Oxaloacetate and malate production in engineered Escherichia coli by expression of codon-optimized phosphoenolpyruvate carboxylase2 gene from Dunaliella salina

A new phosphoenolpyruvate carboxylase (PEPC) gene of Dunaliella salina is identified using homology analysis was conducted using PEPC gene of Chlamydomonas reinhardtii and Arabidopsis thaliana. Recombinant E. coli SGJS115 with increased production of malate and oxaloacetate was developed by introducing codon-optimized phosphoenolpyruvate carboxylase2 (OPDSPEPC2) gene of Dunaliella salina. E. coli SGJS115 yielded a 9.9 % increase in malate production. In addition, E. coli SGJS115 exhibited two times increase in the yield of oxaloacetate over the E. coli SGJS114 having identified PEPC2 gene obtained from Dunaliella salina.     

Molecular biology of C4 phosphoenolpyruvate carboxylase: Structure,regulation and genetic engineering

Three to four families of nuclear genes encode different isoforms of phosphoenolpyruvate (PEP) carboxylase (PEPC): C₄-specific, C₃ or etiolated, CAM and root forms. C₄ leaf PEPC is encoded by a single gene (ppc) in sorghum and maize, but multiple genes in the C₄-dicot Flaveria trinervia. Selective expression of ppc in only C₄-mesophyll cells is proposed to be due to nuclear factors, DNA methylation and a distinct gene promoter. Deduced amino acid sequences of C₄-PEPC pinpoint the phosphorylatable serine near the N-terminus, C₄-specific valine and serine residues near the C-terminus, conserved cysteine, lysine and histidine residues and PEP binding/catalytic sites. During the PEPC reaction, PEP and bicarbonate are first converted into carboxyphosphate and the enolate of pyruvate. Carboxyphosphate decomposes within the active site into Pi and CO₂, the latter combining with the enolate to form oxalacetate. Besides carboxylation, PEPC catalyzes a HCO₃ ^--dependent hydrolysis of PEP to yield pyruvate and Pi. Post-translational regulation of PEPC occurs by a phosphorylation/dephosphorylation cascade in vivo and by reversible enzyme oligomerization in vitro. The interrelation between phosphorylation and oligomerization of the enzyme is not clear. PEPC-protein kinase (PEPC-PK), the enzyme responsible for phosphorylation of PEPC, has been studied extensively while only limited information is available on the protein phosphatase 2A capable of dephosphorylating PEPC. The C₄ ppc was cloned and expressed in Escherichia coli as well as tobacco. The transformed E. coli produced a functional/phosphorylatable C₄ PEPC and the transgenic tobacco plants expressed both C₃ and C₄ isoforms. Site-directed mutagenesis of ppc indicates the importance of His¹³⁸, His⁵⁷⁹ and Arg⁵⁸⁷ in catalysis and/or substrate-binding by the E. coli enzyme, Ser⁸ in the regulation of sorghum PEPC. Important areas for further research on C₄ PEPC are: mechanism of transduction of light signal during photoactivation of PEPC-PK and PEPC in leaves, extensive use of site-directed mutagenesis to precisely identify other key amino acid residues, changes in quarternary structure of PEPC in vivo, a high-resolution crystal structure, and hormonal regulation of PEPC expression.Abbreviations OAA oxalacetate - PEP phosphoenolpyruvate - PEPC PEP carboxylase - PEPC-PK PEPC-protein kinase - PPDK pyruvate, orthophosphate dikinase - Rubisco ribulose 1,5-bis-phosphate carboxylase/oxygenase - CAM Crassulacean acid metabolism     

Molecular and structural analysis of C4-specific PEPC isoform from Pennisetum glaucum plays a role in stress adaptation

