Overexpression of type I L-asparaginase ofBacillus subtilis inEscherichia coli, rapid purification and characterisation of recombinant type I L-asparaginase

Yano, Shigekazu; Minato, Ryota; Thongsanit, Jaruwan; Tachiki, Takashi; Wakayama, Mamoru

doi:10.1007/bf03175579

Cited by 24 publications

(18 citation statements)

References 27 publications

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“…6). By contrast, BsAI retained only 8% of its original activity in the presence of 15% NaCl (Yano et al 2008). For BsAIIS40M and BsAIID49M, N α -acetyl-L-asparagine was a more preferred substrate than L-asparagine (Table 2).…”

Section: Enzyme Characterizationmentioning

confidence: 73%

“…Succinamic acid was also a preferred substrate for both enzymes. While succinamic acid and N α -acetyl-L-asparagine were good substrates for BsAIIS40M and BsAIID49M, they were inert substrates for BsAI (Yano et al 2008). ErAII showed low activities towards D-asparagine, succinamic acid and N α -acetyl-L-asparagine, i.e., 4%, 12%, and 17% of activity towards L-asparagine, respectively (Kotzia and Labrou 2005).…”

Section: Enzyme Characterizationmentioning

confidence: 98%

“…The enzyme assay was performed as described previously by determining the amount of ammonia formed with glutamate dehydrogenase (Yano et al 2008). One unit of enzyme was defined as the amount of enzyme that catalyzed the formation of 1 μmol ammonia per minute.…”

Section: Enzyme and Protein Assaymentioning

confidence: 99%

“…The amino acid sequence deduced from the ansA gene is similar to that deduced from the gene for E. coli asparaginase I (EcAI), whereas the sequence deduced from the ansZ gene closely resembles that deduced from the gene for E. coli L-asparaginase II (EcAII). In our previous study, we reported that the ansA gene product derived from B. subtilis was classified as L-asparaginase I (BsAI) based on its kinetic properties and the quaternary structure of the recombinant enzyme (Yano et al 2008). In this study, we describe the expression, purification, and characterisation of recombinant ansZ products derived from B. subtilis and compare its properties with those of other microbial asparaginases.…”

Section: Introductionmentioning

confidence: 96%

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Expression in Escherichia coli of a gene encoding type II l-asparaginase from Bacillus subtilis, and characterization of its unique properties

et al. 2010

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Expression of the ansZ gene encoding a putative L-asparaginase II (BsAII) from Bacillus subtilis in Escherichia coli was examined. No expression was detected in E. coli transformed with a plasmid containing the full-length ansZ gene. Three N-terminal truncated enzymes (BsAIIT18M, BsAIIS40M, and BsAIID49M) were prepared based on comparison with the N-terminal sequences of other type II L-asparaginases. BsAIIT18M became easily inactivated during DEAE-Toyopearl column chromatography. The purified N-terminal-truncated enzymes BsAIIS40M and BsAIID49M had tetrameric subunit structures and V max values of 45.5 and 45.8 U/mg towards L-asparagine, respectively. Their K m values were 2.06 and 7.02 mM, respectively. The enzymes differed from asparaginase II from E. coli and Erwinia carotovora in substrate specificity and affinity for L-asparagine. BsAIIS40M and BsAIID49M retained over 80% of their original activities in the presence of 15% NaCl, and thus may find application in the food industry for products in which NaCl is used. This study also revealed that BsAII is rather different from the type I enzyme (BsAI) from B. subtilis in substrate specificity and salt-tolerance.

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Section: Enzyme Characterizationmentioning

confidence: 73%

Section: Enzyme Characterizationmentioning

confidence: 98%

Section: Enzyme and Protein Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 96%

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Expression in Escherichia coli of a gene encoding type II l-asparaginase from Bacillus subtilis, and characterization of its unique properties

et al. 2010

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“…The enzyme is considered to play a significant role in L-asparagine metabolism in normal cells. There are two types of Lasparaginases: L-asparaginase I, was used to reduce the level of acrylamide in food industry (Friedman, 2003;Yano et al, 2008). Whereas, L-asparaginase II was used to treat leukemia.…”

Section: Introductionmentioning

confidence: 99%

Optimization, purification and characterization of recombinant L-asparaginase II in Escherichia coli

Nguyen¹,

Nguyen²,

Nguyen³

et al. 2016

Afr. J. Biotechnol.

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We studied optimal L-asparaginase sequence from GenBank accession number X12746 encoding for Lasparaginase from Erwinia chrysanthemi NCPPB1125. The expression level of recombinant Lasparaginase was determined as 78% of the total proteins. The purified L-asparaginase had a molecular mass of 37 kDa with specific activity of 312.8 U/mg. Kinetic parameters, K m , V max , K cat and K cat /K m of purified enzyme were found to be 0.5 mM, 500 U/mg, 14.9  10 3 s-1 , and 29.9  10 3 mM-1 s-1 , respectively. Temperature and pH optimum were observed at 45ºC and pH 7.5, respectively. The enzyme exhibited about 20 and 60% retention of activity following 100 min incubation at 55 or 40°C, respectively. The activity of enzyme was inhibited by EDTA, Hg 2+ , Cu 2+ , Ni 2+ , and enhanced by Mg 2+. Detergents (Tween 20, Tween 80, Triton X-100, and Triton X-114) decreased enzyme activity. DTT and DMSO at appropriate concentrations enhanced enzyme activity. In vitro anti-cancer activity was performed using different tumor cell lines. Concentration of recombinant L-asparaginase at 50 µg/ml inhibited 45.32, 48.22, 53.68, 51.22% with HL-60, P388, P3X63Ag8, SP2/0-Ag14 cell lines. Recombinant L-asparaginase was expressed successfully in Escherichia coli with high expression level, had a high specific activity and antiproliferative effect on several tumor cell lines.

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Recent research progress on microbial l-asparaginases

Zuo

Zou

Jiang

et al. 2014

Appl Microbiol Biotechnol

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L-Asparaginases (EC 3.5.1.1) are enzymes that catalyze the hydrolysis of L-asparagine to L-aspartic acid and found in a variety of organisms from microorganisms to mammals. However, they are mainly expressed and produced by microorganisms. Microbial L-asparaginases have received sustained attention due to their irreplaceable role in the therapy of acute lymphoblastic leukemia and for their inhibition of acrylamide formation during food processing. In this article, we review the application of microbial L-asparaginases in medical treatments and acrylamide mitigation. In addition, we describe in detail recent advances in the existing sources, purification, production, properties, molecular modification, and immobilization of L-asparaginase.

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Overexpression of type I L-asparaginase ofBacillus subtilis inEscherichia coli, rapid purification and characterisation of recombinant type I L-asparaginase

Cited by 24 publications

References 27 publications

Expression in Escherichia coli of a gene encoding type II l-asparaginase from Bacillus subtilis, and characterization of its unique properties

Expression in Escherichia coli of a gene encoding type II l-asparaginase from Bacillus subtilis, and characterization of its unique properties

Optimization, purification and characterization of recombinant L-asparaginase II in Escherichia coli

Recent research progress on microbial l-asparaginases

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