Mutagenesis, heterogeneous gene expression, and purification and amino acid substitution analyses of plant peroxidase, PrxA3a

Yoshinaka, Kyouji; Kawai, Shinya

doi:10.1007/s10086-011-1242-6

Cited by 3 publications

(1 citation statement)

References 36 publications

(37 reference statements)

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“…Plasmid YEpFLAG-1 is a yeast vector for the extracellular secretion of N-terminal FLAG fusion proteins in S. cerevisiae . YEpFLAG-1 also contains the TRP1 gene for selection of yeast transformants (Yoshinaka and Kawai 2012 ). The successful expression and secretion of proteins of prokaryotic origin from this system is well documented (López-López et al 2010 , 2015 ).…”

Section: Resultsmentioning

confidence: 99%

AMWEst, a new thermostable and detergent-tolerant esterase retrieved from the Albian aquifer

Adjeroud,

Kecha,

Escuder-Rodríguez

et al. 2024

Appl Microbiol Biotechnol

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A fosmid library was constructed with the metagenomic DNA from the high-temperature sediment-rich water of the Albian aquifer (Algeria). Functional screening of this library was subsequently done looking for genes encoding lipolytic enzymes. We identified a novel gene named AMWEst (1209 base pairs) encoding a protein of 402 amino acids with a predicted molecular weight of 43.44 kDa and conferring esterase activity. AMWEst was successfully overexpressed in the yeast mesophilic host Saccharomyces cerevisiae, and the expression system used proved to be efficient and produced sufficient activity for its biochemical characterization. Multiple sequence alignment indicated that AMWEst contained a conserved pentapeptide motif (Gly120-His121-Ser122-Gln123-Gly124). The optimum pH and temperature of the recombinant esterase AMWEst were 8 and 80 °C, respectively. Additionally, AMWEst showed higher activity towards short carbon substrates and showed maximum activity for p-nitrophenyl hexanoate (C6). Notably, AMWEst has a remarkable thermostability, and the enzyme retains almost maximum activity at 70 °C after incubation for 1 h. Moreover, enzyme activity was enhanced by high concentrations of SDS and Triton X-100 detergents. Key points • A novel thermostable esterase has been retrieved through functional metagenomics • The esterase is detergent-tolerant, which is attractive for some applications • The esterase can be expressed in a yeast mesophilic host to enhance its yield

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Section: Resultsmentioning

confidence: 99%

AMWEst, a new thermostable and detergent-tolerant esterase retrieved from the Albian aquifer

Adjeroud,

Kecha,

Escuder-Rodríguez

et al. 2024

Appl Microbiol Biotechnol

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Production Technology for Bioenergy Crops and Trees

Hirasawa¹,

Ookawa²,

Kawai³

et al. 2014

Research Approaches to Sustainable Biomass Systems

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Heterologous production and secretion of active-form plant peroxidase in Brevibacillus choshinensis and their monolignol polymerization abilities analysis

et al. 2023

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Plant peroxidases are important for several processes, such as defense against pathogens, and auxin metabolism. In this study, we report the active production and secretion of plant peroxidase and mutated enzymes in the bacterium Brevibacillus choshinensis for the first time in the world. We introduced mutations into prxA3a, an anionic peroxidase gene of hybrid aspen, Populus × kitakamiensis, to substitute the amino acid residues at the surface of the protein and analyzed their modified substrate specificities. We have also reported heterologous expression of PrxA3a and mutated enzymes in yeast. Enzymes secreted in the culture medium by B. chosinensis were purified by Ni affinity chromatography, anion-exchange chromatography, and size-exclusion chromatography. The ability of the mutated enzymes to polymerize sinapyl alcohol, a monolignol, was higher than that of the wild-type enzyme. In particular, the FYAW-mutated enzyme produced by the bacterium showed higher polymerization activity, similar to that of the FYAW-mutated enzyme produced by yeast.

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Mutagenesis, heterogeneous gene expression, and purification and amino acid substitution analyses of plant peroxidase, PrxA3a

Cited by 3 publications

References 36 publications

AMWEst, a new thermostable and detergent-tolerant esterase retrieved from the Albian aquifer

AMWEst, a new thermostable and detergent-tolerant esterase retrieved from the Albian aquifer

Production Technology for Bioenergy Crops and Trees

Heterologous production and secretion of active-form plant peroxidase in Brevibacillus choshinensis and their monolignol polymerization abilities analysis

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