Molecular analysis of the nitrile catabolism operon of the thermophile Bacillus pallidus RAPc8

Cameron, Rory A.; Sayed, Muhammed; Cowan, Don A.

doi:10.1016/j.bbagen.2005.03.019

Cited by 43 publications

(45 citation statements)

References 52 publications

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“…1) indicated that this preparation was approximately 90% homogeneous. The putative amidase band gave an approximate subunit molecular weight of 38 kDa, a value consistent with that derived from the translated gene sequence (38.6 kDa; Cameron et al 2005). The mobility of the active enzyme on native acrylamide gel electrophoresis (data not shown) was consistent with the presence of a homohexameric native structure.…”

Section: Resultssupporting

confidence: 78%

“…With the exception of Bacillus sp. BR449 amidase, and the G. pallidus RAPc8 amidase reported in this study, all nitrile hydratase-coupled amidases belong to the signature amidase class and are typically homodimers with a native molecular weight of approximately 110 kDa (Cameron et al 2005).…”

Section: Introductionmentioning

confidence: 57%

“…In addition, the heating step increased the total yield (U) of the enzyme by about 200% (Table 1). It was previously established that approximately 30% of the expressed protein was soluble (Cameron et al 2005), and thus, this heat treatment presents a suitable method for removing insoluble aggregates. Moreover, this heat treatment effectively denatures mesophilic enzymes including proteases, which may otherwise negatively affect the biocatalyst.…”

Section: Discussionmentioning

confidence: 99%

“…The reactor, constructed from borosilicate 3.3-type glass, was purchased from Glasschem, Stellenbosch (South Africa). The E. coli strain BL 21 (DE3) (Stratagene) harboring plasmid pNH223 encoding the G. pallidus RAPc8 amidase gene was provided by Cameron et al (2005).…”

Section: Methodsmentioning

confidence: 99%

“…The open reading frame (ORF) encoded a 348-amino acid amidase (MW 38.6 kDa), located immediately downstream of ORFs encoding nitrile hydratase α and β subunits, was cloned and over-expressed in E. coli BL21 (DE3) (Cameron et al 2005). Here, we report studies of the biochemical properties of the G. pallidus RAPc8 amidase, including substrate specificity, chiral selectivity, and enzyme kinetic parameters together with operating parameters (i.e., temperature, pH, and thermostability) for the free and immobilized biocatalyst.…”

Section: Introductionmentioning

confidence: 99%

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A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity

Makhongela

Głowacka

Agarkar

et al. 2007

Appl Microbiol Biotechnol

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An amidase (EC 3.5.1.4) in branch 2 of the nitrilase superfamily, from the thermophilic strain Geobacillus pallidus RAPc8, was produced at high expression levels (20 U/mg) in small-scale fermentations of Escherichia coli. The enzyme was purified to 90% homogeneity with specific activity of 1,800 U/mg in just two steps, namely, heat-treatment and gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopic (EM) analysis of the homogenous enzyme showed the native enzyme to be a homohexamer of 38 kDa subunits. Analysis of the biochemical properties of the amidase showed that the optimal temperature and pH for activity were 50 and 7.0°C, respectively. The amidase exhibited high thermal stability at 50 and 60°C, with half-lives greater than 5 h at both temperatures. At 70 and 80°C, the half-life values were 43 and 10 min, respectively. The amidase catalyzed the hydrolysis of low molecular weight aliphatic amides, with D-selectivity towards lactamide. Inhibition studies showed activation/inhibition data consistent with the presence of a catalytically active thiol group. Acyl transfer reactions were demonstrated with acetamide, propionamide, isobutyramide, and acrylamide as substrates and hydroxylamine as the acyl acceptor; the highest reaction rate being with isobutyramide. Immobilization by entrapment in polyacrylamide gels, covalent binding on Eupergit C beads at 4°C and on Amberlite-XAD57 resulted in low protein binding and low activity, but immobilization on Eupergit C beads at 25°C with cross-linking resulted in high protein binding yield and high immobilized specific activity (80% of non-immobilized activity). Characterization of Eupergit C-immobilized preparations showed that the optimum reaction temperature was unchanged, the pH range was somewhat broadened, and stability was enhanced giving half-lives of 52 min at 70°C and 30 min at 80°C. The amidase has potential for application under high temperature conditions as a biocatalyst for D-selective amide hydrolysis producing enantiomerically pure carboxylic acids and for production of novel amides by acyl transfer.

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Section: Resultssupporting

confidence: 78%

Section: Introductionmentioning

confidence: 57%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity

Makhongela

Głowacka

Agarkar

et al. 2007

Appl Microbiol Biotechnol

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Structural insights into the thermostability mechanism of a nitrile hydratase from Caldalkalibacillus thermarum by comparative molecular dynamics simulation

Shen

Cai

et al. 2021

Proteins

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Nitrile hydratase (NHase), an excellent bio‐catalyst for the synthesis of amide compounds, was composed of two heterologous subunits. A thermoalkaliphilic NHase NHCTA1 (Tm = 71.3°C) obtained by in silico screening in our study exhibited high flexibility of α‐subunit but excellent thermostability, as opposed to previous examples. To gain a deeper structural insight into the thermostability of NHCTA1, comparative molecular dynamics simulation of NHCTA1 and reported NHases was carried out. By comparison, we speculated that β‐subunit played a key role in adjusting the flexibility of α‐subunit and the different conformations of linker in “α5‐helix‐coil ring” supersecondary structure of β‐subunit can affect the interaction between β‐subunit and α‐subunit. Mutant NHCTA1‐α6C with a random coil linker and mutant NHCTA1‐αβγ with a truncated linker were therefore constructed to understand the impact on NHCTA1 thermostability by varying the supersecondary structure. The varied thermostability of NHCTA1‐α6C and NHCTA1‐αβγ (Tmα6C = 74.4°C, Tmαβγ = 65.6°C) verified that the flexibility of α‐subunit adjusted by β‐subunit was relevant to the stability of NHCTA1. This study gained an insight into the NNHCTA1 thermostability by virtual dynamics comparison and experimental studies without crystallization, and this approach could be applied to other industrial‐important enzymes.

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Cobalt and Corrinoid Transport and Biochemistry

Cracan

Banerjee

2012

Metal Ions in Life Sciences

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In this chapter, we focus on the biochemistry of non-corrin cobalt and on a subset of corrinoid-containing enzymes. We review the import of cobalt in prokaryotes and discuss two members of the non-corrin cobalt-dependent enzymes, nitrile hydratase and methionine aminopeptidase. Cobalt is best known for its central role in alkylcorrinoid cofactors, where the unique properties of the cobalt-carbon bond are exploited to catalyze chemically challenging biotransformations. We discuss the import of corrinoids and the reactions catalyzed by the acyl-CoA mutases, the -fastest-growing subfamily of adenosylcobalamin (AdoCbl)-dependent enzymes. AdoCbl is used as a radical reservoir to catalyze 1,2 rearrangement reactions. The loading of AdoCbl-dependent enzymes with the correct cofactor form is critically important for their functions and is gated by chaperones that use the chemical energy of GTP hydrolysis to ensure the fidelity of the process. Recent insights into the organization and editing functions of G-protein chaperones in the context of AdoCbl-dependent enzymes that they support, are discussed.

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Molecular analysis of the nitrile catabolism operon of the thermophile Bacillus pallidus RAPc8

Cited by 43 publications

References 52 publications

A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity

A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity

Structural insights into the thermostability mechanism of a nitrile hydratase from Caldalkalibacillus thermarum by comparative molecular dynamics simulation

Cobalt and Corrinoid Transport and Biochemistry

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