Limits...
A novel serine hydroxymethyltransferase from Arthrobacter nicotianae: characterization and improving catalytic efficiency by rational design.

Jiang W, Chen L, Hu N, Yuan S, Li B, Liu Z - BMC Biotechnol. (2014)

Bottom Line: Serine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity.The obtained protein AnSHMT showed the optimal activity at 40 °C and pH 7.5, and was more stable in weakly alkali conditions (pH 6.5-8.5) than Hyphomicrobium methylovorum's SHMT (pH 6.0-7.5), In order to improve the catalytic efficiency of the wild type, the site-directed mutagenesis based on sequences alignment and bioinformatics prediction, was used and the catalytic efficiency of the mutant I249L was found to be 2.78-fold higher than that of the wild-type, with the replacement of isoleucine by leucine at the 249 position.This research provides useful information about the interesting site, and the application of DOP-PCR in cloning a novel glyA gene.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, P. R. China. tianya416@126.com.

ABSTRACT

Background: Serine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity.

Results: Here, a novel SHMT gene, glyA, was obtained through degenerate oligonucleotide-primed PCR and encoded a novel SHMT with 54.3% similarity to the known SHMT from Escherichia coli. The obtained protein AnSHMT showed the optimal activity at 40 °C and pH 7.5, and was more stable in weakly alkali conditions (pH 6.5-8.5) than Hyphomicrobium methylovorum's SHMT (pH 6.0-7.5), In order to improve the catalytic efficiency of the wild type, the site-directed mutagenesis based on sequences alignment and bioinformatics prediction, was used and the catalytic efficiency of the mutant I249L was found to be 2.78-fold higher than that of the wild-type, with the replacement of isoleucine by leucine at the 249 position.

Conclusions: This research provides useful information about the interesting site, and the application of DOP-PCR in cloning a novel glyA gene.

Show MeSH

Related in: MedlinePlus

Design of both end degenerate primers and 12%SDS-PAGE analysis of the purified proteins.(a and b) The glyA gene sequences were obtained from NCBI database and belonged to the genus of Arthrobacter (GI: NC_008541; NC_008711.1; NC_011886.1; NC_014550.1; NC_018531.1). Degenerate primers were designed by the regions in the boxes. (c) 12% SDS-PAGE analysis of the purified proteins. The bands in the ellipses show GST (glutathione-S-transferase) and fusion protein (AnSHMT and GST) respectively. Lane 1: purified I249L without GST. Lane 2: purified AnSHMT (the wild-type enzyme) without GST. Lane 3: protein marker. Lane 4: recombinant bacterium (harboring pGEX-6P-glyA) induced by IPTG. Lane 5: recombinant bacterium (harboring pGEX-6P-glyA) non-induced by 0.1 mM IPTG. Lane 6: bacterium (harboring pGEX-6p-1) induced by IPTG. Lane 7: bacterium (harboring pGEX-6p-1) non-induced by 0.1 mM IPTG. The protein molecular weight ladder is Unstained Protein Molecular Weight Marker (Fermentas, Canada).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4260256&req=5

Fig4: Design of both end degenerate primers and 12%SDS-PAGE analysis of the purified proteins.(a and b) The glyA gene sequences were obtained from NCBI database and belonged to the genus of Arthrobacter (GI: NC_008541; NC_008711.1; NC_011886.1; NC_014550.1; NC_018531.1). Degenerate primers were designed by the regions in the boxes. (c) 12% SDS-PAGE analysis of the purified proteins. The bands in the ellipses show GST (glutathione-S-transferase) and fusion protein (AnSHMT and GST) respectively. Lane 1: purified I249L without GST. Lane 2: purified AnSHMT (the wild-type enzyme) without GST. Lane 3: protein marker. Lane 4: recombinant bacterium (harboring pGEX-6P-glyA) induced by IPTG. Lane 5: recombinant bacterium (harboring pGEX-6P-glyA) non-induced by 0.1 mM IPTG. Lane 6: bacterium (harboring pGEX-6p-1) induced by IPTG. Lane 7: bacterium (harboring pGEX-6p-1) non-induced by 0.1 mM IPTG. The protein molecular weight ladder is Unstained Protein Molecular Weight Marker (Fermentas, Canada).

Mentions: The glyA was cloned into the vector pGEX-6p-1 and expressed in DE3. The induced and non-induced recombinant bacteria (harboring pGEX-6p-glyA) and the induced control bacterium (harboring empty pGEX-6p-1 vector) were examined with SDS-PAGE (Figure 4c). After purification with Glutathione Sepharose 4B and digestion with 3C protease, the recombinant AnSHMT and I249L-SHMT were harvested and resolved to a single band, with the purified AnSHMT exhibiting the expected molecular mass (47.3 kDa) (Figure 4c).Figure 4


A novel serine hydroxymethyltransferase from Arthrobacter nicotianae: characterization and improving catalytic efficiency by rational design.