Phosphoenolpyruvate carboxylase is an ubiquitous cytosolic enzyme that catalyzes the ß-carboxylation of phosphoenolpyruvate (PEP) and is encoded by multigene family in plants. It plays an important role in carbon economy of plants by assimilating CO₂ into organic acids for subsequent C₄ or CAM photosynthesis or to perform several anaplerotic roles in non-photosynthetic tissues. In this study, a cDNA clone encoding for PEPC polypeptide possessing signature motifs characteristic to ZmC₄PEPC was isolated from Pennisetum glaucum (PgPEPC). Deduced amino acid sequence revealed its predicted secondary structure consisting of forty alpha helices and eight beta strands is well conserved among other PEPC homologs irrespective of variation in their primary amino acid sequences. Predicted PgPEPC quartenary structure is a tetramer consisting of a dimer of dimers, which is globally akin to maize PEPC crystal structure with respect to major chain folding wherein catalytically important amino acid residues of active site geometry are conserved. Recombinant PgPEPC protein expressed in E. coli and purified to homogeneity, possessed in vitro ß-carboxylation activity that is determined using a coupled reaction converting PEP into malate. Tetramer is the most active form, however, it exists in various oligomeric forms depending upon the protein concentration, pH, ionic strength of the media and presence of its substrate or effecters. Recombinant PgPEPC protein confers enhanced growth advantage to E. coli under harsh growth conditions in comparison to their respective controls; suggesting that PgPEPC plays a significant role in stress adaptation.     

Cloning,Expression and Characterization of a Lipase Encoding Gene from Human Oral Metagenome

The human oral metagenomic DNA cloned into plasmid pUC19 was used to construct a DNA library in Escherichia coli. Functional screening of 40,000 metagenomic clones led to identification of a clone LIP2 that exhibited halo on tributyrin agar plate. Sequence analysis of LIP2 insert DNA revealed a 939 bp ORF (omlip1) which showed homology to lipase 1 of Acinetobacter junii SH205. The omlip1 ORF was cloned and expressed in E. coli BL21 (DE3) using pET expression system. The recombinant enzyme was purified to homogeneity and the biochemical properties were studied. The purified OMLip1 hydrolyzed p-nitrophenyl esters and triacylglycerol esters of medium and long chain fatty acids, indicating the enzyme is a true lipase. The purified protein exhibited a pH and temperature optima of 7 and 37 °C respectively. The lipase was found to be stable at pH range of 6–7 and at temperatures lower than 40 °C. Importantly, the enzyme activity was unaltered, by the presence or absence of many divalent cations. The metal ion insensitivity of OMLip1offers its potential use in industrial processes.     

Recruiting alternative glucose utilization pathways for improving succinate production

The phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) of Escherichia coli was usually inactivated to increase PEP supply for succinate production. However, cell growth and glucose utilization rate decreased significantly with PTS inactivation. In this work, two glucose transport proteins and two glucokinases (Glk) from E. coli and Zymomonas mobilis were recruited in PTS^? strains, and their impacts on glucose utilization and succinate production were compared. All PTS^? strains recruiting Z. mobilis glucose facilitator Glf had higher glucose utilization rates than PTS^? strains using E. coli galactose permease (GalP), which was suggested to be caused by higher glucose transport velocity and lower energetic cost of Glf. The highest rate obtained by combinatorial modulation of glf and glk _{E. coli} (2.13 g/L?h) was 81 % higher than the wild-type E. coli and 30 % higher than the highest rate obtained by combinatorial modulation of galP and glk _{E. coli} . On the other hand, although glucokinase activities increased after replacing E. coli Glk with isoenzyme of Z. mobilis, glucose utilization rate decreased to 0.58 g/L?h, which was assumed due to tight regulation of Z. mobilis Glk by energy status of the cells. For succinate production, using GalP led to a 20 % increase in succinate productivity, while recruiting Glf led to a 41 % increase. These efficient alternative glucose utilization pathways obtained in this work can also be used for production of many other PEP-derived chemicals, such as malate, fumarate, and aromatic compounds.     

Structure,Regulation and Biosynthesis of Phosphoenolpyruvate Carboxylase from C4 Plants

This review attempts to summarize the large body of information on the structure, regulation and biosynthesis of the enzyme phosphoenolpyruvate carboxylase in C₄ plants which has accumulated particularly since the appearance of the last review in 1987. Among the major discoveries are the involvement of protein phosphorylation-dephosphorylation cascade in the light activation of the enzyme, extraction and characteristics of PEPC-protein serine kinase, dynamic changes in oligomeric state of the enzyme in response to pH or temperature, isolation of multiple cDNAs encoding different forms of PEPC and cloning and expression of maize/sorghum PEPC in transgenic tobacco or transformed E. coli cells. Further experiments using advanced techniques of biochemistry and molecular biology would help in understanding the molecular mechanism of reaction, regulation of enzyme activity, gene expression and evolutionary pattern of C₄ PEPC.     

