Biochemical characterization of Alanine racemase- a spore protein produced by Bacillus anthracis

Kanodia, Shivani; Agarwal, Shivangi; Singh, Priyanka; Singh, Preeti Gunjan; Bhatnagar, Rakesh

doi:10.5483/bmbrep.2009.42.1.047

Cited by 12 publications

(19 citation statements)

References 23 publications

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“…Up to now, it has been shown that many of alanine racemase need PLP as a cofactor [Francois and Kappock, 2007]. However, an alanine racemase from Bacillus anthracis [Kanodia et al, 2008] showed considerable activity without PLP and the activity increased by addition of PLP [Kanodia et al, 2008]. The alanine racemase, in this experiment, is similar to the alanine racemase from B.anthracis in the light of PLP dependency.…”

Section: Resultssupporting

confidence: 51%

Biochemical Characteristics of an Alanine Racemase from Xanthomonas oryzae pv. oryzae

Kang¹,

Yoon²,

Lee³

et al. 2011

Journal of Applied Biological Chemistry

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A gene encoding a putative alanine racemase in Xanthomonas. oryzae pv. oryzae was cloned, expressed and characterized. Expression of the cloned gene was performed in Escherichia coli BL21(DE3)pLys using a pET-21(a) vector harbouring 6 × histidine tag. Purification of the recombinant alanine racemase by affinity chromatography resulted in major one band by sodium dodecyl sulfate polyacryl amide gel electrophoresis analysis, showing about 45 kDa of molecular weight. The alanine racemase gene, cloned in this experiment, appears to be constitutively expressed in X. oryzae, as analyzed by reverse transcriptase polymerase chain reaction. The enzyme was the most active toward L-alanine and secondly D-alanine, showing a racemic reaction, thus the enzyme is considered as an alanine racemase. The enzyme was considerably activated by addition of pyridoxal-5-phosphate (PLP), showing that 75% increase in activity was observed at 0.3 mM, compared with control. D-Cysteine as well as L-cysteine significantly inhibited the enzyme activity. The inhibitions by cysteines were more prominent in the absence of PLP, showing 9 and 5% of control activity at 2 mM of addition, respectively. The enzyme was the most active at pH 8.0 and more stable at alkaline pHs than acidic pH condition.

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Section: Resultssupporting

confidence: 51%

Biochemical Characteristics of an Alanine Racemase from Xanthomonas oryzae pv. oryzae

Kang¹,

Yoon²,

Lee³

et al. 2011

Journal of Applied Biological Chemistry

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“…The primary amino acid sequence of L. salivarius ALR was analyzed and compared with those of previously reported ALRs (Johnston and Diven 1969 ; Inagaki et al 1986 ; Kanda-Nambu et al 2000 ; Ju et al 2009 ; Kanodia et al 2009 ; Liu et al 2012 ). The amino acid sequences of L. salivarius UCC118, B. pseudofirmus OF4 and G. stearothermophilus IFO 12550 ALRs were retrieved from the UniProt data base ( http://www.uniprot.org/ ), and that of P. putida YZ-26 was obtained from the report by Liu et al ( 2012 ).…”

Section: Methodsmentioning

confidence: 99%

“…In the present study, we expressed this L. salivarius ALR gene in Escherichia coli and, interestingly, found that the purified ALR protein was strongly activated in the presence of carboxylates such as acetate and propionate. By contrast, ALRs from Geobacillus and Bacillus species are reportedly inhibited by acetate and propionate (Morollo et al 1999 ; Kanodia et al 2009 ). In addition, ALRs from L. fermentum and Pseudomonas putida are known to be stabilized by acetate, although the mechanism for this effect is not yet known (Johnston and Diven.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Lactobacillus salivarius alanine racemase: short-chain carboxylate-activation and the role of A131

