Molecular Properties and Enhancement of Thermostability by Random Mutagenesis of Glutamate Dehydrogenase from<i>Bacillus subtilis</i>

Khan, Md. Iqbal Hassan; Ito, K; Kim, Hyeung; Ashida, Hiroyuki; Ishikawa, Takahiro; Shibata, Hitoshi; Sawa, Yousuke

doi:10.1271/bbb.69.1861

Cited by 18 publications

(10 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Random mutagenesis techniques such as errorprone PCR and DNA shuffling can be easily applied for the improvement of enzyme thermostability (8,9,12,28) because these techniques do not require detailed structural information or accurate predictions at the substituted residues (14).…”

mentioning

confidence: 99%

Improvement in the Thermostability of d -Psicose 3-Epimerase from Agrobacterium tumefaciens by Random and Site-Directed Mutagenesis

Choi

Yeom

et al. 2011

Appl Environ Microbiol

View full text Add to dashboard Cite

The S213C, I33L, and I33L S213C variants of D-psicose 3-epimerase from Agrobacterium tumefaciens, which were obtained by random and site-directed mutagenesis, displayed increases of 2.5, 5, and 7.5°C in the temperature for maximal enzyme activity, increases of 3.3-, 7.2-, and 29.9-fold in the half-life at 50°C, and increases of 3.1, 4.3, and 7.6°C in apparent melting temperature, respectively, compared with the wild-type enzyme. Molecular modeling suggests that the improvement in thermostability in these variants may have resulted from increased putative hydrogen bonds and formation of new aromatic stacking interactions. The immobilized wild-type enzyme with and without borate maintained activity for 8 days at a conversion yield of 70% (350 g/liter psicose) and for 16 days at a conversion yield of 30% (150 g/liter psicose), respectively. After 8 or 16 days, the enzyme activity gradually decreased, and the conversion yields with and without borate were reduced to 22 and 9.6%, respectively, at 30 days. In contrast, the activities of the immobilized I33L S213C variant with and without borate did not decrease during the operation time of 30 days. These results suggest that the I33L S213C variant may be useful as an industrial producer of D-psicose.D-Psicose (D-allulose), a carbon-3 epimer of D-fructose, is present in small quantities as a nonfermentable constituent of cane molasses (1), as a sugar moiety of the nucleoside antibiotic psicofuranine (4), and as a free sugar in wheat (21) and Itea plants (5). This rare sugar provides suitable sweetness, smooth texture, favorable mouthfeel, and long-time storage stability in food products (23). Psicose is considered to be a potential reduced energy sweetener because the sugar suppresses hepatic lipogenic enzyme activity (18) and does not contribute to calorie production (19).Biological production of psicose from fructose has been studied using D-psicose 3-epimerase from Agrobacterium tumefaciens (10, 13, 16) and D-tagatose 3-epimerases from Pseudomonas cichorii (7,24) and Rhodobacter sphaeroides (26). D-Psicose 3-epimerase from A. tumefaciens has been more effective than the D-tagatose 3-epimerases for the production of psicose. However, D-psicose 3-epimerase is inefficient for the industrial production of psicose because of its short half-life of 63 min at 50°C (10). Thus, improvement in the thermostability of A. tumefaciens D-psicose 3-epimerase is essential for the industrial production of psicose.Random mutagenesis and rational protein design are typical tools for improving enzyme thermostability in the protein engineering field. Random mutagenesis techniques such as errorprone PCR and DNA shuffling can be easily applied for the improvement of enzyme thermostability (8, 9, 12, 28) because these techniques do not require detailed structural information or accurate predictions at the substituted residues (14).In the present study, thermostable variants of D-psicose 3-epimerase from A. tumefaciens were obtained by random and site-directed mutagenesis. The temperature fo...

show abstract

mentioning

confidence: 99%

Improvement in the Thermostability of d -Psicose 3-Epimerase from Agrobacterium tumefaciens by Random and Site-Directed Mutagenesis

Choi

Yeom

et al. 2011

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…GDH catalyzes the reductive amination of 2-oxoglutarate and, together with GOGAT, is involved in the synthesis and degradation of L-glutamate in bacteria (for a review, see reference 17). B. subtilis RocG has been reported to have low K m values for L-glutamate (0.65 mM) and 2-oxoglutarate (0.56 mM) and a much higher K m value for ammonium (55.6 mM) (34). In a separate study, the K m values for RocG and the decryptified GudB for L-glutamate were reported to be 2.9 mM and 17.9 mM, while the K m values for these two proteins for ammonium were 18 mM and 41 mM, respectively (35).…”

Section: Geobacillus Thermodenitrificans (Yp_001125407) Bacillus Licmentioning

confidence: 99%

Functional Characterization of Key Enzymes involved in l -Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