Overexpression in E. coli and Purification of the L. pneumophila Lpp2981 Protein

The Lpp2981 gene from Legionella pneumophila, the causative agent of Legionnaire’s disease, was cloned into the pMWT7 plasmid. The construct was used to express this gene in Escherichia coli. Five different bacterial strains were tested to overexpress the gene but without success. Sequence analysis revealed a cluster of four rare codons near the 5′-end of the gene. These codons were replaced with those commonly used in E. coli. The mutated Lpp2981 gene was successfully expressed in all the E. coli strains tested. The expressed protein (with an apparent molecular mass of 30 kDa) was collected in the insoluble fraction of the cell lysate, purified as inclusion bodies and functionally reconstituted into liposomes. The highest level of overexpression was obtained in E. coli C0214 after 6 h of induction with isopropyl-β-d-thiogalactopyranoside at 37 °C, yielding 74 mg of purified protein per liter of culture. We conclude that the clustering of rare codons at the 5′-end of the open-reading frame is a critical factor for the heterologous expression of Lpp2981 in E. coli.     

Acetophenone reductase with extreme stability against a high concentration of organic compounds or an elevated temperature

The gene encoding acetophenone reductase (APRD), a useful biocatalyst for producing optically pure alcohols, was cloned from the cDNA of Geotrichum candidum NBRC 4597. The gene contained an open reading frame that consisted of 1,029 nucleotides corresponding to 342 amino acid residues. The subunit molecular weight was calculated to be 36.7 kDa. The predicted amino acid sequence did not have significant similarity to those of the acetophenone reductase reported previously. The gene was inserted into the pET-21b(+) expression vector and expressed in Escherichia coli Rosetta?(DE3)pLysS by induction with 1 mM of isopropyl-β-d-thiogalactopyranoside. E. coli cell-free extract gave 21.9 U/mg APRD activity, which was 81 times that of the G. candidum cell-free extract. The enzyme was purified with a HisTrap FF crude column. The enzyme exhibited the highest activity at 60 °C, and optimum reducing and oxidizing activity were observed in a pH range around 7.0–8.0 and 8.5, respectively. The enzyme was most stable at 60 °C and pH?6.5–7.5. The Vmax and the apparent Km value of the reductase were 67.6 μmol/min per milligram of protein and 0.146 mM for acetophenone, respectively. From 4 % (v/v) 4-phenyl-2-butanone, (S)-4-phenyl-2-butanol was obtained with a yield >80 % and an enantiomeric excess >99 % in a 20 h reaction recycling NADH with 15 % (v/v) 2-propanol.     

Purification and characterization of a cis-epoxysuccinic acid hydrolase from Nocardia tartaricans CAS-52, and expression in Escherichia coli

A highly enantioselective cis-epoxysuccinic acid hydrolase from Nocardia tartaricans was purified to electrophoretic homogeneity. The enzyme was purified 184-fold with a yield of 18.8 %. The purified cis-epoxysuccinic acid hydrolase had a monomeric molecular weight of 28 kDa, and its optimum conditions were 37 °C and pH 7–9. With sodium cis-epoxysuccinate as the substrate, Michaelis–Menten enzyme kinetics analysis gave a Km value of 35.71 mM and a Vmax of 2.65 mM min^?1. The enzyme was activated by Ni²⁺ and Al³⁺, while strongly inhibited by Fe³⁺, Fe²⁺, Cu²⁺, and Ag⁺. The cis-epoxysuccinic acid hydrolase gene was cloned, and its open reading frame sequence predicted a protein composed of 253 amino acids. A pET11a expression plasmid carrying the gene under the control of the T7 promoter was introduced into Escherichia coli, and the cis-epoxysuccinic acid hydrolase gene was successfully expressed in the recombinant strains.     

Purification and characterization of a chitinase from Serratia proteamaculans

A chitinase gene from Serratia proteamaculans 18A1 was cloned, sequenced, and expressed in Escherichia coli M15. Recombinant enzyme (ChiA) was purified by Ni-NTA affinity column chromatography. The ChiA gene contains an open reading frame (ORF), encoding an endochitinase with a deduced molecular weight 60 kDa and predicted isoelectric point of 6.35. Comparison of ChiA with other chitinases revealed a modular structure containing an N-terminal PKD-domain, a family 18 catalytic domain and a C-terminal putative chitin-binding domain. Turn over rate (K _cat) of the enzyme was determined using colloidal chitin (49.71 ± 1.15 S^?1) and crystalline β-chitin (17.20 ± 0.83 S^?1) as substrates. The purified enzyme was active over a broad range of pH (pH 4.5–9.0) and temperature (4–70°C) with a peak activity at pH 5.5 and 55°C. However, enzyme activity was found to be stable up to 45°C for longer incubation periods. Purified enzyme was shown to inhibit fungal spore germination and hyphal growth of pathogenic fungi Fusarium oxysporum and Aspergillus niger.     

