Improving thermostability of (R)-selective amine transaminase from Aspergillus terreus through introduction of disulfide bonds.

Abstract:

:To improve the thermostability of (R)-selective amine transaminase from Aspergillus terreus (AT-ATA), we used computer software Disulfide by Design and Modelling of Disulfide Bonds in Proteins to identify mutation sites where the disulfide bonds were most likely to form. We obtained three stabilized mutants (N25C-A28C, R131C-D134C, M150C-M280C) from seven candidates by site-directed mutagenesis. Compared to the wild type, the best two mutants N25C-A28C and M150C-M280C showed improved thermal stability with a 3.1- and 3.6-fold increase in half-life (t1/2 ) at 40 °C and a 4.6 and 5.1 °C increase in T5010 . In addition, the combination of mutant R131C-D134C and M150C-M280C displayed the largest shift in thermostability with a 4.6-fold increase in t1/2 at 40 °C and a 5.5 °C increase in T5010 . Molecular dynamics simulation indicated that mutations of N25C-A28C and M150C-M280C lowered the overall root mean square deviation for the overall residues at elevated temperature and consequently increased the protein rigidity. The stabilized mutation of R131C-D134C was in the region of high mobility and on the protein surface, and the disulfide bond constraints the flexibility of loop 121-136.

authors

Xie DF,Fang H,Mei JQ,Gong JY,Wang HP,Shen XY,Huang J,Mei LH

doi

10.1002/bab.1572

subject

Has Abstract

pub_date

2018-03-01 00:00:00

pages

255-262

issue

2

eissn

0885-4513

issn

1470-8744

journal_volume

65

pub_type

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