ABSTRACT
AIMS: The objective of the study was to produce and characterize the cinnamoyl esterase EstA from the anaerobic fungus Piromyces equi for potential industrial applications. METHODS AND RESULTS: The catalytic domain EstA was produced in Trichoderma reesei. Because the two fungi displayed different genome features, including different codon usage and GC content, a synthetic gene was designed and expressed, leading to the production of the corresponding protein at around 33 mg per litre in the T. reesei culture medium. After the recombinant protein was purified, biochemical characterization showed that EstA presents peak activity at pH 6.5 and at 50-60 degrees C. Furthermore, EstA remained stable at pH 6-8 and below 50 degrees C. EstA was compared to cinnamoyl esterases FaeA and FaeB from Aspergillus niger in terms of ferulic acid (FA) release from wheat bran (WB), maize bran (MB) and sugar beet pulp (SBP). CONCLUSION: The synthetic gene was successfully cloned and overexpressed in T. reesei. EstA from P. equi was demonstrated to efficiently release FA from various natural substrates. SIGNIFICANCE AND IMPACT OF THE STUDY: Recombinant EstA produced in an industrial enzyme producer, T. reesei, was biochemically characterized, and its capacity to release an aromatic compound (FA) for biotechnological applications was demonstrated.
Subject(s)
Carboxylic Ester Hydrolases/metabolism , Fungal Proteins/metabolism , Industrial Microbiology , Piromyces/enzymology , Trichoderma/metabolism , Aspergillus niger/enzymology , Carboxylic Ester Hydrolases/genetics , Cloning, Molecular , Coumaric Acids/metabolism , Fungal Proteins/genetics , Hydrogen-Ion Concentration , Piromyces/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Temperature , Trichoderma/geneticsABSTRACT
AIMS: A molecular tool for extensive detection of prokaryotic alkane hydroxylase genes (alkB) was developed. AlkB genotypes involved in the degradation of short-chain alkanes were quantified in environmental samples in order to assess their occurrence and ecological importance. METHODS AND RESULTS: Four primer pairs specific for distinct clusters of alkane hydroxylase genes were designed, allowing amplification of alkB-related genes from all tested alkane-degrading strains and from six of seven microcosms. For the primer pair detecting alkB genes related to the Pseudomonas putida GPo1 alkB gene and the one targeting alkB genes of Gram-positive strains, both involved in short-chain alkane degradation (Subject(s)
Cytochrome P-450 CYP4A/genetics
, Ecology
, Environmental Microbiology
, Environmental Pollution
, Alkanes/metabolism
, Animals
, Biodegradation, Environmental
, Cytochrome P-450 CYP4A/metabolism
, DNA Primers/genetics
, Environmental Monitoring/methods
, Fresh Water
, Genotype
, Microscopy, Fluorescence
, Polymerase Chain Reaction/methods
, Soil Microbiology
, Water Microbiology
