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Strategic single point mutation yields a solvent- and salt-stable transaminase from Virgibacillus sp. in soluble form.

ABSTRACT: A new transaminase (VbTA) was identified from the genome of the halotolerant marine bacterium Virgibacillus 21D. Following heterologous expression in Escherichia coli, it was located entirely in the insoluble fraction. After a single mutation, identified via sequence homology analyses, the VbTA T16F mutant was successfully expressed in soluble form and characterised. VbTA T16F showed high stability towards polar organic solvents and salt exposure, accepting mainly hydrophobic aromatic amine and carbonyl substrates. The 2.0 Å resolution crystal structure of VbTA T16F is here reported, and together with computational calculations, revealed that this mutation is crucial for correct dimerisation and thus correct folding, leading to soluble protein expression.

SUBMITTER: Guidi B

PROVIDER: S-EPMC6219536 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Strategic single point mutation yields a solvent- and salt-stable transaminase from Virgibacillus sp. in soluble form.

Guidi Benedetta B Planchestainer Matteo M Contente Martina Letizia ML Laurenzi Tommaso T Eberini Ivano I Gourlay Louise J LJ Romano Diego D Paradisi Francesca F Molinari Francesco F

Scientific reports 20181106 1

A new transaminase (VbTA) was identified from the genome of the halotolerant marine bacterium Virgibacillus 21D. Following heterologous expression in Escherichia coli, it was located entirely in the insoluble fraction. After a single mutation, identified via sequence homology analyses, the VbTA T16F mutant was successfully expressed in soluble form and characterised. VbTA T16F showed high stability towards polar organic solvents and salt exposure, accepting mainly hydrophobic aromatic amine and ...[more]

PMID: 30401905

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Project description:BackgroundMicrobial proteases are one of the most commercially valuable enzymes, of which the largest market share has been taken by subtilases or alkaline proteases of the Bacillus species. Despite a large amount of information on microbial proteases, a search for novel proteases with unique properties is still of interest for both basic and applied aspects of this highly complex class of enzymes. Oxidant stable proteases (OSPs) have been shown to have a wide application in the detergent and bleaching industries and recently have become one of the most attractive enzymes in various biotechnological applications.ResultsA gene encoding a novel member of the subtilase superfamily was isolated from Virgibacillus sp. SK37, a protease-producing bacterium isolated from Thai fish sauce fermentation. The gene was cloned by an activity-based screening of a genomic DNA expression library on Luria-Bertani (LB) agar plates containing 1 mM IPTG and 3% skim milk. Of the 100,000 clones screened, all six isolated positive clones comprised one overlapping open reading frame of 45% identity to the aprX gene from Bacillus species. This gene, designated aprX-sk37 was cloned into pET21d(+) and over-expressed in E. coli BL21(DE3). The enzyme product, designated AprX-SK37, was purified by an immobilized metal ion affinity chromatography to apparent homogeneity and characterized. The AprX-SK37 enzyme showed optimal catalytic conditions at pH 9.5 and 55°C, based on the azocasein assay containing 5 mM CaCl2. Maximum catalytic activity was found at 1 M NaCl with residual activity of 30% at 3 M NaCl. Thermal stability of the enzyme was also enhanced by 1 M NaCl. The enzyme was absolutely calcium-dependent, with optimal concentration of CaCl2 at 15 mM. Inhibitory effects by phenylmethanesulfonyl fluoride and ethylenediaminetetraacetic acid indicated that this enzyme is a metal-dependent serine protease. The enzyme activity was sensitive towards reducing agents, urea, and SDS, but relatively stable up to 5% of H2O2. Phylogenetic analysis suggested that AprX-SK37 belongs to a novel family of the subtilase superfamily. We propose the name of this new family as alkaline serine protease-X (AprX).ConclusionsThe stability towards H2O2 and moderately halo- and thermo-tolerant properties of the AprX-SK37 enzyme are attractive for various biotechnological applications.

Project description:Amine transaminases (ATA) convert ketones into optically active amines and are used to prepare active pharmaceutical ingredients and building blocks. Novel ATA can be identified in protein databases due to the extensive knowledge of sequence-function relationships. However, predicting thermo- and operational stability from the amino acid sequence is a persisting challenge and a vital step towards identifying efficient ATA biocatalysts for industrial applications. In this study, we performed a database mining and characterized selected putative enzymes of the β-alanine:pyruvate transaminase cluster (3N5M) - a subfamily with so far only a few described members, whose tetrameric structure was suggested to positively affect operational stability. Four putative transaminases (TA-1: Bilophilia wadsworthia, TA-5: Halomonas elongata, TA-9: Burkholderia cepacia, and TA-10: Burkholderia multivorans) were obtained in a soluble form as tetramers in E. coli. During comparison of these tetrameric with known dimeric transaminases we found that indeed novel ATA with high operational stabilities can be identified in this protein subfamily, but we also found exceptions to the hypothesized correlation that a tetrameric assembly leads to increased stability. The discovered ATA from Burkholderia multivorans features a broad substrate specificity, including isopropylamine acceptance, is highly active (6 U/mg) in the conversion of 1-phenylethylamine with pyruvate and shows a thermostability of up to 70 °C under both, storage and operating conditions. In addition, 50% (v/v) of isopropanol or DMSO can be employed as co-solvents without a destabilizing effect on the enzyme during an incubation time of 16 h at 30 °C. KEY POINTS: • Database mining identified a thermostable amine transaminase in the β-alanine:pyruvate transaminase subfamily. • The tetrameric transaminase tolerates 50% DMSO and isopropanol under operating conditions at 30 °C. • A tetrameric structure is not necessarily associated with a higher operational stability.

Dataset Information

Strategic single point mutation yields a solvent- and salt-stable transaminase from Virgibacillus sp. in soluble form.

Publications

Strategic single point mutation yields a solvent- and salt-stable transaminase from Virgibacillus sp. in soluble form.

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