• The Korean Journal of Zoology
• /
• v.34 no.2
• /
• pp.203-208
• /
• 1991

Body weight gain in the niadn deficient group of quail was markedly lowered as compared to that of the control group, but heart, kidney and liver weight were slighdy reduced reladve to the body weight. Nicotinic acid deficiency resulted in the significant increase of serum glucose level but the serum cholesterol, albumin and total protein levels were not affected to any extent. Glutamic oxaloacetate iransaminase and glutamic pyruvate transaminase activities were significandy enhanced but alkaline phosphatase and lactic dehydrogenase activities were not influenced. Tryptophan and tyrosine levels were remarkably reduced and a similar observation was also made with aspartic acid, glutamic acid and alanine plus serine. However, the levels of basic amino acids such as arginine, histidine and lysine plus branched chain amino acids such as isoleucine, leucine and valine were not affected.

• Journal of Animal Science and Technology
• /
• v.51 no.2
• /
• pp.177-182
• /
• 2009

The glycosylated human MINPP (multiple inositol polyphosphate phosphatase), which was recombinantly over-expressed by using industrial host, Pichia pastoris, showed the phytase activity against phytate ($InsP_6$) and the enzyme activity of the unglycosylated counterpart was decreased to 30%. The optimal phytase activity occurred at pH 7.4. The human MINPP showed high substrate specificity for $InsP_6$ with little activity on other organic phosphate conjugates such as para-nitrophenylphosphate (pNPP), ATP, and ribose-1-phosphate (R-1-P). The phosphatase activity against 2,3-bisphosphoglycerate (2,3-BPG) by human MINPP was increased to 1.2-fold in the presence of stimulator, 1 mM 2-phosphoglycolate (2-PG) but the phytase activity against $InsP_6$ was not affected by addition of 1 mM 2-PG. The phosphatase activity against 2,3-BPG by human MINPP was not increased in the presence of 2 mM $Mg^{2+}$ or 100 mM $Cl^-$.

• Korean journal of applied entomology
• /
• v.15 no.4 s.29
• /
• pp.169-178
• /
• 1976

The concentration of amino acids, total nitrogen, trehalose, lipids and the activities of respiratory, acid$\cdot$alkaline phosphatase, glutamic oxalozcetic transaminase and glutamic pyruvic transaminase during larval stage in Pine leaf gall midge, Thecodiplosis janensis Uchi. et Inouye were measured using Paper chromatographic method, micro-Kjeldahl method, Thin layer chromatographic method, Warburg's manometric method, Bessey-Lowry method and Reitman-Frankel method, respectively. Healthy specimens )yore chosen as samples of each larval stages; alrva in gall and larva in soil. Amino acids present in the alcoholic extracts were alanine, glutamic acid, glycine, histidine, methionine, proline, threonine, tryptophan and valine. The total nitrogen concentration reached to 31.348mg/g during the larva in gall and the larval stage in soil of the value was decreased to 29.027mg/g. The hemolymph sugar, trehalose value for larva in soil was about two times of the value for larva in gall. Total lipid, phospholipid,monoacylglycerol, triacylglycerol, sterol, free fatty acid and ester cholesterol were identified at larval stages in gall and soil. Triacylglycerol concentration reached high level in contrast with other lipid contents during larvae in gall and larva in soil. Free fatty acid, sterol except decreased lipids during larval stage in soil. Endogenous respiration, succinate of respiratory activities decreased at larval stage in soil compare with larva in gall. The activities of acid phosphatase decreased larval stage in soil but the activities of alkaline phosphatase increased remarkably. The activities of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase reached high level of the larva in gall.

• Microbiology and Biotechnology Letters
• /
• v.19 no.1
• /
• pp.37-44
• /
• 1991

- I aimed to isolate trans-acting factors involved in the basal expression level of eukaryotic genes. One of the yeast histidine biosynthetic gene, HIS5 was taken as a model for this study. HIS5 gene has a substantial basal level in amino acid rich medium and is derepressed if starved for any single amino acid. The derepression is mediated by cis-acting DNA sequences 5'-TGACTC-3' found in 5' non-transcribed region of the gene and trans-acting factors including GCN4 as positive factor and its negative factor GCDI 7, and GCNZ as a negative factor of GCD17. I first investigated the role of these trans-acting factors in HIS5 basal expression level by using HIS5-pH05 fusion in which expression of pH05 gene encoding inorganic phosphate-repressible acid phosphatase (APase) is regulated by HIS5 promoter. Strain with gcn2 or gcn4 mutation showed 3 to 4 fold lower APase activity than wild type. The level of APase activity was similar in gcn2 and gcn4 mutants. Trans-acting factors involved in basal level were identified by isolating 14 mutants showing increased expression of HISSPH05 fusion from gcn4 background. All the mutants carry a single nuclear recessive mutation and fall into four complementation groups, designated as bell (basal expression level), be12, be23 and be14.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.7
• /
• pp.1221-1226
• /
• 2008

