over 3,000 expressed sequence tags (ESTs) from a cDNA library constructed from F. palustris. Clustering of 3,095 high-quality ESTs resulted in a set of 1,403 putative unigenes comprising 485 contigs and 918 singlets. Homology searches based on BlastX analysis revealed that 78% of the F. palustris unigenes had a significant match to proteins deposited in the nonredundant databases. A subset of F. palustris unigenes showed similarity to the carbohydrateactive enzymes (CAZymes), including a range of glycosyl hydrolase (GH) family proteins. Some of these CAZyme-encoded genes were previously undescribed for F. palustris but predicted to have potential roles in biodegradation of wood. Among them, we identified and characterized a gene (FpCel45A) encoding the GH family 45 endoglucanase. Moreover, we also provided functional classification of 473 (34%) of F. palustris unigenes using the Gene Ontology hierarchy. The annotated EST data sets and related analysis may be useful in providing an initial insight into the genetic background of F. palustris.

• Journal of Life Science
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• v.31 no.3
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• pp.356-365
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• 2021

Recently, we sequenced the entire genome of a freshwater agar-degrading bacterium Cellvibrio sp. KY-GH-1 (KCTC13629BP) to explore genetic information encoding agarases that hydrolyze agarose into monomers 3,6-anhydro-L-galactose (L-AHG) and D-galactose. The KY-GH-1 strain appeared to possess nine β-agarase genes and two α-neoagarobiose hydrolase (α-NABH) genes in a 77-kb agarase gene cluster. Based on these genetic information, the KY-GH-1 strain-caused agarose degradation into L-AHG and D-galactose was predicted to be initiated by both endolytic GH16 and GH86 β-agarases to generate NAOS (NA4/NA6/NA8), and further processed by exolytic GH50 β-agarases to generate NA2, and then terminated by GH117 α-NABHs which degrade NA2 into L-AHG and D-galactose. More recently, by employing E. coli expression system with pET-30a vector we obtained three recombinant His-tagged GH50 family β-agarases (GH50A, GH50B, and GH50C) derived from Cellvibrio sp. KY-GH-1 to compare their enzymatic properties. GH50A β-agarase turned out to have the highest exolytic β-agarase activity among the three GH50 isozymes, catalyzing efficient NA2 production from the substrate (agarose, NAOS or AOS). Additionally, we determined that GH117A α-NABH, but not GH117B α-NABH, could potently degrade NA2 into L-AHG and D-galactose. Sequentially, we examined the enzymatic characteristics of GH50A β-agarase and GH117A α-NABH, and assessed their efficiency for NA2 production from agarose and for production of L-AHG and D-galactose from NA2, respectively. In this review, we describe the benefits of recombinant GH50A β-agarase and GH117A α-NABH originated from Cellvibrio sp. KY-GH-1, which may be useful for the enzymatic hydrolysis of agarose for mass production of L-AHG and D-galactose.

• Journal of Aquaculture
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• v.16 no.1
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• pp.51-58
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• 2003

The first Indian transgenic fish was generated in 1991 using borrowed constructs from foreign sources. To construct transformation vectors for the indigenous fishes, growth hormone genes of rohu (r-CH), Labeo rohita and catfish, Heteropneustes fossilis were isolated, cloned and sequenced; their fidelity was confirmed in prokaryotic and eukaryotic systems. A vector was constructed with grass carp b-actin promoter driving the expression of r-GH. Rohu eggs are large. fragile and swell 2~3 times. when fertilized. Hence they were amenable only for electroporated sperm-mediated gene transfer. Accordingly, the sperm electroporation technique was standardized to ensure 25% hatchling survival and 37% Presumptive transgenics without suffering any deformity. Southern analysis confirmed genomic integration in 15% of the tested individuals (Ti) belonging to family lines 2 and 3: another 25% of the Juveniles (Te) were also proved transgenic but with the transgene persisting extrachromosomally for longer than 1 to 2 years. perhaps due to the presence of replicon in the vector. Transgenics belonging to different family lines grew 6~8 times faster than the respective controls. Difference in growth trends of Ti and Te within a family line was not significant. In the Ti family 3 remarkable growth acceleration was sustained for a period longer than 36 weeks but in those of family 2, it gradually decreased. All transgenic fishes including the rohu converted the food at a significantly higher efficiency. Barring the transgenic mudloach, all the other transgenic fishes consumed food at significantly reduced rate.

• BMB Reports
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• v.40 no.3
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• pp.325-332
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• 2007

The mitogen-activated protein kinase (MAPK) cascade is one of the major and evolutionally conserved signaling pathways and plays pivotal role in the regulation of stress and developmental signals in plants. Here, a novel gene, termed Gossypium hirsutum MAPK (GhMAPK), was isolated from cotton. The full-length cDNA of GhMAPK encodes for a 372 amino acid protein that contains all 11 of the MAPK conserved subdomains and the phosphorylationactivation motif, TEY. Amino acid sequence alignment revealed that GhMAPK shared high identity with group-C MAPK in plants and showed 83~89% similarities with MAPKs from Arabidopsis, apricot, pea, petunia, and tobacco. Southern blot analysis indicated that the GhMAPK belonged to a multygene family in cotton. Two introns were found within the region of genomic sequence. Northern blot analysis revealed that the transcripts of GhMAPK accumulated markedly when the cotton seedlings were subjected to various abiotic stimuli such as wounding, cold (4$^{\circ}C$), or salinity stress; Furthermore, GhMAPK was upregulated by the exogenous signaling molecules, such as salicylic acid (SA) and hydrogen peroxide ($H_2O_2C$), as well as pathogen attacks. These results indicate that the GhMAPK, which has a high degree of identity with group-C plant MAPKs, may also play an important role in response to environmental stresses.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.5
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• pp.757-766
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• 2002

Growth hormone-releasing peptide-2 (GHRP-2, also named KP102) is a new hexapeptide of a series of synthetic growth hormone-releasing peptides (GHRPs) which stimulates the secretion of growth hormone (GH) in vitro and in vivo in several species including calf, sheep and pig. The GH-releasing activity of GHRP-2 is two to three times more effective than that of the original GHRP-6, and GHRP-1 in the rats and humans. To date, GHRP-2 seems to be the most potent member of the family of GHRPs. Since the GHRPs are short peptides (5-7 amino acid residues), they are synthesized easily and are not as readily degraded in plasma as GHreleasing hormone (GHRH). These features ameliorate their potential on domestic animals because of their chemical nature the GHRPs are efficacious when administered i.v. orally or orally. However, studies in cow, pig and sheep do not indicate such a close relationship between GHRH, somatostatin (SS) and GH, calling into question the general applicability of the human and rat models. Perhaps there is an important role for an endogenous GHRP in the regulation of GH secretion in domestic animals. This review provides an overview on the current knowledge of physiological role of GHRP-2 in domestic animals.