Molecular cloning and sequencing of the glycogen phosphorylase gene from Escherichia coli

AbstractThe glgP gene, which codes for glycogen phosphorylase, was cloned from a genomic library of Escherichia coli. The nucleotide sequence of the glgP gene contained a single open reading frame encoding a protein consisting of 790 amino acid residues. The glgP gene product, a polypeptide of Mr 87 000, was confirmed by SDS-polyacrylamide gel electrophoresis. The deduced amino acid sequence showed that homology between glgP of E. coli and rabbit glgP, human glgP, potato glgP, and E. coli malP was 48.6, 48.6, 42.3, and 46.1%, respectively. Within this homologous region, the active site, glycogen storage site, and pyridoxal-5′-phosphate binding site are well conserved. The enzyme activity of glycogen phosphorylase increased after introduction on a multicopy of the glgP gene

Gene expression and characterization of 2-keto-3-deoxy-gluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of Serratia marcescens KCTC 2172

We cloned 2-keto-3-deoxy-gluconate kinase (KDGK), which catalyzes the phosphorylation of 2-keto-3-deoxygluconate (KDG) to 2-keto-3-deoxy-6-phophogluconate (KDPG) from Serratia marcescens KCTC 2172. The nucleotide sequence revealed a single open reading frame containing 1,208 bp and encoding for 309 amino acids, with a molecular weight of 33,993 Da. The enzyme was purified via GST affinity chromatography. The putative KdgT binding site was detected upstream of the initial codon. The KDG kinase utilized 2-ketogluconate (KG) and KDG as substrates. The optimal temperature and pH for KDGK activity were 50ºC and 8.0, respectively

Cloning, purification, and characterization of an organic solvent-tolerant chitinase, MtCh509, from Microbulbifer thermotolerans DAU221

Abstract Background The ability to use organic solvents in enzyme reactions offers a number of industrially useful advantages. However, most enzymes are almost completely inactive in the presence of organic solvents. Thus, organic solvent-tolerant enzymes have potential applications in industrial processes. Results A chitinase gene from Microbulbifer thermotolerans DAU221 (mtch509) was cloned and expressed in Escherichia coli BL21 (DE3). The molecular weight of the expressed MtCh509 protein was approximately 60 kDa, and it was purified by His-tag affinity chromatography. Enzymatic assays showed that the optimum temperature for MtCh509 chitinase activity was 55 °C, and the enzyme was stable for 2 h at up to 50 °C. The optimum pH for MtCh509 activity was in the sub-acidic range, at pH 4.6 and 5.0. The activity of MtCh509 was maintained in presence of 1 M salt, gradually decreasing at higher concentrations, with residual activity (20%) detected after incubation in 5 M salt. Some organic solvents (benzene, DMSO, hexane, isoamyl alcohol, isopropyl alcohol, and toluene; 10–20%, v/v) increased the reactivity of MtCh509 relative to the aqueous system. When using NAG3, as a substrate, MtCh509 produced NAG2 as the major product, as well as NAG4, demonstrating that MtCh509 has transglycosylation activity. The K m and V max values for MtCh509 using colloidal chitin as a substrate were 9.275 mg/mL and 20.4 U/mg, respectively. Thus, MtCh509 could be used in extreme industrial conditions. Conclusion The results of the hydrolysate analysis and the observed increase in enzyme activity in the presence of organic solvents show that MtCh509 has industrially attractive advantages. This is the first report on an organic solvent-tolerant and transglycosylating chitinase from Microbulbifer species

Molecular cloning, purification and characterization of thermostable <img src='/image/spc_char/beta.gif' border=0>-1,3-1,4 glucanase from <i>Bacillus subtilis </i>A8-8