Removal of Feedback Inhibition of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> Phosphoenolpyruvate Carboxylase by Addition of a Short Terminal Peptide

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the carboxylation of phosphoenolpyruvate (PEP) in the presence of bicarbonate to form oxaloacetate (OAA), and it plays an important role in high-efficient production of OAA-derived metabolites such as lysine, glutamate and succinate. However, PEPCs often suffered from serious feedback inhibition by various metabolic effectors like aspartate. Here, the feedback inhibition of PEPC from Corynebacterium glutamicum was removed by adding a short terminal peptide like His-tag. The effect of His-tag location on the structure and important properties such as activity, thermostability and feedback inhibition of PEPC has been investigated. The purified untagged PEPC, Nterminal His-tagged PEPC (PEPC-N-His) and C-terminal His-tagged PEPC (PEPC-C-His) were characterized. PEPCN- His (439.71/sec/mM) showed a 1.26 and 186-fold higher catalytic efficiency than untagged PEPC (348.59/sec/mM) and PEPC-C-His (2.36/sec/mM), respectively. Both PEPCN- His and untagged PEPC were significantly inhibited by aspartate at the concentrations above 4 mM (residual activities < 10%), while PEPC-C-His was almost desensitized to aspartate within 10 mM (around 90% of residual activity). Structural analysis showed that the extension of C-terminus may cause steric hindrance for aspartate binding with enzymes, leading to the deregulation of feedback inhibition of PEPC-C-His. This study provides a deeper understanding of the effect of terminal fragments on the structure and function of PEPCs, and helps to engineer the feedback inhibition of PEPCs and structurally similar enzymes.     

Recombinant thermoactive phosphoenolpyruvate carboxylase (PEPC) from Thermosynechococcus elongatus and its coupling with mesophilic/thermophilic bacterial carbonic anhydrases (CAs) for the conversion of CO2 to oxaloacetate

With the continuous increase of atmospheric CO₂ in the last decades, efficient methods for carbon capture, sequestration, and utilization are urgently required. The possibility of converting CO₂ into useful chemicals could be a good strategy to both decreasing the CO₂ concentration and for achieving an efficient exploitation of this cheap carbon source. Recently, several single- and multi-enzyme systems for the catalytic conversion of CO₂ mainly to bicarbonate have been implemented. In order to design and construct a catalytic system for the conversion of CO₂ to organic molecules, we implemented an in vitro multienzyme system using mesophilic and thermophilic enzymes. The system, in fact, was constituted by a recombinant phosphoenolpyruvate carboxylase (PEPC) from the thermophilic cyanobacterium Thermosynechococcus elongatus, in combination with mesophilic/thermophilic bacterial carbonic anhydrases (CAs), for converting CO₂ into oxaloacetate, a compound of potential utility in industrial processes. The catalytic procedure is in two steps: the conversion of CO₂ into bicarbonate by CA, followed by the carboxylation of phosphoenolpyruvate with bicarbonate, catalyzed by PEPC, with formation of oxaloacetate (OAA). All tested CAs, belonging to α-, β-, and γ-CA classes, were able to increase OAA production compared to procedures when only PEPC was used. Interestingly, the efficiency of the CAs tested in OAA production was in good agreement with the kinetic parameters for the CO₂ hydration reaction of these enzymes. This PEPC also revealed to be thermoactive and thermostable, and when coupled with the extremely thermostable CA from Sulphurhydrogenibium azorense (SazCA) the production of OAA was achieved even if the two enzymes were exposed to temperatures up to 60 °C, suggesting a possible role of the two coupled enzymes in biotechnological processes.     

Molecular cloning, characterization, and heterologous expression of a new κ-carrageenase gene from marine bacterium Zobellia sp. ZM-2

κ-Carrageenases exhibit apparent distinctions in gene sequence, molecular weight, enzyme properties, and posttranslational processes. In this study, a new κ-carrageenase gene named cgkZ was cloned from the marine bacterium Zobellia sp. ZM-2. The gene comprised an open reading frame of 1,638 bp and encoded 545 amino acids. The natural signal peptide of κ-carrageenase was used successfully for the secretory production of the recombinant enzyme in Escherichia coli. A posttranslational process that removes an amino acid sequence of about 20 kDa from the C-terminal end of κ-carrageenase was first discovered in E. coli. An increase in enzyme activity by 167.3 % in the presence of 5 mM DTT was discovered, and Na⁺ at a certain concentration range was positively correlated with enzyme activity. The κ-carrageenase production of E. coli was 9.0 times higher than that of ZM-2. These results indicate the potential use of the enzyme in the biotechnological industry.     