et al. 2015

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Many strains of lactic acid bacteria produce high concentrations of d-amino acids. Among them, Lactobacillus salivarius UCC 118 produces d-alanine at a relative concentration much greater than 50 % of the total d, l-alanine (100d/d, l-alanine). We characterized the L. salivarius alanine racemase (ALR) likely responsible for this d-alanine production and found that the enzyme was activated by carboxylates, which is an unique characteristic among ALRs. In addition, alignment of the amino acid sequences of several ALRs revealed that A131 of L. salivarius ALR is likely involved in the activation. To confirm that finding, an L. salivarius ALR variant with an A131K (ALRA131K) substitution was prepared, and its properties were compared with those of ALR. The activity of ALRA131K was about three times greater than that of ALR. In addition, whereas L. salivarius ALR was strongly activated by low concentrations (e.g., 1 mM) of short chain carboxylates, and was inhibited at higher concentrations (e.g., 10 mM), ALRA131K was clearly inhibited at all carboxylate concentrations tested (1–40 mM). Acetate also increased the stability of ALR such that maximum activity was observed at 35 °C and pH 8.0 without acetate, but at 50 °C in the presence of 1 mM acetate. On the other hand, maximum ALRA131K activity was observed at 45 °C and around pH 9.0 with or without acetate. It thus appears that A131 mediates the activation and stabilization of L. salivarius ALR by short chain carboxylates.

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“…Assays were performed to measure alanine racemase on the surface of B. anthracis spores (66,67). Two individual preparations of B. anthracis Sterne spores were diluted to a working stock concentration of 1 ϫ 10 8 CFU/ml.…”

Section: Bacillus Anthracismentioning

confidence: 99%

A Standard Method To Inactivate Bacillus anthracis Spores to Sterility via Gamma Irradiation

Cote

Buhr

Bernhards

et al. 2018

Appl Environ Microbiol

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In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped preparations of gamma-irradiated spores of Bacillus anthracis that contained live spores. In response, a systematic evidence-based method for preparing, concentrating, irradiating, and verifying the inactivation of spore materials was developed. We demonstrate the consistency of spore preparations across multiple biological replicates and show that two different DoD institutions independently obtained comparable dose-inactivation curves for a monodisperse suspension of B. anthracis spores containing 3 ϫ 10 10 CFU. Spore preparations from three different institutions and three strain backgrounds yielded similar decimal reduction (D 10 ) values and irradiation doses required to ensure sterility (D SAL ) to the point at which the probability of detecting a viable spore is 10 Ϫ6 . Furthermore, spores of a genetically tagged strain of B. anthracis strain Sterne were used to show that high densities of dead spores suppress the recovery of viable spores. Together, we present an integrated method for preparing, irradiating, and verifying the inactivation of spores of B. anthracis for use as standard reagents for testing and evaluating detection and diagnostic devices and techniques. IMPORTANCEThe inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of live Bacillus anthracis (anthrax) spores to U.S. and international destinations revealed the need to standardize inactivation methods for materials derived from biological select agents and toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of such an event. Following a retrospective analysis of the procedures previously employed to generate inactivated B. anthracis spores, a study was commissioned by the DoD to provide data required to support the production of inactivated spores for the biodefense community. The results of this work are presented in this publication, which details the method by which spores can be prepared, irradiated, and tested, such that the chance of finding residual living spores in any given preparation is 1/1,000,000. These irradiated spores are used to test equipment and methods for the detection of agents of biological warfare and bioterrorism.

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Biochemical characterization of Alanine racemase- a spore protein produced by Bacillus anthracis

Cited by 12 publications

References 23 publications

Biochemical Characteristics of an Alanine Racemase from Xanthomonas oryzae pv. oryzae

Biochemical Characteristics of an Alanine Racemase from Xanthomonas oryzae pv. oryzae

Characterization of Lactobacillus salivarius alanine racemase: short-chain carboxylate-activation and the role of A131

A Standard Method To Inactivate Bacillus anthracis Spores to Sterility via Gamma Irradiation

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