The soft rot bacterium Pectobacterium wasabiae is an economically important pathogen of many crops. A new phytase gene, appA, was cloned from P. wasabiae by degenerate PCR and TAIL-PCR. The open reading frame of appA consisted of 1,302 bp encoding 433 amino acid residues, including 27 residues of a putative signal peptide. The mature protein had a molecular mass of 45 kDa and a theoretical pI of 5.5. The amino acid sequence contained the conserved active site residues RHGXRXP and HDTN of typical histidine acid phosphatases, and showed the highest identity of 48.5% to PhyM from Pseudomonas syringae. The gene fragment encoding the mature phytase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant phytase had a specific activity of 1,072$\pm$47 U/mg for phytate substrate. The optimum pH and temperature for the purified phytase were pH 5.0 and 50$^{\circ}C$, respectively. The $K_m$ value was 0.17 mM, with a $V_{max}$ of 1,714 $\mu$mol/min/mg. This is the first report of the identification and isolation of phytase from Pectobacterium.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.6
• /
• pp.930-935
• /
• 2015

A phytase gene was identified in a publicly available metagenome derived from subsurface groundwater, which was deduced to encode for a protein of the histidine acid phosphatase (HAP) family. The nucleotide sequence of the phytase gene was chemically synthesized and cloned, in order to further overexpress the phytase in Escherichia coli. Purified protein of the recombinant phytase demonstrated an activity for phytic acid of 298 ± 17 µmol P/min/mg, at the pH optimum of 2.0 with the temperature of 37℃. Interestingly, the pH optimum of this phytase is much lower in comparison with most HAP phytases known to date. It suggests that the phytase could possess improved adaptability to the low pH condition caused by the gastric acid in livestock and poultry stomachs.

• Journal of Microbiology and Biotechnology
• /
• v.29 no.3
• /
• pp.419-428
• /
• 2019

Phytases are enzymes that can hydrolyze phytate and its salts into inositol and phosphoric acid, and have been utilized to increase the availability of nutrients in animal feed and mitigate environmental pollution. However, the enzymes' low thermostability has limited their application during the feed palletization process. In this study, a combination of B-value calculation and protein surface engineering was applied to rationally evolve the heat stability of Escherichia coli phytase. After systematic alignment and mining for homologs of the original phytase from the histidine acid phosphatase family, the two models 1DKL and 1DKQ were chosen and used to identify the B-values and spatial distribution of key amino acid residues. Consequently, thirteen potential amino acid mutation sites were obtained and categorized into six domains to construct mutant libraries. After five rounds of iterative mutation screening, the thermophilic phytase mutant P56214 was finally yielded. Compared with the wild-type, the residual enzyme activity of the mutant increased from 20% to 75% after incubation at $90^{\circ}C$ for 5 min. Compared with traditional methods, the rational engineering approach used in this study reduces the screening workload and provides a reference for future applications of phytases as green catalysts.

• Microbiology and Biotechnology Letters
• /
• v.30 no.1
• /
• pp.1-7
• /
• 2002

Phytase from Escherichia coli WC7 was purified from cell extracts and its molecular mass was estimated to be 45 kDa by SDS-PAGE. Its optimum temperature and pH for phytate hydrolysis was 6$0^{\circ}C$ and pH 5.0, respectively. The enzyme was stable up to 6$0^{\circ}C$ and over broad pH range (pH 2-12). The enzyme had higher affinity for sodium phytate than p-nitrophenylphosphate (pNPP). That is, the apparent Km value for sodium phytate and pNPP were $0.15\pm$0.02 mM and 2.82$\pm$0.05 mM, respectively. The gene encoding the phytase was cloned in E. coli XL1-Blue. Sequence analysis showed an open reading frame of 1241 Up encoding a signal peptide (22 aa) and a mature enzyme (410 aa). WC7 phytase was expressed up to 17.5 U/ml in the transformed E. coli XL1-Blue/pUEP, which was 23-fold higher than the activity from wild strain.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.1
• /
• pp.113-118
• /
• 2014

Environmental microorganisms are emerging as an important source of new enzymes for wide-scale industrial application. In this study, novel phytase genes were identified from a soil microbial community. For this, a function-based screening approach was utilized for the identification of phytase activity in a metagenomic library derived from an agricultural soil. Two novel phytases were identified. Interestingly, one of these phytases is an unusual histidine acid phosphatase family phytase, as the conserved motif of the active site of PhyX possesses an additional amino acid residue. The second phytase belongs to a new type, which is encoded by multiple open reading frames (ORFs) and is different to all phytases known to date, which are encoded by a single ORF.

• Journal of Microbiology and Biotechnology
• /
• v.11 no.4
• /
• pp.592-597
• /
• 2001

The pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate with the formation of $CO_2$, acetyl-CoA, NADH, and H+. This complex contains multiple copies of three catalytic components including pyruvate dehydrogenase(E1), dihydrolipoamide acetyltransferase(E2), and dihydrolipoamide dehydrogenase (E3). Two regulatory components (E1-kinase and phospho-E1 phosphatase) and functionally less-understood protein (protein X, E3BP) are also involved in the formation of the complex. In this study, cloning and characterization of a gene for human E3BP have been carried out. A cDNA encoding the human E3BP was isolated by database search and cDNA library screening. The primary structure of E3BP has some similar characteristics with that of E2 in the lipoyl domain and the carboxyl-terminal domain, based on the nucleotide sequence and the deduced amino acid sequence. However, the conserved amino acid moiety including the histidine residue for acetyltransferase activity in E2 is not conserved in the case of human E3BP. The human E3BP was expressed and purified in E. coli. The molecular weight of the protein, excluding the mitochondrial target sequence, was about 50 kDa as determined by SDS-PAGE. Cloning of human E3BP and expression of the recombinant E3BP will facilitate the understanding of the role(s) of E3BP in mammalian PDC.