• Microbiology and Biotechnology Letters
• /
• v.36 no.1
• /
• pp.49-54
• /
• 2008

The gene encoding human pancreatic pro-carboxypeptidase B (CPB) was cloned and fused to Saccharomyces cerevisiae mating factor alpha-1 secretion signal $(MF{\alpha}1)$, in which the transcription of $MF{\alpha}1$-pro-CPB was under the control of GAL10 promoter. The constructed plasmid $pY{\alpha}$-hproCPB(7.72 kb) was transformed into S. cerevisiae 2805. The recombinant human pro-CPB (hproCPB) was successfully expressed in S. cerevisiae after induction of galactose, and could be secreted into the culture medium. By analyses of SDS-PAGE and western blotting, the molecular weight of the purified hproCPB was estimated to be a 45.9kDa. The activity of extracellular hCPB after removal of pro-region by trypsin treatment reached about 10.16 unit/ml at batch culture of S. cerevisiae $2805/pY{\alpha}$-hproCPB for 60 h. Also, the Km value of partially purified recombinant hCPB is about 0.43 mM.

• Journal of the Korean Chemical Society
• /
• v.22 no.3
• /
• pp.164-172
• /
• 1978

Carboxypeptidase A-catalyzed hydrolysis of ester substrates was carried out at room temperature in the presence of a number of external reagents. If the acyl-enzyme intermediate, an anhydride, is attacked by the external reagents, products formed by trapping at the acyl portion or at the enzyme portion of the anhydride group can be obtained. Examination of the uv/vis spectral properties of the reaction products and of changes in enzyme activity indicated that such trapping reactions did not occur. Also performed was evaluation of enzymatic rate parameters for the the hydrolysis of O-(o-hydroxyphenylacetyl)-L-${\beta}$-phenyllactate. Detection of 2-coumaranone possibly formed by attack of the o-hydroxy group as an intramolecular trapping group at the acyl-enzyme intermediate was tried, but no evidences for the intramolecular trapping reaction were obtained. Failure to trap the intermediate was discussed in terms of steric hindrance imposed on the approach of the trapping reagents to the anhydride group of the acyl-enzyme intermediate and of the fast enzymatic breakdown of the intermediate.

• Journal of the Korean Chemical Society
• /
• v.34 no.3
• /
• pp.232-238
• /
• 1990

To study the biological roles of Zn, we investigated simple model systems of $Zn^{++}, coordinated with OH_2 or NH_3,$ or with O=C- in peptide. The geometrical structures and net atomic charges were calculated by the ab initio HF-SCF theory using double zeta basis sets. The ligands of O-H, N-H, and O=C- are very polar due to $Zn^{++}$. Therefore, the carbon atom in peptide becomes so electrophilic that it can be easily attacked by other nucleophiles. In addition, to understand how $Zn^{++}$ is coordinated with ligands in enzyme, a molecular mechanics method is applied to the system of the enzyme of carboxypeptidase A (CPA) with the substrate of glycyltyrosine. From our results, it appears that the Zn ion is coordinated not only by four ligands in enzyme and substrate but also by one water molecule.

• Journal of Life Science
• /
• v.12 no.6
• /
• pp.682-687
• /
• 2002

We analyzed improvement on the enzyme activity of CPase Taq by addition of various metal ions. The enzyme activity was increased more then four times by 1 mM cobalt ion and almost three times by 1 mM calcium ion. However, the active center metal zinc ion did not affect the enzyme activity. In order to investigate whether the active center metal affects the enzyme activity, zinc ion which is occupied the active center of the enzyme was replaced by cobalt ion which activates the enzyme activity very effectively. Since the cobalt ion in the active center of the cobalt-substituted CPase Taq did not affect the enzyme activity, it could act as the natal metal ion in the active center of the enzyme.

• Asian Pacific Journal of Cancer Prevention
• /
• v.16 no.10
• /
• pp.4383-4386
• /
• 2015

Background: The C1561T variant of the glutamate carboxypeptidase II (GCPII) gene is critical for natural methylfolylpolyglutamte (methylfolate) absorption, and has been associated with perturbations in folate metabolism and disease susceptibility. However, little is known on C1561T-GCPII as a risk factor for colorectal cancer. Therefore, this study examined whether C1561T-GCPII influences folate metabolism and adenomatous polyp occurrence. Materials and Methods: 164 controls and 38 adenomatous polyp cases were analysed to determine blood folate and plasma homocysteine (Hcy) level, dietary intake of natural methylfolate, synthetic pteroylglutamic acid (PteGlu), vitamin C and C1561T-GCPII genotype. Results: In controls and cases, 7.3 and 18.4 percent of subjects respectively, were found to have the CT genotype, increasing the risk for adenomatous polyp occurrence 2.86 times (95% CI:1.37-8.0, p=0.035). Total dietary folate, methylfolate and PteGlu intake and the level of erythrocyte folate and plasma Hcy did not predict the occurrence of an adenomatous polyp. However, dietary natural vitamin C intake was associated with adenomatous polyp risk within C1561T-GCPII CT genotype subjects (p=0.037). Conclusions: The findings suggest that C1561T-GCPII variation may be associated with risk for adenomatous polyp, and vitamin C may modify risk by interacting with the variant gene, its expression product and/or folate substrates.

