Arginase from Bacillus thuringiensis SK 20.001: Purification, characteristics, and implications for l-ornithine biosynthesis

Zhang, Tao; Guo, Yujie; Zhang, Hao; Mu, Wanmeng; Miao, Ming

doi:10.1016/j.procbio.2013.02.023

Cited by 24 publications

(14 citation statements)

References 22 publications

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“…The addition of Mn 2+ increased enzyme activity by eightfold. This result agreed well with results from most organisms, such as human liver , B. caldovelox , and B. thuringiensis . It is reported that the active site of l ‐arginase contains two Mn 2+ ions (Mn 2+ A and Mn 2+ B ), which are coordinated by Asp and His residues .…”

Section: Resultssupporting

confidence: 90%

“…The mixture was then incubated at pH 9.0 and 40 °C for 2 H. The yield of l ‐ornithine without addition of Mn 2+ was determined to be 9.18 g/L, and the molar yield was 24.2%. However, the yield of l ‐ornithine with addition of Mn 2+ was determined to be 36.9 g/L and the molar yield was 97.2%, which was higher than the molar yield from calf liver and B. thuringiensis .…”

Section: Resultsmentioning

confidence: 76%

“…The molecular mass of the native enzyme was estimated to be approximately 192.0 kDa as determined by gel‐filtration chromatography. This result indicated that the recombinant l ‐arginase formed a hexamer, which was like l ‐arginase of B. caldovelox and Bacillus thuringiensis . However, the enzymes from the animals were usually trimers, and the molecular mass was ranging from 94 to 120 kDa .…”

Section: Resultsmentioning

confidence: 93%

“…The effects of buffers were also determined, and showed that glycine–NaOH buffer was superior to the other buffers. The optimal pH for other l ‐arginases, such as B. thuringiensis is pH 10.0, S. cerevisiae is pH 9.5, Bacillus brevis and B. caldovelox are pH 9.0, and human liver is pH 8.0. However, the maximal activity of H. pylori is at pH 6.0.…”

Section: Resultsmentioning

confidence: 99%

“…The final concentrations of Mn 2+ were 0 and 2 mM, respectively. The conversion was performed in a shaking water bath at pH 9.0 and 40 °C for 2 H. The products and substrate were analyzed by high‐performance liquid chromatography .…”

Section: Methodsmentioning

confidence: 99%

See 4 more Smart Citations

Cloning, expression, and characterization of a thermostablel‐arginase fromGeobacillus thermodenitrificansNG80‐2 forl‐ornithine production

Huang

Zhang

et al. 2015

Biotech and App Biochem

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Arginase (l-arginine amidinohydrolase, EC 3.5.3.1) can efficiently catalyze conversion of arginine to ornithine. Therefore, this enzyme can be used to produce l-ornithine from l-arginine. In this article, the l-arginase gene encoding the Geobacillus thermodenitrificans NG80-2 was cloned and overexpressed in Escherichia coli. The specific activity of the purified enzyme was 138.3 U/mg. The molecular mass of the l-arginase was approximately 33.0 kDa as estimated by SDS-PAGE and 192.0 kDa as determined by gel-filtration chromatography. Manganese ions were the optimum metal cofactor for activity, whereas the enzyme was slightly inhibited by Mg(2+) , Cu(2+) , Ba(2+) , Ca(2+) , and Zn(2+) . Activity was optimal at pH 9.0 and 80 °C, and the protein was stable at 40 and 50 °C. The recombinant enzyme was a uricotelic arginase. Using arginine as the substrate, the Michaelis-Menten constant (Km ) and catalytic efficiency (kcat /Km ) were measured to be 171.9 mM and 3.8 mM(-1) s(-1) , respectively. Trp and His residues were directly involved in the l-arginase activity evaluated by inactivation agents. The biosynthesis yield of l-ornithine by the purified enzyme was 36.9 g/L, and the molar yield was 97.2%.

