Improving the Specific Activity and Thermostability of Psychrophilic Xylosidase AX543 by Comparative Mutagenesis

Abstract

Improving the specific activity and thermostability of psychrophilic xylosidase is important for improving its enzymatic performance and promoting its industrial application. Herein, a psychrophilic xylosidase AX543 exhibited activity in the temperature range between 0 and 35 °C, with optimum activity at 20 °C, which is lower than that of other reported psychrophilic xylosidases. The thermostability, specific activity, and catalytic efficiency of the site-directed variants G110S, Q201R, and L2 were significantly enhanced, without affecting the optimal reaction temperature. Comparative protein structural analysis and molecular dynamics simulation indicated that these improvements might be the result of the increased hydrogen bonds interaction and improved structural rigidity. Furthermore, homologous module substitution with four segments demonstrated that the psychrophilic characteristics of AX543 are the results of the whole protein structure, and the C-terminal segment A4 appears to be more essential in determining psychrophilic characteristics, exhibiting potentiality to produce more psychrophilic xylosidases. This study provides valuable structural information on psychrophilic xylosidases and also offers attractive modification strategies to modify catalytic activity, thermostability, and optimal reaction temperature.

Improving the catalytic properties of xylanase from Alteromones Macleadii H35 through evolution analysis

“…Numerous publications have demonstrated that xylanase characteristics from the GH10 and GH11 families can be improved using protein engineering methods, and few publications have explored improving the catalytic performance of enzymes from the GH43 family using site-directed mutagenesis (10,20). In this work, protein engineering methods were used to improve the catalytic performance of Xyn ZT-2 based on evolutionary analysis.…”

Structures, Biochemical Characteristics, and Functions of β-Xylosidases

“…JB13 showed a relative activity of 20% at 20 °C owing to the high flexibility resulting from its high frequency of small amino acids and high proportion of random coils . Conversely, the increased rigidity of structure could improve the thermostability of enzyme . Some Loops are critical for the structural rigidity of the enzyme .…”

“…45 Conversely, the increased rigidity of structure could improve the thermostability of enzyme. 97 Some Loops are critical for the structural rigidity of the enzyme. 98 Moreover, an oligomeric state, relatively longer asparagine ladder, and glycosylated amino acids can enhance the thermostability of enzymes.…”