| Glucose dehydrogenase could potentially be used for coenzyme regeneration in practical applications due to the advantages of its high activity and dual-cofactor specificity. We have cloned several genes encoding glucose dehydrogenase from strains of Bacillus and related species, and glucose dehydrogenase of highest activity from Lysinibacillus sphaericus G10 was chosen for further study. The enzymatic properties of recombinant LsGDH and alkali resistance mechanism was studyed; a mutant with improved thermal stability was obtained by rational design; and a strategy for one-step cloning of glucose dehydrogenase directly from the soil metagenome was invented. The main results are as follows:1) Cloning, expression and characterization of LsGDHThe full-length nucleotide sequence of LsGDH from L. sphaericus G10 was obtained by using degenerate PCR and chromosome walking. The gene constituted by 786 bp nucleotides, which encoding 262 amino acids. The activity of recombinant LsGDH was 205.7±6.9 U/mg at 25℃. LsGDH was determined to be a homologous tetramer by SDS-PAGE and size exclusion chromatography. The recombinant LsGDH exhibited maximum activity at pH 9.5 and 50℃; the enzyme was stable just at the temperature below 35℃, but very stable at a wide pH ranging from 6.5 to 10.5. The stability of LsGDH in the chain alkanol can be described by the Gaussian distribution model (R2≥0.94).2) The alkali-resistant mechanism of LsGDHThe alkali-resistant mechanism was investigated by theoretical analysis and functional experiment. The results of site-directed mutagenesis showed that the type of the 17th amino acid played a crucial role for the pH stability of LsGDH and the 114th amino acids accounted for about 20% contribution to the pH stability. The analysis of electrostatic interaction energy and hydrophobic interaction energy after energy minimization showed that electrostatic interactions was the main factor affected the pH stability of LsGDH, rather than the hydrophobic interactions.3) The rational design to improve the thermal stability of LsGDHA mutant with signicicantly improved thermal stability, DS255, has been designed by introducing disulfide bonds at the subunits intersurface. About 60% of the mutant formed disulfide bonds during the purification process, and almost all the DS255 formed disulfide bonds through the natural air oxidation after placing at 4℃for one week. The Tm value of DS255 is 40.5℃higher than wild-type LsGDH. The affinity to substrate and coenzyme of DS255 was about three times lower than wild-type, but the kcat value increased about 50%, meanwhile the soluble expression was little lower than wild-type. These results indicated that the strategy for increasing the thermal stability of LsGDH by introduction of disulfide bonds between subunits was successful, and greatly enhanced the practical value of LsGDH.4) one-step strategy for cloning of glucose dehydrogenase from soil metagenomic DNA.A strategy for one-step cloning of glucose dehydrogenase directly from soil metagenomic DNA was invented. Study on the conditions of the amplification process showed that the purity and complexity of the metagenomic DNA is essential to amplication. Furthermore, the degeneracy of the primers should be as low as possible and the desired length of the amplified fragment had better less than 2 kb. This method obviates the cumbersome chromosome walking process, and the obtained DNA fragments can be directly used for directed evolution mediated by DNA shuffling without sequencing.
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