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Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger.

ABSTRACT: The full-length gene that encodes the chlorogenic acid hydrolase from Aspergillus niger CIRM BRFM 131 was cloned by PCR based on the genome of the strain A. niger CBS 513.88. The complete gene consists of 1,715 bp and codes for a deduced protein of 512 amino acids with a molecular mass of 55,264 Da and an acidic pI of 4.6. The gene was successfully cloned and overexpressed in A. niger to yield 1.25 g liter(-1), i.e., 330-fold higher than the production of wild-type strain A. niger CIRM BRFM131. The histidine-tagged recombinant ChlE protein was purified to homogeneity via a single chromatography step, and its main biochemical properties were characterized. The molecular size of the protein checked by mass spectroscopy was 74,553 Da, suggesting the presence of glycosylation. ChlE is assembled in a tetrameric form with several acidic isoforms with pIs of around 4.55 and 5.2. Other characteristics, such as optimal pH and temperature, were found to be similar to those determined for the previously characterized chlorogenic acid hydrolase of A. niger CIRM BRFM 131. However, there was a significant temperature stability difference in favor of the recombinant protein. ChlE exhibits a catalytic efficiency of 12.5 x 10(6) M(-1) s(-1) toward chlorogenic acid (CGA), and its ability to release caffeic acid from CGA present in agricultural by-products such as apple marc and coffee pulp was clearly demonstrated, confirming the high potential of this enzyme.

SUBMITTER: Benoit I

PROVIDER: S-EPMC2042068 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger.

Benoit Isabelle I Asther Michèle M Bourne Yves Y Navarro David D Canaan Stéphane S Lesage-Meessen Laurence L Herweijer Marga M Coutinho Pedro M PM Asther Marcel M Record Eric E

Applied and environmental microbiology 20070713 17

The full-length gene that encodes the chlorogenic acid hydrolase from Aspergillus niger CIRM BRFM 131 was cloned by PCR based on the genome of the strain A. niger CBS 513.88. The complete gene consists of 1,715 bp and codes for a deduced protein of 512 amino acids with a molecular mass of 55,264 Da and an acidic pI of 4.6. The gene was successfully cloned and overexpressed in A. niger to yield 1.25 g liter(-1), i.e., 330-fold higher than the production of wild-type strain A. niger CIRM BRFM131. ...[more]

PMID: 17630312

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Project description:BackgroundExtracellular production of fungal lipases especially the lipases obtained from the Aspergilli has gained immense interest in recent years due to its diverse biotechnological applications. In this study, we focused on determining the fermentation parameters required for the optimal lipase production.MethodsA total of 256 fungal isolates were obtained from oil seeds. From each genus, one isolate was selected to evaluate lipase production using phenol red and tributyrin plate assays. Lipase activity was estimated using the spectrophotometric pNPP hydrolysis assay. The highest lipase producer isolates were identified using 18S ribosomal RNA gene sequencing. The genetic variability was determined by random amplified polymorphic DNA (RAPD) analysis and the dendrogram was constructed using the unweighted pair group method with arithmetic averages method. The isolates were examined in a submerged fermentation culture (Smf) to measure the effect of temperature, pH, incubation time, carbon source, nitrogen source, inoculum volume, and lipid source on lipase production.ResultsEleven isolates belonging to the genus Aspergillus were analyzed for lipase production where they were found to be the highest lipase producers among various fungal genera. All the tested isolates were identified as A. niger using 18s rRNA sequencing. Genetic diversity was evaluated among all of the studied A. niger isolates using RAPD primers. The RAPD primers were used to amplify 285 loci, of which five were polymorphic (1.75%) and seven were monomorphic (2.45%). Thus, a high level of genetic diversity was observed among all isolates. The tributyrin test and the lipase activity assay identified five strains of A. niger as high lipase producers, and their optimal enzyme activities were 709.74, 532.54, 735.64, 794.62, and 787.69 U/ml. The optimal conditions for lipase production were as follows: 40 °C, pH 7.5, 1% fructose as the carbon source, 1% yeast extract as the nitrogen source, 2% palm oil, 2.5 × 107 spores/ml suspension, and 3 days of incubation.ConclusionsThe current study provides a comprehensive characterization of the optimal conditions, which are essential to enhance lipase production in five A. niger isolates.

Dataset Information

Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger.

Publications

Gene overexpression and biochemical characterization of the biotechnologically relevant chlorogenic acid hydrolase from Aspergillus niger.

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