• Microbiology and Biotechnology Letters
• /
• v.29 no.1
• /
• pp.50-55
• /
• 2001

A new bacteriocin produced by Bacillus subtilis cx1, was partially purified and characterized. The bactericoin from B. subtilis cx1 was stable in the range of pH 2.5-9.5. B. subtilis csx1 retained its antimicrobial activity to long-term exposure at $-20^{\circ}C$ and $-70^{\circ}C$. However, B. subtilis cx1 was inactivated completely within 15 min over $60^{\circ}C$ and lost 50% of its antimicrobial activity within 15 min at $50^{\circ}C$, B. subtilis cx1 was inactivated by protease, trypsin, proteinase K and carboxypeptidase, which indi-cates its protein nature. Direct detection of the antimicrobial activity on Tricine -SDS-PAGE suggested an apparent molecular mass of about 9,500 dalton.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.6
• /
• pp.542-546
• /
• 2009

Leaves are the major biomass-producing organs in herbaceous plants and mainly develop during vegetative stage by activities of shoot apical meristem. There is a strong correlation between leaf number and bolting, a characteristic phenotype during the transition to reproductive phase in Arabidopsis thaliana. In order to study interactions between leaf number and bolting, we isolated a Landsberg erecta-derived mutant named multifolial (mfo1) that produces increased number of leaves and bolts at the same time as the wild type. Through positional cloning and allelism test, mfo1 was found to be an allele of a previously reported mutant, altered meristem program1-1 (amp1-1) that is defective in a glutamate carboxypeptidase and bolts earlier than its wild type, Columbia ecotype, with the increased number of leaves. The bolting time differences between mfo1 and amp1, despite the same phenotype of many leaves, suggest the existence of genetic factor(s) differently function in each ecotype in the presence of mfo1/amp1 mutation.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.6 no.4
• /
• pp.244-251
• /
• 2001

Over half of all biologically active peptide and peptide hormones are $\alpha$-amidated at their C-terminus, which is essential for their full biological activities. Amidation is accomplished through the sequential reaction of the two enzymes encoded by the single bifunctional, peptidyl-glycine $\alpha$-amidating monooxygenase (PAM or an $\alpha$-amidating enzyme). PAM catalyze the forma - tion of a peptide amide from peptide precursors that include a C-terminal glycine, and requires copper molecular oxygen and ascorbate. PAM is the only enzyme that produces peptide amides in vivo. However various strategies utilizing PAM, carboxypeptidase-Y enzymes, and chemical syn-thesis have been developed for producing peptide amides in vitro. The growing need and impor-tance of peptide amide drugs has highlighted the necessity for a efficient in vitro amidating sys-tem for industrial application for the production of peptide hormones, like calcitonin and oxytocin. This review presents the current situation regarding amidation with a special emphasis on the in-dustrial production or peptide hormones.

• Journal of Microbiology and Biotechnology
• /
• v.2 no.1
• /
• pp.1-6
• /
• 1992

The aspartame, $\alpha$-L-aspartyl-L-phenylalanine methylester, is an artificial sweetener. Taking advantage of the fact that the aspartame is a derivative of dipeptide, synthesis of aspartame from the artificial polypeptide made by an artificial gene has been attempted. The artificial polypeptide (LAP32), a polymer of tripeptide (aspartyl-phenylalanyl-lysine), was purified from the E. coli cells harboring a recombinant plasmid containing the artificial gene. This polypeptide was then digested with trypsin and carboxypeptidase B to produce dipeptide (Asp-Phe). Using the esterase activity of $\alpha$-chymotrypsin, the dipeptide was directly converted into Asp-Phe methylester in a water-methanol system. When the methanol concentration in reaction mixture was 25%, 50% of dipeptide was converted to the dipeptide methylester without producing any by-products.

• Journal of Dairy Science and Biotechnology
• /
• v.40 no.2
• /
• pp.86-91
• /
• 2022

Chryseobacterium mulctrae KACC 21234^T is a novel species isolated from raw bovine milk. Psychrotrophic bacteria are considered contaminants and are hypothesized to originate from the environment. In this investigation, the C. mulctrae KACC 21234^T genome was determined to be 4,868,651 bp long and assembled into four contigs with a G+C ratio of 33.8%. In silico genomic analyses revealed the presence of genes encoding proteases (endopeptidase Clp, oligopeptidase b, carboxypeptidase) and lipases (phospholipase A(2), phospholipase C, acylglycerol lipase) that can catalyze the degradation of the proteins and lipids in milk, causing its quality to deteriorate. Additionally, antimicrobial resistance and putative bacteriocin genes were detected, potentially intensifying the pathogenicity of the strain. The genomic evidence presented highlights the need for improved screening protocols to minimize the potential contamination of milk by proteolytic and lipolytic psychrotrophic bacteria.