• Asian-Australasian Journal of Animal Sciences
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• 제22권2호
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• pp.155-161
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• 2009

Mammal microsomal glutathione transferase 1 (MGST1) can conjugate many toxic or carcinogenic substances and depress oxidative stress. In this study, Chicken MGST1 and its variants were cloned for the first time and were composed of 956 or 944 nucleotides. The 12 nt deletion in the exon 2 did not alter the GT-AG rule and the ORFs for the two MGST1 variants were the same, which both comprised 465 nucletides and encoded a peptide with 155 amino acids. It was found that the two different splice variants identified using RT-PCR expressed in all three organs investigated of Dwarf Brown Chicken, namely liver, spleen and shell gland. Moreover, the expression level of MGST1 mRNA in the liver of Dwarf Brown chickens was the highest (p<0.01), and there were no significant differences between the spleen and the shell gland. These results provide a base for studying the biological function of Chicken MGST1.

• Journal of Microbiology and Biotechnology
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• 제19권11호
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• pp.1295-1300
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• 2009

A 2,172-bp full-length gene (aga-F78), encoding a protease-resistant $\alpha$-galactosidase, was cloned from Rhizopus sp. F78 and expressed in Escherichia coli. The deduced amino acid sequence shared highest identity (45.0%) with an $\alpha$-galactosidase of glycoside hydrolase family 36 from Absidia corymbifera. After one-step purification with a Ni-NTA chelating column, the recombinant Aga-F78 migrated as a single band of ~82 and ~210 kDa on SDS-PAGE and nondenaturing gradient PAGE, respectively, indicating that the native structure of the recombinant Aga-F78 was a trimer. Exhibiting the similar properties as the authentic protein, purified recombinant Aga-F78 was optimally active at $50^{\circ}C$ and pH 4.8, highly pH stable over the pH range 5.0-10.0, more resistant to some cations and proteases, and had wide substrate specificity (pNPG, melidiose, raffinose, and stachyose). The recombinant enzyme also showed good hydrolytic ability to soybean meal, releasing galactose of $415.58\;{\mu}g/g$ soybean meal. When combined with trypsin, the enzyme retained over 90% degradability to soybean meal. These favorable properties make Aga-F78 a potential candidate for applications in the food and feed industries.

• Journal of Microbiology and Biotechnology
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• 제31권10호
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• pp.1455-1464
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• 2021

Trehalose is a non-reducing disaccharide in increasing demand for applications in food, nutraceutical, and pharmaceutical industries. Single-step trehalose production by trehalose synthase (TreS) using maltose as a starting material is a promising alternative process for industrial application due to its simplicity and cost advantage. Pseudomonas monteilii TBRC 1196 was identified using the developed screening method as a potent strain for TreS production. The TreS gene from P. monteilii TBRC 1196 was first cloned and expressed in Escherichia coli. Purified recombinant trehalose synthase (PmTreS) had a molecular weight of 76 kDa and showed optimal pH and temperature at 9.0 and 40℃, respectively. The enzyme exhibited >90% residual activity under mesophilic condition under a broad pH range of 7-10 for 6 h. Maximum trehalose yield by PmTreS was 68.1% with low yield of glucose (4%) as a byproduct under optimal conditions, equivalent to productivity of 4.5 g/l/h using enzyme loading of 2 mg/g substrate and high concentration maltose solution (100 g/l) in a lab-scale bioreactor. The enzyme represents a potent biocatalyst for energy-saving trehalose production with potential for inhibiting microbial contamination by alkaline condition.

• Journal of Microbiology and Biotechnology
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• 제26권6호
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• pp.1067-1076
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• 2016

The gene encoding lipase (Lip98) from Aeromicrobium sp. SCSIO 25071 was cloned and functionally expressed in Escherichia coli. Lip98 amino acid sequence shares the highest (49%) identity to Rhodococcus jostii RHA1 lipase and contains a novel motif (GHSEG), which is different from other clusters in the lipase superfamily. The recombinant lipase was purified to homogeneity with Ni-NTA affinity chromatography. Lip98 showed an apparent molecular mass of 30 kDa on SDS gel. The optimal temperature and pH value for enzymatic activity were recorded at 30℃ and 7.5, respectively. Lip98 exhibited high activity at low temperatures with 35% maximum activity at 0℃ and good stability at temperatures below 35℃. Its calculated activation energy was 4.12 kcal/mol at the low temperature range of 15-30℃. Its activity was slightly affected by some metal ions such as K⁺, Ca²⁺, and Na⁺. The activity of Lip98 was increased by various organic solvents such as DMSO, ethanol, acetone, and hexane with the concentration of 30% (v/v) and retained more than 30% residual activity in neat organic solvent. The unique characteristics of Lip98 imply that it is a promising candidate for industrial application as a nonaqueous biocatalyst and food additive.

