Purification and Characterization of Thermostable Aspartase fromBacillussp. YM55-1

Kawata, Yasushi; Tamura, Koichi; Yano, Shigeru; Mizobata, Tomohiro; Nagai, Jun; Esaki, Nobuyoshi; Soda, Kenji; Tokushige, Masanobu; Yumoto, Noboru

doi:10.1006/abbi.1999.1186

Cited by 29 publications

(51 citation statements)

References 23 publications

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“…The aspartase has one tryptophan residue at position 86, which was unique for Bacillus YM55‐1 aspartase. This tryptophan was utilized previously as a fluorescence probe in structural stability experiments [18].…”

Section: Resultsmentioning

confidence: 99%

“…Very recently, we found a novel thermostable aspartase from Bacillus sp. YM55‐1, and purified and characterized the enzyme using biochemical and biophysical methods [18]. The enzyme showed a high activity and shared a high sequence homology in the N‐terminal region between various bacterial aspartases, while at the same time displaying a much higher resistance to thermal and chemical denaturation.…”

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confidence: 99%

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Cloning and over‐expression of thermostable Bacillus sp. YM55‐1 aspartase and site‐directed mutagenesis for probing a catalytic residue

Kawata

Tamura

Kawamura

et al. 2000

European Journal of Biochemistry

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A thermostable aspartase gene (aspB) from Bacillus sp. YM55-1 was cloned and the gene sequenced. The aspB gene (1407 bp ORF) encodes a protein with a molecular mass of 51 627 Da, consisting of 468 amino-acid residues. An amino-acid sequence comparison revealed that Bacillus YM55-1 aspartase shared 71% homology with Bacillus subtilis aspartase and 49% with Escherichia coli and Pseudomonas fluorescens aspartases. The E. coli TK237/pUCASPB strain, which was obtained by transforming E. coli TK237 (aspartase-null strain) with a vector plasmid (pUCASPB) containing the cloned aspB gene, produced a large amount of the enzyme corresponding to . 10% of the total soluble protein. The over-expressed recombinant enzyme (native molecular mass: 200 kDa) was purified effectively and rapidly using heat treatment and affinity chromatography. In order to probe the catalytic residues of this enzyme, two conserved amino-acid residues, Lys183 and His134, were individually mutated to alanine. Although the tertiary structure of each mutant was estimated to be the same as that of wild-type aspartase in CD and fluorescence measurements, the Lys183Ala mutant lost its activity completely, whereas His134Ala retained full activity. This finding suggests that Lys183 may be involved in the catalytic activity of this thermostable Bacillus YM55-1 aspartase.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Cloning and over‐expression of thermostable Bacillus sp. YM55‐1 aspartase and site‐directed mutagenesis for probing a catalytic residue

Kawata

Tamura

Kawamura

et al. 2000

European Journal of Biochemistry

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“…To prevent partial inactivation of aspartase at 50 °C, higher temperature-resistant enzymes such as Bacillus sp. YM55-1 [8] or Cytophaga sp. KUC-1 [9] aspartases could be used, although the activity of recombinant E. coli aspartase in this study was stable after heat treatment at 50 °C for 15 min and remained so in immobilized PSCat over 9 reuse cycles.…”

Section: Discussionmentioning

confidence: 98%

“…The relative activity of native aspartase in E. coli was decreased to 40-47 % of that in control cells by heat treatment at 50 °C for 10 min [4,8]. To prevent partial inactivation of aspartase at 50 °C, higher temperature-resistant enzymes such as Bacillus sp.…”

Section: Discussionmentioning

confidence: 99%

Efficient aspartic acid production by a psychrophile-based simple biocatalyst

Tajima

Hamada

Nakashimada

et al. 2015

Journal of Industrial Microbiology and Biotechnology

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We previously constructed a Psychrophile-based Simple bioCatalyst (PSCat) reaction system, in which psychrophilic metabolic enzymes are inactivated by heat treatment, and used it here to study the conversion of aspartic acid from fumaric acid mediated by the activity of aspartate ammonia-lyase (aspartase). In Escherichia coli, the biosynthesis of aspartic acid competes with that of L-malic acid produced from fumaric acid by fumarase. In this study, E. coli aspartase was expressed in psychrophilic Shewanella livingstonensis Ac10 heat treated at 50 °C for 15 min. The resultant PSCat could convert fumaric acid to aspartic acid without the formation of L-malic acid because of heat inactivation of psychrophilic fumarase activity. Furthermore, alginate-immobilized PSCat produced high yields of aspartic acid and could be re-used nine times. The results of our study suggest that PSCat can be applied in biotechnological production as a new approach to increase the yield of target compounds.

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“…A highly thermostable aspartase has been identified in Bacillus sp. YM55-1 [124]. On-going engineering projects will certainly benefit from the recently elucidated X-ray crystal structures of the E. coli aspartase [125] and of the thermostable aspartase from Bacillus sp.…”

Section: Ammonia Lyase Processesmentioning

confidence: 99%