Krog

Heggeset

Ellingsen

et al. 2013

Appl Environ Microbiol

View full text Add to dashboard Cite

bBacillus methanolicus wild-type strain MGA3 secretes 59 g/liter ؊1 of L-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in L-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low K m value for ammonium (10 mM) and a high K m value for L-glutamate (250 mM), and the V max value is 7-fold higher for L-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar K m values (1 to 1.4 mM) and V max values (4 U/mg) for both L-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating L-glutamate production in this organism.T he production of L-glutamate is a fast-growing industry, and approximately 2 million tons are produced annually, almost exclusively by Corynebacterium glutamicum fermentation using sugar-based raw materials (1). Genetic and biochemical insight into L-glutamate synthesis and degradation is of fundamental importance to understand nitrogen metabolism in the cells, as well as for metabolic engineering of L-glutamate overproduction. In C. glutamicum, L-glutamate is synthesized by both glutamate dehydrogenase (GDH) and glutamate synthase (GOGAT) (2), and L-glutamate overproduction can be achieved by downregulating the 2-oxoglutarate dehydrogenase (OGDH) activity (3, 4).In Bacillus subtilis, the gltAB operon encodes the large (GltA) and the small (GltB) subunit of GOGAT. GOGAT catalyzes a reaction where the NH 2 group from the amide group in L-glutamine is transferred to 2-oxoglutarate, forming two molecules of L-glutamate. Glutamine synthetase (GS) is encoded by glnA and catalyzes the synthesis of L-glutamine from L-glutamate and ammonia. Together, these two enzymes form the GOGAT/GS cycle ( Fig. 1) that is responsible for ammonium assimilation and L-glutamate degradation and synthesis, and B. subtilis mutants deficient in GOGAT are L-glutamate auxotrophs (5, 6). To our knowledge, the kinetic properties of GOGAT in B. subtilis have not been reported in the scientific literature.B. subtilis harbors two genes, rocG and gudB, that both encode GDHs. In the most-studied B. subtilis strain, strain 168, GudB is found to be...

show abstract

“…Khan et al described enhancement of thermostability of a glutamate dehydrogenase from Bacillus subtilis by random mutagenesis. The mutant enzyme Q144R has a Tm value of 61 o C in contrast to the wild-type, 41 o C and a high kcat for amination reaction at 37 o C, 1.3 times higher than that of the wild-type enzyme [46]. Alcade et al created a mutant library of fungal laccases by error-prone PCR and the mutant 6C9 displayed about 3.5-fold higher activities than parent type in the presence of organic solvents [47].…”

Section: Directed Evolutionmentioning

confidence: 99%

Combinatorial Synthesis and Directed Evolution Applied to the Production of α-Helix Forming Antimicrobial Peptides Analogues

Castro¹,

Cilli²,

Fontes

2006

CPPS

View full text Add to dashboard Cite

Antimicrobial peptides (AMPs) are effector molecules of innate immune systems found in different groups of organisms, including microorganisms, plants, insects, amphibians and humans. These peptides exhibit several structural motifs but the most abundant AMPs assume an amphipathic alpha-helical structure. The alpha-helix forming antimicrobial peptides are excellent candidates for protein engineering leading to an optimization of their biological activity and target specificity. Nowadays several approaches are available and this review deals with the use of combinatorial synthesis and directed evolution in order to provide a high-throughput source of antimicrobial peptides analogues with enhanced lytic activity and specificity.

show abstract

Molecular Properties and Enhancement of Thermostability by Random Mutagenesis of Glutamate Dehydrogenase fromBacillus subtilis

Cited by 18 publications

References 41 publications

Improvement in the Thermostability of d -Psicose 3-Epimerase from Agrobacterium tumefaciens by Random and Site-Directed Mutagenesis

Improvement in the Thermostability of d -Psicose 3-Epimerase from Agrobacterium tumefaciens by Random and Site-Directed Mutagenesis

Functional Characterization of Key Enzymes involved in l -Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

Combinatorial Synthesis and Directed Evolution Applied to the Production of α-Helix Forming Antimicrobial Peptides Analogues

Contact Info

Product

Resources

About

Molecular Properties and Enhancement of Thermostability by Random Mutagenesis of Glutamate Dehydrogenase fromBacillus subtilis

Cited by 18 publications

References 41 publications

Improvement in the Thermostability of d -Psicose 3-Epimerase from Agrobacterium tumefaciens by Random and Site-Directed Mutagenesis

Improvement in the Thermostability of d -Psicose 3-Epimerase from Agrobacterium tumefaciens by Random and Site-Directed Mutagenesis

Functional Characterization of Key Enzymes involved in l -Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

Combinatorial Synthesis and Directed Evolution Applied to the Production of &#945;-Helix Forming Antimicrobial Peptides Analogues

Contact Info

Product

Resources

About

Combinatorial Synthesis and Directed Evolution Applied to the Production of α-Helix Forming Antimicrobial Peptides Analogues