• Asian-Australasian Journal of Animal Sciences
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• 제21권8호
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• pp.1134-1142
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• 2008

Cationic amino acid transporter $b^{0,+}AT$ (HGMW-approved gene symbol SLC7A9, solute carrier family 7, member 9) plays a crucial role in amino acid nutrition. In the present study, we describe the cloning and sequencing of porcine $b^{0,+}AT$. Based on the sequence of porcine $b^{0,+}AT$ deposited in the NCBI (National Center for Biotechnological Information), we identified a putative porcine homologue. Using rapid amplification of cDNA ends (RACE), the full-length cDNA encoding porcine $b^{0,+}AT$ was isolated. The porcine $b^{0,+}AT$ cDNA was 1,680 bp long, encoding a 487 amino acid trans-membrane protein. The predicted amino acid sequence was found to have 88.9% and 87.1% identity with human and mouse $b^{0,+}AT$, respectively. Real-time RT-PCR indicated porcine $b^{0,+}AT$ transcripts expressed in heart, kidney, muscle and small intestine. The small intestine had the highest $b^{0,+}AT$ mRNA abundance while the muscle had the lowest (p<0.05). Along the longitudinal axis, the ileum had the highest $b^{0,+}AT$ mRNA abundance while the colon had the lowest (p<0.05). The $b^{0,+}AT$ mRNA level was highest on day 7 and 90 in the duodenum (p<0.05). It increased from day 1 to day 26 in the jejunum (p>0.05) and had the highest abundance on day 60 (p<0.05). There was, however, no difference between day 1, 7, 26, 30, 90 and 150 (p>0.05). The strongest $b^{0,+}AT$ expression appeared on day 7 in the ileum before weaning, and then decreased till day 30 but rose gradually again from day 60 to 150 (p<0.05).

• 한국양식학회지
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• 제8권3호
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• pp.209-220
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• 1995

미꾸라지의 간으로부터 핵을 분리하여, 저농도 염추출 및 제한효소 처리로 핵기질(nuclear matrix)을 분리하였다. 분리된 핵기질을 Proteinase K로 분해한 후, phenol-chloroform 추출로 크기가 약 0.3kb-15kb의 분포를 나타내는 핵기질 부착 DNA (nuclear matrix attachment regions : MARs)를 얻었다. 효모 URA 3 유전자를 가진 2.13 kb Eco47 III 단편을 제한효소 Ssp I 으로 절단된 pUC19 플라즈미드 벡타에 결합시켜, ARS (autonomously replication sequence) 클로닝을 위한 pURY19 플라즈미드 벡타를 만들었다. 이 pURY19 벡타는 Saccharomyces cerevisiae내에서 독립적으로 복제할 수 없기 때문에, 물고기의 효율적인 발현 벡타 개발을 위해, 이 system을 이용하여, S. cerevisiae내에서 독립적으로 복제 가능한 미꾸라지의 ARS를 클로닝하고자 하였다. 분리 된 MARs를 pURY19 벡타에 결합시 킨 다음, E. coli $DH5\alpha$에 형질전환시켜 $pURY19N_{l-62}$를 얻었다. MAR Libraries $(pURY19N_{1-62})$를 각각 $Ura^-\;S.\;cerevisiae$에 형질전환시켜, S. cerevisiae내에서 독립적으로 복제 가능한 M. mizolepis 유래의 복제원점들 (ARSs)을 분리하여, Sanger's dideoxy-chain termination method로 염기서열을 분석하였다. 염기서열 분석결과 모든 clones들은 AT-rich하였으며, 특히 $pURY19N_6$에는 ARS concensus sequence, Topoisomerase II consensus, near A-box, 그리고 T-box들이 존재하였다.

• BMB Reports
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• 제39권5호
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• pp.502-510
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• 2006

2C-methyl-D-erythritol 2, 4-cyclodiphosphate synthase (MECPS, EC: 4.6.1.12) is the fifth enzyme of the non-mevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and is involved in the methylerythritol phosphate (MEP) pathway for ginkgolide biosynthesis. The full-length mecps cDNA sequence (designated as Gbmecps) was cloned and characterized for the first time from gymnosperm plant species, Ginkgo biloba, using RACE (rapid amplification of cDNA ends) technique. The full-length cDNA of Gbmecps was 874 bp containing a 720 bp open reading frame (ORF) encoding a peptide of 239 amino acids with a calculated molecular mass of 26.03 kDa and an isoelectric point of 8.83. Comparative and bioinformatic analyses revealed that GbMECPS showed extensive homology with MECPSs from other species and contained conserved residues owned by the MECPS protein family. Phylogenetic analysis indicated that GbMECPS was more ancient than other plant MECPSs. Tissue expression pattern analysis indicated that GbMECPS expressed the highest in roots, followed by in leaves, and the lowest in seeds. The color complementation assay indicated that GbMECPS could accelerate the accumulation of $\beta$-carotene. The cloning, characterization and functional analysis of GbMECPS will be helpful to understand more about the role of MECPS involved in the ginkgolides biosynthesis at the molecular level.

