Modeling and re-engineering of Azotobacter vinelandii alginate lyase to enhance its catalytic efficiency for accelerating biofilm degradation

Chul Ho Jang, Yu Lan Piao, Xiaoqin Huang, Eun Jeong Yoon, So Hee Park, Kyoung Lee, Chang Guo Zhan, Hoon Cho

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20 Scopus citations

Abstract

Alginate is known to prevent elimination of Pseudomonas aeruginosa biofilms. Alginate lyase (AlgL) might therefore facilitate treatment of Pseudomonas aeruginosa-infected cystic fibrosis patients. However, the catalytic activity of wild-type AlgL is not sufficiently high. Therefore, molecular modeling and site-directed mutagenesis of AlgL might assist in enzyme engineering for therapeutic development. AlgL, isolated from Azotobacter vinelandii, catalyzes depolymerization of alginate via a β-elimination reaction. AlgL was modeled based on the crystal structure template of Sphingomonas AlgL species A1-III. Based on this computational analysis, AlgL was subjected to site-directed mutagenesis to improve its catalytic activity. The kcat/Km of the K194E mutant showed a nearly 5-fold increase against the acetylated alginate substrate, as compared to the wild-type. Double and triple mutants (K194E/K245D, K245D/K319A, K194E/K245D/E312D, and K194E/K245D/K319A) were also prepared. The most potent mutant was observed to be K194E/K245D/K319A, which has a 10-fold improved kcat value (against acetylated alginate) compared to the wild-type enzyme. The antibiofilm effect of both AlgL forms was identified in combination with piperacillin/tazobactam (PT) and the disruption effect was significantly higher in mutant AlgL combined with PT than wild-type AlgL. However, for both the wild-type and K194E/K245D/K319A mutant, the use of the AlgL enzyme alone did not show significant antibiofilm effect.

Original languageEnglish
Article numbere0156197
JournalPLoS ONE
Volume11
Issue number6
DOIs
StatePublished - Jun 1 2016

Bibliographical note

Funding Information:
This work was supported in part by the Human Resource Training Program for Regional Innovation and Creativity through the Ministry of Education and National Research Foundation of Korea (NRF-2014H1C1A1067030) and the National Science Foundation (NSF grant CHE-1111761).

Publisher Copyright:
© 2016, Public Library of Science. All rights reserved. This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

ASJC Scopus subject areas

  • General

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