Jiang W, Chen L, Hu N, Yuan S, Li B, Liu Z - BMC Biotechnol. (2014)

Design of both end degenerate primers and 12%SDS-PAGE analysis of the purified proteins.(a and b) The glyA gene sequences were obtained from NCBI database and belonged to the genus of Arthrobacter (GI: NC_008541; NC_008711.1; NC_011886.1; NC_014550.1; NC_018531.1). Degenerate primers were designed by the regions in the boxes. (c) 12% SDS-PAGE analysis of the purified proteins. The bands in the ellipses show GST (glutathione-S-transferase) and fusion protein (AnSHMT and GST) respectively. Lane 1: purified I249L without GST. Lane 2: purified AnSHMT (the wild-type enzyme) without GST. Lane 3: protein marker. Lane 4: recombinant bacterium (harboring pGEX-6P-glyA) induced by IPTG. Lane 5: recombinant bacterium (harboring pGEX-6P-glyA) non-induced by 0.1 mM IPTG. Lane 6: bacterium (harboring pGEX-6p-1) induced by IPTG. Lane 7: bacterium (harboring pGEX-6p-1) non-induced by 0.1 mM IPTG. The protein molecular weight ladder is Unstained Protein Molecular Weight Marker (Fermentas, Canada).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4260256&req=5

Fig4: Design of both end degenerate primers and 12%SDS-PAGE analysis of the purified proteins.(a and b) The glyA gene sequences were obtained from NCBI database and belonged to the genus of Arthrobacter (GI: NC_008541; NC_008711.1; NC_011886.1; NC_014550.1; NC_018531.1). Degenerate primers were designed by the regions in the boxes. (c) 12% SDS-PAGE analysis of the purified proteins. The bands in the ellipses show GST (glutathione-S-transferase) and fusion protein (AnSHMT and GST) respectively. Lane 1: purified I249L without GST. Lane 2: purified AnSHMT (the wild-type enzyme) without GST. Lane 3: protein marker. Lane 4: recombinant bacterium (harboring pGEX-6P-glyA) induced by IPTG. Lane 5: recombinant bacterium (harboring pGEX-6P-glyA) non-induced by 0.1 mM IPTG. Lane 6: bacterium (harboring pGEX-6p-1) induced by IPTG. Lane 7: bacterium (harboring pGEX-6p-1) non-induced by 0.1 mM IPTG. The protein molecular weight ladder is Unstained Protein Molecular Weight Marker (Fermentas, Canada).
Mentions: The glyA was cloned into the vector pGEX-6p-1 and expressed in DE3. The induced and non-induced recombinant bacteria (harboring pGEX-6p-glyA) and the induced control bacterium (harboring empty pGEX-6p-1 vector) were examined with SDS-PAGE (Figure 4c). After purification with Glutathione Sepharose 4B and digestion with 3C protease, the recombinant AnSHMT and I249L-SHMT were harvested and resolved to a single band, with the purified AnSHMT exhibiting the expected molecular mass (47.3 kDa) (Figure 4c).Figure 4

Bottom Line: Serine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity.The obtained protein AnSHMT showed the optimal activity at 40 °C and pH 7.5, and was more stable in weakly alkali conditions (pH 6.5-8.5) than Hyphomicrobium methylovorum's SHMT (pH 6.0-7.5), In order to improve the catalytic efficiency of the wild type, the site-directed mutagenesis based on sequences alignment and bioinformatics prediction, was used and the catalytic efficiency of the mutant I249L was found to be 2.78-fold higher than that of the wild-type, with the replacement of isoleucine by leucine at the 249 position.This research provides useful information about the interesting site, and the application of DOP-PCR in cloning a novel glyA gene.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, P. R. China. tianya416@126.com.

ABSTRACT

Background: Serine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity.

Results: Here, a novel SHMT gene, glyA, was obtained through degenerate oligonucleotide-primed PCR and encoded a novel SHMT with 54.3% similarity to the known SHMT from Escherichia coli. The obtained protein AnSHMT showed the optimal activity at 40 °C and pH 7.5, and was more stable in weakly alkali conditions (pH 6.5-8.5) than Hyphomicrobium methylovorum's SHMT (pH 6.0-7.5), In order to improve the catalytic efficiency of the wild type, the site-directed mutagenesis based on sequences alignment and bioinformatics prediction, was used and the catalytic efficiency of the mutant I249L was found to be 2.78-fold higher than that of the wild-type, with the replacement of isoleucine by leucine at the 249 position.

Conclusions: This research provides useful information about the interesting site, and the application of DOP-PCR in cloning a novel glyA gene.

Show MeSH
Related in: MedlinePlus