| A Streptomyces griseus RX-17 which produce high amount of lysozyme was isolated form soil by double layer plate containing Streptococcus mutans.Someβ-1,4-N,6-O-diacetylmuramidase components were isolated and purified after CM Sepharose Fast Flow ion exchange chromatography.In order to isolate the genes of theseβ-1,4-N,6-O-diacetylmuramidase components,Consensus-Degenerate Hybrid Oligonucleotide Primers(CODEHOP)were designed.A knownβ-1,4-N,6-O-diacetylmuramidase protein sequence named M1 in the GenBank was selected to submitted to Blast program to get 50 most homologous protein sequences. After aligned of these 50 sequences,two most conserved short amino acid sequences was determined.Meanwhile,CODEHOP PCR Primers(CD primers)were designed based on the characteristic of base in the genomic DNA of Streptomyces by an internet database program.The conserved region of gene of aβ-1,4-N,6-O-diacetylmuramidase component named R2 was amplified and isolated. Thermal Asymmetric Interlaced PCR(TAIL PCR)was performed to get the whole sequence of R2 gene.Expression ofβ-1,4-N,6-O-diacetylmuramidase R2 gene was performed in Escherichia coli.We used such plasmids as pET-28a(+),pBV220, pGEX-4T-1,pMAL-c2E and pET-26b(+),and the host cell was BL21(DE3)pLysS, Rosetta(DE3)and Rosetta-gamiTM(DE3).The result showed that no expression was detected using plasmids pET-28a(+)or pBV220.Fusion expression using pGEX-4T-1 showed that obvious protein was detected by SDS-PAGE,but nearly all the fusion protein was in the form of infusion body,which had no biological activity;only a few fusion protein was soluble.The purification of fusion protein was performed by GST Bind Resin.The purified protein was digested by thrombin,there was still no enzyme activity was detected.It is maybe the error folding of the soluble fusion protein that leads to the no activity of the expressed protein.The product of expression using plasmid pMAL-c2E was purified by Amylose Resin and claved by enterokinase,and it could be detected that the final product had biological activity.The optimum pH of recombinantβ-1,4-N,6-O-diacetylmuramidase was the same as the wide type,which was around 7.0.The optimum temperature of recombinantβ-1,4-N,6-O-diacetylmuramidase R2 was around 50℃,which is lower than the wide type.But the activity of recombinant enzyme on 37℃was much higher than the wide type.The expression in BL21(DE3)pLysS with plasmid pET-26b(+)was weak and the production had biological activity in the periplasmic space.But it was undetectable for the expression production by SDS-PAGE or the measurement of activity by turbidimetry.But the enzyme activity can be detected by means of agar plate diffusions and directed observation of the decrease of the density of host cells after induction of IPTG.β-1,4-N,6-O-diacetylmuramidase has the ability to decompose Streptococcus mutans,and it has good effect on the prevention and treatment of dental curies.But the weak acidic oral microenvironment was not suitable for theβ-1,4-N,6-O-diacetylmuramidase to play its role.So molecular modification was performed to change the optimum pH ofβ-1,4-N,6-O-diacetylmuramidase in order to meet the oral environment.The catalytically active residues ofβ-1,4-N,6-O-diacetylmuramidase were Asp-9,Asp-98,and Glu-100,which was reported by the analysis of the crystal structure ofβ-1,4-N,6-O-diacetylmuramidase Cellosyl produced by Streptomyces coelicolor.The 183-Gln was selected to be the target residue for the site-directed mutagenesis.Because it lies near the catalytically active residues,and does not take part in the substrate binding nor the catalysis. Results showed that the Gln183Glu mutation shift the optimum pH to acidic direction. The mutation Gln183Ala did not cause any changes to the optimum pH of the enzyme. The mutation Gln183Lys brought a complete vanish of protein activity.Another two amino acids were selected with 183Gln together to perform saturation mutagenesis. The result indicated that 62Tyr and 138Tyr played little effect on the optimum pH of the enzyme.The mutation of 62Tyr and 138Tyr changed the spatial structure of the active center of the protein molecular,but there was no obvious change of electric charge.It is the same to the site-directed mutagenesis that 183Gln played an important effect on the optimum pH of the enzyme catalysis.The acidic amino acid after mutation could be the proton donor which provided H+ that changes the pKa of the active center region,so the optimum pH altered.The gene of anotherβ-1,4-N,6-O-diacetylmuramidase component named R5 was isolated by a novel PCR protocol called Homologous restraint PCR(HRPCR).The primers of this PCR(HRPP)were designed to add a restriction sequence before the 5' ends of CD primers.After two steps amplification,the conserved region of R5 gene was isolated,and the whole gene was got by TAIL PCR.The whole gene of R5 protein was composed of 825 nucleotides.The homology of these two genes was only 79%.The protein sequence of R5 was submitted to SignalP3.0 server to predict the signal peptides.The result showed that the protein was a typical secret one.The phylogenetic tree showed that the genetic relationship ofβ-1,4-N,6-O-diacetylmuramidase proteins R2 and R5 produced by RX-17 was quite large.The expression of the mature peptide gene which was based on the SignalP 3.0 software ofβ-1,4-N,6-O-diacetylmuramidase R5 was performed using pMAL-c2E, and finally the product with biological activity was got.The optimum pH of recombinantβ-1,4-N,6-O-diacetylmuramidase R5 was 7.0 and the optimum temperature was 70℃.
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