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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.

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Related in: MedlinePlus

Effects of temperature and pH on activity. (a) Effect of temperature on the activity of the AnSHMT and the I249L-SHMT. The optimal temperature was determined by measuring the activity at temperatures from 0 to 70°C. The maximal activity was taken as 100%. (b) Effect of pH on the activity of the enzymes. The activity was measured over a pH values ranging from 2.5 to 10.5, and the maximal activity was taken as 100%. (c) Effect of temperature on the stability of the AnSHMT and the I249L-SHMT. At the optimal pH 7.5, the purified enzyme was pre-treated at a different temperature for 1 h. The activity of the enzyme without pre-incubation was defined as 100%. (d) The pH stability of the enzymes was determined by incubating the enzymes at a different pH at 4°C for 24 h. Then assays were conducted in the standard conditions and the enzyme activity without pre-treatment was taken as 100%. Error bars represent the standard deviation. Black circles represent the AnSHMT (●), and black squares represent the I249L-SHMT (■).
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Fig5: Effects of temperature and pH on activity. (a) Effect of temperature on the activity of the AnSHMT and the I249L-SHMT. The optimal temperature was determined by measuring the activity at temperatures from 0 to 70°C. The maximal activity was taken as 100%. (b) Effect of pH on the activity of the enzymes. The activity was measured over a pH values ranging from 2.5 to 10.5, and the maximal activity was taken as 100%. (c) Effect of temperature on the stability of the AnSHMT and the I249L-SHMT. At the optimal pH 7.5, the purified enzyme was pre-treated at a different temperature for 1 h. The activity of the enzyme without pre-incubation was defined as 100%. (d) The pH stability of the enzymes was determined by incubating the enzymes at a different pH at 4°C for 24 h. Then assays were conducted in the standard conditions and the enzyme activity without pre-treatment was taken as 100%. Error bars represent the standard deviation. Black circles represent the AnSHMT (●), and black squares represent the I249L-SHMT (■).

Mentions: The maximal activity of the AnSHMT was observed at 40°C, retaining over 50% of the maximal activity at temperatures from 30°C to 65°C (Figure 5a). The I249L-SHMT diaplayed the same optimum temperature (40°C) with the AnSHMT (Figure 5a). After 1 h incubation under pH 7.5 (Figure 5c), the AnSHMT retained over 35% of its maximal activity from 35°C to 45°C, but less than 15% at 55°C.Figure 5


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)

Effects of temperature and pH on activity. (a) Effect of temperature on the activity of the AnSHMT and the I249L-SHMT. The optimal temperature was determined by measuring the activity at temperatures from 0 to 70°C. The maximal activity was taken as 100%. (b) Effect of pH on the activity of the enzymes. The activity was measured over a pH values ranging from 2.5 to 10.5, and the maximal activity was taken as 100%. (c) Effect of temperature on the stability of the AnSHMT and the I249L-SHMT. At the optimal pH 7.5, the purified enzyme was pre-treated at a different temperature for 1 h. The activity of the enzyme without pre-incubation was defined as 100%. (d) The pH stability of the enzymes was determined by incubating the enzymes at a different pH at 4°C for 24 h. Then assays were conducted in the standard conditions and the enzyme activity without pre-treatment was taken as 100%. Error bars represent the standard deviation. Black circles represent the AnSHMT (●), and black squares represent the I249L-SHMT (■).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Effects of temperature and pH on activity. (a) Effect of temperature on the activity of the AnSHMT and the I249L-SHMT. The optimal temperature was determined by measuring the activity at temperatures from 0 to 70°C. The maximal activity was taken as 100%. (b) Effect of pH on the activity of the enzymes. The activity was measured over a pH values ranging from 2.5 to 10.5, and the maximal activity was taken as 100%. (c) Effect of temperature on the stability of the AnSHMT and the I249L-SHMT. At the optimal pH 7.5, the purified enzyme was pre-treated at a different temperature for 1 h. The activity of the enzyme without pre-incubation was defined as 100%. (d) The pH stability of the enzymes was determined by incubating the enzymes at a different pH at 4°C for 24 h. Then assays were conducted in the standard conditions and the enzyme activity without pre-treatment was taken as 100%. Error bars represent the standard deviation. Black circles represent the AnSHMT (●), and black squares represent the I249L-SHMT (■).
Mentions: The maximal activity of the AnSHMT was observed at 40°C, retaining over 50% of the maximal activity at temperatures from 30°C to 65°C (Figure 5a). The I249L-SHMT diaplayed the same optimum temperature (40°C) with the AnSHMT (Figure 5a). After 1 h incubation under pH 7.5 (Figure 5c), the AnSHMT retained over 35% of its maximal activity from 35°C to 45°C, but less than 15% at 55°C.Figure 5

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