Expression of Secreted His-Tagged S-adenosylmethionine Synthetase in the Methylotrophic Yeast Pichia pastoris and Its Characterization, One-Step Purification, and Immobilization

Luo, Yunxing; Yuan, Zhongyi; Luo, Gaoxing; Zhao, Fukun

doi:10.1021/bp0702727

Cited by 25 publications

(31 citation statements)

References 33 publications

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“…High expression of the recombinant enzyme in a 2-L shake flask was achieved (~150 mg/L wild-type MAT, ~100 mg/L I303V MAT). The expression level of the MAT gene from E. coli is much higher than that of the MAT gene from S. cerevisiae or B. subtilis [17,26]. Compared with the low specific activity of yeast MAT (~0.4 U/mg) [17], the specific activity of wild-type MAT and its variant was 4.1 ± 0.19 U/mg and 15.8 ± 0.84 U/mg, respectively.…”

Section: Purification and Characterization Of Wild-type And I303v Matmentioning

confidence: 90%

“…For example, after incubation of the recombinant yeast MAT at 40 • C for 6 h the enzyme lost its activity completely [17]. The residual activity of the recombinant E. coli MAT incubated at 37 • C for 12 h was approximately 70%, and after 36-48 h of incubation at 37 • C the enzyme lost its activity completely (Figure 5a).…”

Section: Thermal Inactivation Of Soluble and Immobilized Enzymementioning

confidence: 98%

“…The enzyme accumulated as the induction time increased. The highest yield of the recombinant enzyme was~800 mg/L after a 16 h induction, much higher than the highest yield of recombinant yeast MAT expressed in P. pastoris KM71 strains after a 160 h induction (~200 mg/L in a bioreactor) [17]. Moreover, in order to reduce the cost of producing the enzyme, the recombinant enzyme was partially purified by ammonium sulfate (1 M) precipitation ( Figure 3b).…”

Section: Production Of Recombinant I303v Mat In a Bioreactormentioning

confidence: 99%

“…However, the production of SAM by yeast fermentation has some obvious drawbacks, such as a long fermentation period, low SAM content and yield, and low conversion rate of the substrate. Furthermore, the extraction of SAM from yeast cells is complicated, since the yeast cells have to be disrupted first and SAM is purified from the many other cell components [17].…”

Section: Introductionmentioning

confidence: 99%

“…In contrast to the MAT isoenzymes derived from animal tissues and microorganisms [17,19,20], the MAT encoded by the metK gene in Escherichia coli has some advantages, such as high specific activity and conversion efficiency, a low Km value, and a high level of expression in E. coli [21,22]. The lysates that contain the recombinant E. coli MAT have been used to synthesize SAM on the 30-mM scale.…”

Section: Introductionmentioning

confidence: 99%

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Enzymatic Synthesis of S-Adenosylmethionine Using Immobilized Methionine Adenosyltransferase Variants on the 50-mM Scale

et al. 2017

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S-adenosylmethionine (SAM), an important metabolite in all living organisms, has been widely used to treat various diseases. To develop a simple and efficient method to produce SAM, an engineered variant of the methionine adenosyltransferase (MAT) from Escherichia coli was investigated for its potential use in the enzymatic synthesis of SAM due to its significantly decreased product inhibition. The recombinant I303V MAT variant was successfully produced at a high level (~800 mg/L) with approximately four-fold higher specific activity than the wild-type MAT. The recombinant I303V MAT was covalently immobilized onto the amino resin and epoxy resin in order to obtain a robust biocatalyst to be used in industrial bioreactors. The immobilized preparation using amino resin exhibited the highest activity coupling yield (~84%), compared with approximately 3% for epoxy resin. The immobilized enzyme was more stable than the soluble enzyme under the reactive conditions, with a half-life of 229.5 h at 37 • C. The Km ATP value (0.18 mM) of the immobilized enzyme was ca. two-fold lower than that of the soluble enzyme. Furthermore, the immobilized enzyme showed high operational stability during 10 consecutive 8 h batches, with the substrate adenosine triphosphate (ATP) conversion rate above 95% on the 50-mM scale.

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Section: Purification and Characterization Of Wild-type And I303v Matmentioning

confidence: 90%

Section: Thermal Inactivation Of Soluble and Immobilized Enzymementioning

confidence: 98%

Section: Production Of Recombinant I303v Mat In a Bioreactormentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Enzymatic Synthesis of S-Adenosylmethionine Using Immobilized Methionine Adenosyltransferase Variants on the 50-mM Scale

et al. 2017

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Overexpression of methionine adenosyltransferase in Corynebacterium glutamicum for production of S‐adenosyl‐l‐methionine

Han

Wang

2015

Biotech and App Biochem

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Two genes encoding methionine adenosyltransferase, SAM2 from Saccharomyces cerevisiae and metK from Corynebacterium glutamicum, were individually cloned into pDXW-8, the shuttle vector between Escherichia coli and C. glutamicum, and overexpressed in E. coli DH5α and C. glutamicum ATCC13032. In DH5α, both genes were overexpressed and their protein products showed the activity of methionine adenosyltransferase. In ATCC13032, metK was overexpressed, its product MetK showed the enzyme activity and could convert l-methionine to S-adenosyl-l-methionine (SAM). However, when SAM2 was overexpressed in ATCC13032, neither the enzyme activity nor the conversion of SAM from l-methionine was observed. Reverse transcription PCR analysis and SDS-PAGE showed that SAM2 was transcribed but not translated in C. glutamicum. Therefore, SAM2-C, a mutant SAM2, was constructed by codon optimization, and overexpressed in ATCC13032; it was well transcribed and translated, and could convert l-methionine to SAM. Finally, SAM2-C and metK were individually overexpressed in E. coli BL21(DE3), and their products SAM2-C and MetK were purified and characterized. The optimum activity for both enzymes was found at pH 8.5 and 35 °C; SAM2-C and MetK have similar K for ATP, but quite different K for l-methionine. These results suggest that SAM2-C and MetK can be useful for developing C. glutamicum to produce SAM.

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Current awareness on yeast

2008

Yeast

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In order to keep subscribers up‐to‐date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly‐published material on yeasts. Each bibliography is divided into 10 sections. 1 Reviews; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (5 weeks journals ‐ search completed 7th. May 2008)

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Expression of Secreted His-Tagged S-adenosylmethionine Synthetase in the Methylotrophic Yeast Pichia pastoris and Its Characterization, One-Step Purification, and Immobilization

Cited by 25 publications

References 33 publications

Enzymatic Synthesis of S-Adenosylmethionine Using Immobilized Methionine Adenosyltransferase Variants on the 50-mM Scale

Enzymatic Synthesis of S-Adenosylmethionine Using Immobilized Methionine Adenosyltransferase Variants on the 50-mM Scale

Overexpression of methionine adenosyltransferase in Corynebacterium glutamicum for production of S‐adenosyl‐l‐methionine

Current awareness on yeast

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