Journal Article

Rational design and structure-based engineering of alkaline pectate lyase from Paenibacillus sp. 0602 to improve thermostability

Zhou, Zhanping, Wang, Xiao

2021

Overview

Background Ramie degumming is often carried out at high temperatures; therefore, thermostable alkaline pectate lyase (PL) is beneficial for ramie degumming for industrial applications. Thermostable PLs are usually obtained by exploring new enzymes or reconstructing existing enzyme by rational design. Here, we improved the thermostability of an alkaline pectate lyase (PelN) from Paenibacillus sp. 0602 with rational design and structure-based engineering. Results From 26 mutants, two mutants of G241A and G241V showed a higher thermostability compared with the wild-type PL. The mutant K93I showed increasing specific activity at 45 °C. Subsequently, we obtained combinational mutations (K93I/G241A) and found that their thermostability and specific activity improved simultaneously. The K93I/G241A mutant showed a half-life time of 15.9 min longer at 60 °C and a melting temperature of 1.6 °C higher than those of the wild PL. The optimum temperature decreased remarkably from 67.5 °C to 60 °C, accompanied by a 57% decrease in Km compared with the Km value of the wild-type strain. Finally, we found that the intramolecular interaction in PelN was the source in the improvements of molecular properties by comparing the model structures. Rational design of PelN was performed by stabilizing the α-helices with high conservation and increasing the stability of the overall structure of the protein. Two engineering strategies were applied by decreasing the mutation energy calculated by Discovery Studio and predicting the free energy in the process of protein folding by the PoPMuSiC algorithm. Conclusions The results demonstrated that the K93I/G241A mutant was more suitable for industrial production than the wild-type enzyme. Furthermore, the two forementioned strategies could be extended to reveal engineering of other kinds of industrial enzymes.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Algorithms / Alkaline pectate lyase / Amino Acid Sequence / Applied Microbiology / Bacillus (Bacteria) / Bacterial Proteins - chemistry / Bacterial Proteins - genetics / Bacterial Proteins - metabolism / Biochemical Engineering / Bioengineering / Biomedical Engineering/Biotechnology / Biotechnology / Chemistry / Chemistry and Materials Science / Cost control / Degumming / Design / Efficiency / energy / Engineering / Enzyme Stability / Enzymes / Free energy / Genetic Engineering / Gibbs free energy / half life / Helices / High temperature / Hydrogen-Ion Concentration / Industrial applications / Industrial microbiology / Industrial production / Kinetics / Lyases / Melt temperature / Methods / Microbial enzymes / Models, Molecular / Mutagenesis / Mutants / Mutation / Paenibacillus / Paenibacillus - chemistry / Paenibacillus - enzymology / Paenibacillus - genetics / Pectate lyase / Physiological aspects / Plant Breeding/Biotechnology / Polysaccharide-Lyases - chemistry / Polysaccharide-Lyases - genetics / Polysaccharide-Lyases - metabolism / PoPMuSiC / Production processes / Protein Engineering / Protein folding / Protein structure / Proteins / ramie / Site-directed mutagenesis / Temperature / Textiles / Thermal properties / Thermal stability / Thermostability