Characterization of a flavin-containing monooxygenase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and its application to production of indigo and indirubin

Objective

To examine the role of a gene encoding flavin-containing monooxygenase (cFMO) from Corynebacterium glutamicum ATCC13032 when cloned and expressed in Escherichia coli for the production of indigo pigments.

Results

The blue pigments produced by recombinant E. coli were identified as indigo and indirubin. The cFMO was purified as a fused form with maltose-binding protein (MBP). The enzyme was optimal at 25 °C and pH 8. From absorption spectrum analysis, the cFMO was classified as a flavoprotein. FMO activity was strongly inhibited by 1 mM Cu²⁺ and recovered by adding 1–10 mM EDTA. The enzyme catalyzed the oxidation of TMA, thiourea, and cysteamine, but not glutathione or cysteine. MBP-cFMO had an indole oxygenase activity through oxygenation of indole to indoxyl. The recombinant E. coli produced 685 mg indigo l^?1 and 103 mg indirubin l^?1 from 2.5 g l-tryptophan l^?1.

Conclusion

The results suggest the cFMO can be used for the microbial production of both indigo and indirubin.

     

Physical and kinetic properties of photosynthetic phosphoenolpyruvate carboxylase in developing apple fruit

Phosphoenolpyruvate carboxylase (PEPC) was partially purified from young developing apple fruit, cultivars Golden Delicious and Cox's Orange Pippin. Freeze-drying of tissue reduced the yield of PEPC activity compared to samples stored at 4°. Activities measured by H¹⁴CO₃⁻ incorporation exceeded the spectrophotometric assay for the enzyme with coupled NADH-malate dehydrogenase (MDH) by up to 60%. The enzyme could be stored at −16° with glycerol and bovine serum albumin for several months without loss of activity. Thermal inactivation of PEPC occurred after heating to 75° for 3 min when MDH was still slightly active. Inhibition of PEPC activity by endogenous phenolics could be prevented by grinding in liquid nitrogen in the presence of polyvinylpyrrolidine and dithiothreitol. Apparent K_m (PEP) and V_max values compared more favourably with those obtained from a C₃-species (spinach) than from a C₄-species (maize). l-Malate (5 mM) inhibited fruit PEPC by 22%; this was decreased to 12% by addition of glucose-6-phosphate (2 mM). From kinetic and effector experiments PEPC in the apple fruit is concluded to be a non-C₄ photosynthetic enzyme.     

Structural Characterization and Functional Validation of Aldose Reductase from the Resurrection Plant Xerophyta viscosa

Aldose reductases are key enzymes in the detoxification of reactive aldehyde compounds like methylglyoxal (MG) and malondialdehyde. The present study describes for first time the preliminary biochemical and structural characterization of the aldose reductase (ALDRXV4) enzyme from the resurrection plant Xerophyta viscosa. The ALDRXV4 cDNA was expressed in E. coli using pET28a expression vector, and the protein was purified using affinity chromatography. The recombinant protein showed a molecular mass of ~36 kDa. The K _M (1.2 mM) and k _cat (14.5 s^?1) of the protein determined using MG as substrate was found to be comparable with other reported homologs. Three-dimensional structure prediction based on homology modeling suggested several similarities with the other aldose reductases reported from plants. Circular dichroism spectroscopy results supported the bioinformatic prediction of alpha–beta helix nature of aldose reductase proteins. Subcellular localization studies revealed that the ALDRXV4-GFP fusion protein was localized both in the nucleus and the cytoplasm. The E. coli cells overexpressing ALDRXV4 exhibited improved growth and showed tolerance against diverse abiotic stresses induced by high salt (500 mM NaCl), osmoticum (10 % PEG 6000), heavy metal (20 mM CdCl₂), and MG (5 mM). Based on these results, we propose that ALDRXV4 gene from X. viscosa could be a potential candidate for developing stress-tolerant crop plants.     