show abstract

Section: Resultssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 76%

Section: Resultsmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Cloning, expression, and characterization of a thermostablel‐arginase fromGeobacillus thermodenitrificansNG80‐2 forl‐ornithine production

Huang

Zhang

et al. 2015

Biotech and App Biochem

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Ratiometric fluorescent l‐arginine and l‐asparagine biosensors based on the oxazine 170 perchlorate‐ethyl cellulose membrane

Duong

Rhee

2017

Engineering in Life Sciences

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Ratiometric fluorescent l-arginine (Arg) and l-asparagine (Asn) biosensors were developed using an oxazine 170 perchlorate (O17) ethyl cellulose (EC) membrane and the enzymes entrapped into the matrix of EC and hydrogel polyurethane. The sensing principles were based on the hydrolysis reactions of urea and l-Arg under the catalysis of the urease and arginase to produce ammonia in the case of an l-Argsensing membrane and also on the hydrolysis reaction of l-Asn under the catalysis of asparaginase in the case of an l-Asn-sensing membrane. The O17-EC membrane reacted with the ammonia produced from the hydrolysis reactions and changed the fluorescence intensities at λ em = 565 and 625 nm. The ratio of the fluorescence intensities at λ em = 565 and 625 nm was proportional to the concentrations of l-Arg or l-Asn in the range of 0.1-10 mM. The LOD of the l-Arg-and l-Asn-sensing membranes was 0.082 ± 0.0014 and 0.074 ± 0.0023 mM, respectively. The sensing membranes also showed good quality in terms of response time, reversibility, and stability. The interference study demonstrated that some components such as amino acids had little negative effects on the performance of the sensing membranes for the detection of l-Arg and l-Asn. These simple and sensitive ratiometric fluorescent sensing membranes provide a basic or comprehensive method for detecting l-Arg and l-Asn in blood and urine samples as well as in the fermentation processes.Keywords: l-Arginine / l-Asparagine / O17-EC membrane / Ratiometric fluorescent biosensor Additional supporting information may be found in the online version of this article at the publisher's web-site

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Overproduction of Rummeliibacillus pycnus arginase with multi-copy insertion of the arg R.pyc cassette into the Bacillus subtilis chromosome

Huang

Zhang

Yan

et al. 2017

Appl Microbiol Biotechnol

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A plasmid-less and marker-less strain with multi-copy integration of the arginase gene from Rummeliibacillus pycnus was constructed using Bacillus subtilis 168 as a host. A total of nine copies of the arg cassettes, in which the R. pycnus arginase gene was fused with the strong promoter P43, were inserted into the recipient chromosome. These multiple insertions were completed via step-by-step integrations into designed (2 copies) and random (9 copies) sites, respectively. A strategy for random site integration was developed based on the construction of the arg cassette sandwiched between "front" and "back" homologous arms which were randomly restricted from chromosomal DNA. An antibiotic resistance marker was applied in transformant selection and was eliminated via the Cre/lox system. Performance showed that the highest enzyme activity (14.5 U/mL) was obtained after culture in flasks, and this segregation stable strain could efficiently hydrolyze L-arginine with a 97.2% molar yield, showing potential application in the food industry.

show abstract

Arginase from Bacillus thuringiensis SK 20.001: Purification, characteristics, and implications for l-ornithine biosynthesis

Cited by 24 publications

References 22 publications

Cloning, expression, and characterization of a thermostablel‐arginase fromGeobacillus thermodenitrificansNG80‐2 forl‐ornithine production

Cloning, expression, and characterization of a thermostablel‐arginase fromGeobacillus thermodenitrificansNG80‐2 forl‐ornithine production

Ratiometric fluorescent l‐arginine and l‐asparagine biosensors based on the oxazine 170 perchlorate‐ethyl cellulose membrane

Overproduction of Rummeliibacillus pycnus arginase with multi-copy insertion of the arg R.pyc cassette into the Bacillus subtilis chromosome

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