Expression and characterization of a pectin methylesterase from Aspergillus niger ZJ5 and its application in fruit processing

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A pectin methylesterase gene, pme-zj5a, from Aspergillus niger ZJ5 was cloned and high-level expressed in Pichia pastoris. The highest PME activity was 71.11 U/ml after induction with methanol for 20 h at 30 °C. The molecular mass of purified PME-ZJ5A was estimated to be 37 kDa by SDS-PAGE, and its Km, Vmax and kcat values of PME-ZJ5A were determined to be 3.27 mg/ml, 5.36 μmol/min/mg, and 22.33 s−1 with pectin. Purified recombinant PME-ZJ5A exhibited optimal activity at pH 3.8 and 45 °C. It retained more than 60% of its maximum activity at 10 °C. Moreover, recombinant PME-ZJ5A can increase the transmittance of pineapple juice by 60.8%, and increase the firmness of pineapple cubes nearly double when combined with CaCl2, which showed good potential in fruit processing.

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Reagents, strains and media

Substrate citrus pectin, citrus pectin with 77% esterified (DE77) and citrus pectin with 85% esterified (DE85) were purchased from Sigma–Aldrich (San Diego, CA, USA). The pEASY-T3 vector, Escherichia coli Trans1-T1 competent cells and Fastpfu DNA polymerase (TransGen, Beijing, China) were used for gene cloning and sequencing. The plasmid pPIC9 and P. pastoris GS115 (Invitrogen, Carlsbad, CA, USA) were used to construct the expression vector and for heterologous expression. T4-DNA ligase and

Cloning and sequence analysis of pme-zj5a

Full-length DNA and cDNA corresponding to the pme-zj5a gene (GenBank accession number: MG397069) (Supplementary Fig. S1) were cloned from A. niger ZJ5 by PCR using specific primers. The DNA was 1088 bp in length and included two introns. Its cDNA was 984 bp long and encoded a polypeptide of 327 amino acids and a stop codon. The deduced PME-ZJ5A protein contained a catalytic domain and a putative N-terminal signal peptide (residues 1–19) of carbohydrate esterase family 8 of Carbohydrate-Active

Discussion

More than 200 amino acid sequences of PMEs from fungal were collected in the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/protein). The majority of them have a mesophilic temperature optimum (40–60 °C) and an acidic pH optimum, such as PME from A. flavus whose optimal activity occurs at 55 °C and pH 4.8 (25), PME from A. repens maximum activity occurred at 30 °C and pH 6.5 (26), and recombinant PME from A. niger showed maximum activity at pH 4.7 and 50 °C

Acknowledgments

This research was supported by the National High Technology Research and Development Program of China (2012AA022207) and the National Natural Science Foundation of China (grant number 31402007 and 31201313). We thank Angie Deng (Molecular Physiology and Biophysics, Vanderbilt University) for her valuable comments during manuscript writing and revising.

The authors declare no competing financial interest.

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    The first two authors contributed equally to this work.

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