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Overexpression and characterization of laccase from Trametes versicolor in Pichia pastoris

Overexpression and characterization of laccase from Trametes versicolor in Pichia pastoris A laccase-encoding gene of Trametes versicolor, lccA, was cloned and expressed in Pichia pastoris X33. The lccA gene consists of a 1560 bp open reading frame encoding 519 amino acids, which was classified into family copper blue oxidase. To improve the expression level of recombinant laccase in P. pastoris, conditions of the fermentation were optimized by the single factor experiments. The optimal fermentation conditions for the laccase production in shake flask cultivation using BMGY medium were obtained: the optimal initial pH 7.0, the presence of 0.5 mM Cu2+, 0.6% methanol added into the culture every 24 h. The laccase activity was up to 11, 972 U/L under optimal conditions after 16 days of induction in a medium with 4% peptone. After 100 h of large scale production in 5 L fermenter the enzyme activity reached 18, 123 U/L. The recombinant laccase was purified by ultrafiltration and (NH4)2SO4 precipitation showing a single band on SDS-PAGE, which had a molecular mass of 58 kDa. The optimum pH and temperature for the laccase were pH 2.0 and 50°C with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate. The recombinant laccase was stable over a pH range of 2.0–7.0. The Km and the Vmax value of LccA were 0.43 mM and 82.3 U/mg for ABTS, respectively. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Biochemistry and Microbiology Springer Journals

Overexpression and characterization of laccase from Trametes versicolor in Pichia pastoris

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References (34)

Publisher
Springer Journals
Copyright
Copyright © 2014 by Pleiades Publishing, Inc.
Subject
Life Sciences; Biochemistry, general; Microbiology; Medical Microbiology
ISSN
0003-6838
eISSN
1608-3024
DOI
10.1134/S0003683814020124
Publisher site
See Article on Publisher Site

Abstract

A laccase-encoding gene of Trametes versicolor, lccA, was cloned and expressed in Pichia pastoris X33. The lccA gene consists of a 1560 bp open reading frame encoding 519 amino acids, which was classified into family copper blue oxidase. To improve the expression level of recombinant laccase in P. pastoris, conditions of the fermentation were optimized by the single factor experiments. The optimal fermentation conditions for the laccase production in shake flask cultivation using BMGY medium were obtained: the optimal initial pH 7.0, the presence of 0.5 mM Cu2+, 0.6% methanol added into the culture every 24 h. The laccase activity was up to 11, 972 U/L under optimal conditions after 16 days of induction in a medium with 4% peptone. After 100 h of large scale production in 5 L fermenter the enzyme activity reached 18, 123 U/L. The recombinant laccase was purified by ultrafiltration and (NH4)2SO4 precipitation showing a single band on SDS-PAGE, which had a molecular mass of 58 kDa. The optimum pH and temperature for the laccase were pH 2.0 and 50°C with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate. The recombinant laccase was stable over a pH range of 2.0–7.0. The Km and the Vmax value of LccA were 0.43 mM and 82.3 U/mg for ABTS, respectively.

Journal

Applied Biochemistry and MicrobiologySpringer Journals

Published: Mar 6, 2014

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