203-210A gene encoding a -1,3-1,4-glucanase (CelA) belonging to family 5 of glycoside hydrolases was cloned and sequenced from the Bacillus subtilis A8-8. The open-reading-frame of celA comprised 1499 base pairs and the enzyme was composed of 500 amino acids with a molecular mass of 55 kDa. The recombinant -1,3-1,4 glucanase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 8.0 and 60oC, respectively. The enzyme was stable within pH 6.0-9.0. It was stable up to 60oC and retained 30% of its original activity at 70oC for 60 min. It hydrolyzed lichenan, CMC, xylan, laminarin, avicel and pNPC, but was inactive towards cellobiose. The enzyme activity was markedly activated by Co2+ and Mn2+, but was strongly inactivated by Fe3+. The truncated gene, devoid of cellulose-binding domain (CBD) showed 60% of activity and bound to avicel

Cellulolytic Enzymes Production from Submerged Fermentation of Different Substrates by Newly Isolated Bacillus spp. FME

Newly isolated strains Bacillus sp. FME 1 and FME 2 were evaluated for the cellulolytic enzymes production during submerged fermentation (SmF) of different substrates including rice husk, Whatman filter paper and cellulose powder CF 11. Extracellular enzyme assays for CMCase, FPase and β-glucosidase were examined up to 8 days of submerged fermentation. Among the three substrates, rice husk was the most suitable substrate for higher production of cellulolytic enzymes. Maximum titers of 100, 45, and 3.5 U/mL in respect of CMCase, FPase and β-glucosidase in Bacillus sp. FME 2 were recovered as against 45, 12, and 0.39 U/mL in Bacillus sp. FME 1 respectively, at their respective peak time intervals. Bacillus sp. FME 2 was found to produce higher cellulolytic enzyme activities than Bacillus sp. FME 1

B-cell translocation gene-2 increases hepatic gluconeogenesis via induction of CREB

Hepatic gluconeogenesis is mediated by the network of transcriptional factors and cofactors. Here, we show that B-cell translocation gene-2 (BTG2) plays a crucial cofactor in hepatic gluconeogenesis via upregulation of the cyclic AMP (cAMP) response element binding (CREB) in hepatocytes. cAMP/dexamethasone (Dex) significantly increased BTG2 and other gluconeogenic genes such as Nur77, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase) in hepatocytes. In contrast, insulin treatment completely blocks their expressions. Interestingly, overexpression of BTG2 using adenoviral system (Ad-BTG2) significantly elevated hepatic glucose production via the increase of transcriptional activity and gene expression of CREB, PEPCK, and G6Pase in hepatocytes, suggesting that BTG2 is the key player on hepatic glucose production. Physiological interaction studies demonstrated that BTG2 correlated CREB recruitment on the PEPCK gene promoter via a direct interaction. Finally, knockdown of endogenous BTG2 expression markedly inhibits the cAMP/Dex-induced transcriptional activity of gluconeogenic genes and glucose production in hepatocytes. Overall, the present study provides us with a novel molecular mechanism of BTG2-mediated induction of hepatic gluconeogenesis and suggests that BTG2 plays an important role in hepatic glucose metabolism.close3

Overexpression and characterization of a novel chitinase gene from a marine bacterium Pseudomonas sp. BK1

339-344The chitinase A (ChiA)-coding gene of Pseudomonas sp. BK1, which was isolated from a marine red alga Porphyra dentata, was cloned and expressed in Escherichia coli. The structural gene consists of 1602 bp encoding a protein of 534 amino acids, with a predicted molecular weight of 55,370 Da. The deduced amino acid sequence of ChiA showed low identity (less than 32%) with other bacterial chitinases. The ChiA was composed of multiple domains, unlike the arrangement of domains in other bacterial chitinases. Recombinant ChiA overproduced as inclusion bodies was solubilized in the presence of 8 M urea, purified in a urea-denatured form and re-folded by removing urea. The purified enzyme showed maximum activity at pH 5.0 and 40ºC. It exhibited high activity towards glycol chitosan and glycol chitin, and lower activity towards colloidal chitin. The enzyme hydrolyzed the oligosaccharides from (GlcNAc)₄ to (GlcNAc)₆, but not GlcNAc to (GlcNAc)3. The results suggest that the ChiA is a novel enzyme, with different domain structure and action mode from bacterial family 18 chitinases