Rational Design of Alginate Lyase from Microbulbifer sp. Q7 to Improve Thermal Stability.

Min Yang,Li Li,Su-Xiao Yang,Nan-Nan Li,Hai-Jin Mou,Zhe-Min Liu

doi:10.3390/md17060378

Abstract

Alginate lyase degrades alginate by the β-elimination mechanism to produce oligosaccharides with special bioactivities. The low thermal stability of alginate lyase limits its industrial application. In this study, introducing the disulfide bonds while using the rational design methodology enhanced the thermal stability of alginate lyase cAlyM from Microbulbifer sp. Q7. Enzyme catalytic sites, secondary structure, spatial configuration, and molecular dynamic simulation were comprehensively analyzed. When compared with cAlyM, the mutants D102C-A300C and G103C-T113C showed an increase by 2.25 and 1.16 h, respectively, in half-life time at 45 °C, in addition to increases by 1.7 °C and 0.4 °C in the melting temperature, respectively. The enzyme-specific activity and kcat/Km values of D102C-A300C were 1.8- and 1.5-times higher than those of cAlyM, respectively. The rational design strategy that was used in this study provides a valuable method for improving the thermal stability of the alginate lyase.

Highlights

Alginate lyases are derived from abundant sources, including bacteria, brown seaweed, and marine mollusks [1]
The rational design strategy that was used in this study provides a valuable method for improving the thermal stability of the alginate lyase
His185 corresponded to the catalytic residues proposed to exist in polysaccharide lyase (PL)-7 alginate lyases [23,24]

Summary

Introduction

Alginate lyases are derived from abundant sources, including bacteria, brown seaweed, and marine mollusks [1]. Alginate lyase degrades alginate by the β-elimination mechanism to form a double bond between C4 and C5 at the non-reducing end [2]. The degradation products, alginate oligosaccharides, and 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) are used for various functions in medical and energy industries [3,4,5]. Previous studies have demonstrated that alginate lyase exhibits poor thermal stability when being incubated with enzymes at above 40 ◦ C for more than 0.5 h (residual enzyme activity < 50%) [6,7,8]. It is important for alginate lyase to maintain enzyme activity during the process of degrading alginate

Methods

Results

Conclusion