The effect of sodium bicarbonate on plant performance and iron acquisition system of FA-5 (Forner-Alcaide 5) citrus seedlings

This work studies the effect of bicarbonate on plant performance and the iron acquisition system of Forner-Alcaide 5 (FA-5) seedlings, a citrus genotype known for its tolerance to calcareous soils. Plants were irrigated for 6 weeks with or without 10 mM NaHCO₃. Treatment significantly decreased shoot growth, photosynthetic levels and iron concentration in shoots and roots. o,o-⁵⁷FeEDDHA experiments indicated that ⁵⁷Fe uptake by roots was inhibited in treated plants. Moreover, those seedlings accumulated more ⁵⁷Fe in roots, and enhanced mRNA accumulation of ferric reductase genes FRO1 and FRO2 and FC-R activity in roots. H⁺-ATPase activity and HA1 gene expression were also increased, while HA2 was not affected. In addition, expression of the iron transporter gene IRT1 was increased, while IRT2 was not significantly affected. Finally, according to PEPC enzymatic activity, PEPC1 gene expression was higher in treated roots. In conclusion, it appears that bicarbonate prevents medium acidification by roots, thus reducing Fe²⁺ uptake. Accordingly, Fe deficiency enhanced the expression of some genes related with the Fe acquisition system (IRT1, FRO1, FRO2, HA1 and PEPC1) and the activity of the corresponding enzymes, which appear to constitute an adaptive mechanism of FA-5 in these soils.     

Combination of site-directed mutagenesis and calcium ion addition for enhanced production of thermostable MBP-fused heparinase I in recombinant Escherichia coli

Heparinase I (HepI), which specifically cleaves heparin and heparan sulfate, is one of the most extensively studied glycosaminoglycan lyases. Low productivity of HepI has largely hindered its industrial and pharmaceutical applications. Loss of bacterial HepI enzyme activity through poor thermostability during its expression and purification process in production can be an important issue. In this study, using a thermostabilization strategy combining site-directed mutagenesis and calcium ion addition during its production markedly improved the yield of maltose-binding protein-fused HepI (MBP–HepI) from recombinant Escherichia coli. Substitution of Cys297 to serine in MBP–HepI offered a 30.6 % increase in the recovered total enzyme activity due to a mutation-induced thermostabilizing effect. Furthermore, upon addition of Ca²⁺ as a stabilizer at optimized concentrations throughout its expression, extraction, and purification process, purified mutant MBP–HepI showed a specific activity of 56.3 IU/mg, 206 % higher than that of the wild type obtained without Ca²⁺ addition, along with a 177 % increase in the recovered total enzyme activity. The enzyme obtained through this novel approach also exhibited significantly enhanced thermostability, as indicated by both experimental data and the kinetic modeling. High-yield production of thermostable MBP–HepI using the present system will facilitate its applications in laboratory-scale heparin analysis as well as industrial-scale production of low molecular weight heparin as an improved anticoagulant substitute.     

Characterization and application of a newly synthesized 2-deoxyribose-5-phosphate aldolase

A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0–7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni²⁺, Ba²⁺ and Fe²⁺. The apparent K _m and V _max values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 μmol min^?1 mg^?1, for 2-deoxyribose were 0.033 mM and 2.59 μmol min^?1 mg^?1, respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.     

Gene cloning and characterization of thermostable peptidyl prolyl cis-trans isomerase (PPIase) from Bacillus stearothermophilus SIC1

The peptidyl prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) gene (ppiT) from Bacillus stearothermophilus SIC1 was cloned on the basis of a partial amino acid sequence of the purified enzyme. ppiT was found as an open reading frame (501 bases) which coded for a protein consisting of 167 amino acid residues (molecular weight, 18,349) (GenBank accession number D42050). The cloned ppiT was overexpressed in Escherichia coli cells using pET-8c as an expression vector. The enzyme was purified by heat treatment and column chromatography on DEAE-Sepharose CL-6B. Purification was about 148-fold and the molecular weight of the enzyme was estimated to be about 18.0 kDa by SDS-PAGE. PPIase activity was determined using synthetic peptide as a substrate in a 2-step reaction coupled with chymotrypsin treatment. The enzyme was stable at pH 7.5–8.0. No heat denaturation was observed when the enzyme was treated at 60°C for 30 min. The PPIase purified from recombinant E. coli has almost the same characteristics as that from B. stearothermophilus SIC1. In refolding solution, the PPIase enhanced the isomerization rate of unfolded RNase T1.