A De Novo Designed Esterase with p-Nitrophenyl Acetate Hydrolysis Activity

Houjin Zhang,Yunjun Yan,Junjun Liu,Li Xu,Guanlin Li,Jinyong Yan

doi:10.3390/molecules25204658

Houjin Zhang, Yunjun Yan + Show 4 more

Open Access

https://doi.org/10.3390/molecules25204658

Copy DOI

Journal: Molecules	Publication Date: Oct 13, 2020
Citations: 7	License type: CC BY 4.0

Affiliation: Huazhong University of Science and Technology

Abstract

Esterases are a large family of enzymes with wide applications in the industry. However, all esterases originated from natural sources, limiting their use in harsh environments or newly- emerged reactions. In this study, we designed a new esterase to develop a new protocol to satisfy the needs for better biocatalysts. The ideal spatial conformation of the serine catalytic triad and the oxygen anion hole at the substrate-binding site was constructed by quantum mechanical calculation. The catalytic triad and oxygen anion holes were then embedded in the protein scaffold using the new enzyme protocol in Rosetta 3. The design results were subsequently evaluated, and optimized designs were used for expression and purification. The designed esterase had significant lytic activities towards p-nitrophenyl acetate, which was confirmed by point mutations. Thus, this study developed a new protocol to obtain novel enzymes that may be useful in unforgiving environments or novel reactions.

Highlights

Esterases are commonly used in the food, pharmaceutical, agricultural and chemical industries.The methods for obtaining new esterases mainly include mining existing natural biological resources such as extreme environment organisms, isolated bacterial genomes, and uncultured metagenomes [1,2].esterases in nature are usually not suitable for the application in harsh environments or newly-emerged reactions.Esterases account for a small portion of the protein sequence space [3]
A new protocol is needed to facilitate the design of novel enzymes
This study focused on the most critical step in esterase catalysis—the nucleophilic attack of serine on the substrate’s carbonyl carbon

Summary

Introduction

Esterases are commonly used in the food, pharmaceutical, agricultural and chemical industries.The methods for obtaining new esterases mainly include mining existing natural biological resources such as extreme environment organisms, isolated bacterial genomes, and uncultured metagenomes [1,2].esterases in nature are usually not suitable for the application in harsh environments or newly-emerged reactions.Esterases account for a small portion of the protein sequence space [3]. Because evolution is driven by progressive mutations and selection pressure, the sequences of the native proteins are not evenly distributed throughout the sequence space. Instead, they form a family of proteins with similar amino acid sequences, structures, and functions [4]. Exploring the large sequence space outside the evolutionary path requires the de novo design of proteins [5,6,7]. To this end, a new protocol is needed to facilitate the design of novel enzymes

Methods

Results

Conclusion