The gene encoding thermostable lactate dehydrogenase (Tm-LDH) was cloned into the plasmid pHsh from Thermotoga maritima, and expressed in Escherichia coli JM 109. The recombinant protein was purified to homogeneity by a simple step, heat treatment. The recombinant enzyme had a molecular mass of 33 kDa. The optimal temperature and pH of Tm-LDH were observed 95 degrees C and 7.0. The purified enzyme had a half-life of 2 h at 90 degrees C, and exhibited better stability over a pH range from 5.5 to 8.0. The K(m) and V(max) values were 1.7 mmol/L, 3.8 x 10(4) U/mg of protein for pyruvate, and 7.2 mmol/L and 1.1 x 10(5) U/mg for NADH, respectively. The expression of Tm-LDH in T7 system could not obtain high efficiency, but it has been soluble over-expression in pHsh system and reached 340 mg/L. The superior stability and productivity of Tm-LDH will lay the foundation of its industrial-scale fermentation and application in the NAD regeneration.
Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; metabolism ; L-Lactate Dehydrogenase ; biosynthesis ; Molecular Weight ; Recombinant Proteins ; biosynthesis ; Temperature ; Thermotoga maritima ; enzymology

The xylanolytic enzymes found in Thermotoga maritima showed extremely high thermostability and considerable potential in industrial application. Yet expression level of the genes encoding these enzymes was very low. The alpha-glucuronidase gene aguA from T. maritima ATCC 43589 was cloned and expressed in several E. coli strains with different vector. The alpha-glucuronidase was overexpressed in E. coli BL21-CodonPlus(DE3)-RIL with plasmid pET-28a(+), and made up about 20% of the total proteins present in the intracellular soluble fraction. The results proved the assumption that rare codons for arginine (AGA/AGG) and isoleucine (AUA) affect the expression of aguA gene from hyperthermophilic bacterium T. maritima in E. coli. Purification procedure included two steps, heat treatment and immobilized metal affinity chromatography, and over 13.5mg of pure enzyme was obrained from 1L of induced culture. The purified enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a purification of 5.1 fold, and a yield of 55.1%. The optimum activity of recombinant alpha-glucuronidase was found at pH 6.0 and 85 degrees C, the enzyme retained 70% of its activity after 1 h of incubation at 85 degrees C. The induction conditions for expression of recombinant strain BL21-CodonPlus(DE3)-RIL/pET-28a-aguA were studied on induction time and duration by IPTG. The results showed that the activity of thermostable alpha-glucuronidase reach the maximum in 5-hour after inducted at the exponential phase (OD600 of 0.7 - 0.8).
Escherichia coli ; genetics ; Glycoside Hydrolases ; genetics ; isolation & purification ; metabolism ; Plasmids ; Recombinant Proteins ; biosynthesis ; isolation & purification ; Thermotoga maritima ; enzymology

The structure gene pelA from Thermotoga maritima MSB8 encoding pectate lyase was amplified and ligated into pHsh, resulting pHsh-pelA. Through structural optimization on pHsh-pelA, the ultimate plasmid, pHsh-pelC, which possessed the most appropriate structure and free energy of mRNA, was obtained. Pectate lyase C (PelC) was obtained after expressing pHsh-pelC in Escherichia coli JM109. The optimum activity of PelC was determined at pH 8.5 at 90 degrees C, with a half-life for almost 2 h at 95 degrees C. PelC was stable at the pH range of 8.2-9.8, and was dependent on Ca2+ for activity and stability. The enzyme kept stable for a long time and possessed a high level of activity at 60 degrees C. The kinetic assay using polygalacturonic acid (PGA) as substrate gave K(m) and V(max) of 0.11 mmol/L and 327 U per mg of protein. SDS-PAGE analysis showed that the molecular mass of the expressed recombinant PelC was about 43 kD, which was exactly the size predicted. The expression vector system of the heat shock plasmid pHsh owned such advantages as high expression level and cheap induction. Moreover, the superior stability of the recombinant enzyme laid the base for large-scale fermentation application.
Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Hot Temperature ; Polysaccharide-Lyases ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Recombination, Genetic ; Thermotoga maritima ; enzymology

The gene of beta-glucuronidase from Thermotoga maritima was cloned into the plasmid pHsh, and expressed in Escherichia coli JM109. The recombinant protein was purified to homogeneity by a simple step, heat treatment. The recombinant enzyme had a molecular mass of 65.9 kD. The optimal activity of beta-glucuronidase was found at pH 5.0 and 80 degrees C. The purified enzyme was stable over a pH range from 5.8 to 8.2 and had a half life of 2 h at 80 degrees C. The kinetic experiments at 80 degrees C with p-nitrophenyl-beta- glucuronide as substrate gave a K(m) and V(max) of 0.18 mmol/L and 312 u per mg of protein. The purified enzyme could transform glycyrrhizin to glycyrrhetinic acid.
Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; enzymology ; genetics ; Glucuronidase ; biosynthesis ; genetics ; metabolism ; Glycyrrhetinic Acid ; metabolism ; Glycyrrhizic Acid ; metabolism ; Hot Temperature ; Kinetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Thermotoga maritima ; enzymology ; genetics