• Journal of Microbiology and Biotechnology
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• 제21권8호
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• pp.830-837
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• 2011

A genomic DNA fragment encoding a putative maltooligosyltrehalose trehalohydrolase (NfMTH) for trehalose biosynthesis was cloned by the degenerate primer- PCR from cyanobacterium Nostoc flagelliforme. The ORF of NfMTH is 1,848 bp in length and encodes 615 amino acid residues, constituting a 70 kDa protein. The deduced amino acid sequence of NfMTH contains 4 regions highly conserved for MTHs. By expression of NfMTH in E. coli, the function of this protein was demonstrated, where the recombinant protein catalyzed the hydrolysis of maltooligosyl trehalose to trehalose. The expressions of MTH and maltooligosyltrehalose synthase in the filaments of N. flagelliforme were upregulated significantly under dehydration stress, NaCl stress, and high temperature-drought stress. The accumulations of both trehalose and sucrose in the filaments of N. flagelliforme were also improved significantly under the above stresses. Furthermore, trehalose accumulated in smaller quantities than sucrose did when under NaCl stress, but accumulated in higher quantities than sucrose did when under temperature-drought stress, indicating that both trehalose and sucrose were involved in N. flagelliforme adapted to stresses and different strategies conducted in response to various stress conditions.

• Asian-Australasian Journal of Animal Sciences
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• 제26권12호
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• pp.1680-1688
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• 2013

Many different approaches have been developed to improve the efficiency of animal cloning by somatic cell nuclear transfer (SCNT), one of which is to modify histone acetylation levels using histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA). In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from SCNT. We found that TSA treatment (50 nM) for 24 h following oocyte activation improved blastocyst formation rates (to 22.0%) compared with 8.9% in the non-treatment group and total cell number of the blastocysts for determining embryo quality also increased significantly ($88.9{\rightarrow}114.4$). Changes in histone acetylation levels as a result of TSA treatment were examined using indirect immunofluorescence and confocal microscopy scanning. Results showed that the histone acetylation level in TSA-treated embryos was higher than that in controls at both acetylated histone H3 lysine 9 (AcH3K9) and acetylated histone H4 lysine 12 (AcH4K12). Next, we compared the expression patterns of seven genes (OCT4, ID1; the pluripotent genes, H19, NNAT, PEG1; the imprinting genes, cytokeratin 8 and 18; the trophoblast marker genes). The SCNT blastocysts both with and without TSA treatment showed lower levels of OCT4, ID1, cytokeratin 8 and 18 than those of the in vivo blastocysts. In the case of the imprinting genes H19 and NNAT, except PEG1, the SCNT blastocysts both with and without TSA treatment showed higher levels than those of the in vivo blastocysts. Although the gene expression patterns between cloned blastocysts and their in vivo counterparts were different regardless of TSA treatment, it appears that several genes in NT blastocysts after TSA treatment showed a slight tendency toward expression patterns of in vivo blastocysts. Our results suggest that TSA treatment may improve preimplantation porcine embryo development following SCNT.

• Journal of Ginseng Research
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• 제41권3호
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• pp.403-410
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• 2017

Background: Prenyltransferases catalyze the sequential addition of isopentenyl diphosphate units to allylic prenyl diphosphate acceptors and are classified as either trans-prenyltransferases (TPTs) or cis-prenyltransferases (CPTs). The functions of CPTs have been well characterized in bacteria, yeast, and mammals compared to plants. The characterization of CPTs also has been less studied than TPTs. In the present study, molecular cloning and functional characterization of a CPT from a medicinal plant, Panax ginseng Mayer were addressed. Methods: Gene expression patterns of PgCPT1 were analyzed by quantitative reverse transcription polymerase chain reaction. In planta transformation was generated by floral dipping using Agrobacterium tumefaciens. Yeast transformation was performed by lithium acetate and heat-shock for $rer2{\Delta}$ complementation and yeast-two-hybrid assay. Results: The ginseng genome contains at least one family of three putative CPT genes. PgCPT1 is expressed in all organs, but more predominantly in the leaves. Overexpression of PgCPT1 did not show any plant growth defect, and its protein can complement yeast mutant $rer2{\Delta}$ via possible protein-protein interaction with PgCPTL2. Conclusion: Partial complementation of the yeast dolichol biosynthesis mutant $rer2{\Delta}$ suggested that PgCPT1 is involved in dolichol biosynthesis. Direct protein interaction between PgCPT1 and a human Nogo-B receptor homolog suggests that PgCPT1 requires an accessory component for proper function.