Structural Characterization and Directed Evolution of a Novel Acetyl Xylan Esterase Reveals Thermostability Determinants of the Carbohydrate Esterase 7 Family

Abstract

A hot desert hypolith metagenomic DNA sequence data set was screened for genes annotated as acetyl xylan esterases (AcXEs). One of the genes identified encoded an ∼36-kDa protein (Axe1). The synthesized gene was cloned and expressed, and the resulting protein was purified. NaM1 was optimally active at pH 8.5 and 30°C and functionally stable at salt concentrations of up to 5 M. The specific activity and catalytic efficiency were 488.9 U mg and 3.26 × 10 M s, respectively. The crystal structure of wild-type NaM1 was solved at a resolution of 2.03 Å, and a comparison with the structures and models of more thermostable carbohydrate esterase 7 (CE7) family enzymes and variants of NaM1 from a directed evolution experiment suggests that reduced side-chain volume of protein core residues is relevant to the thermal stability of NaM1. Surprisingly, a single point mutation (N96S) not only resulted in a simultaneous improvement in thermal stability and catalytic efficiency but also increased the acyl moiety substrate range of NaM1. AcXEs belong to nine carbohydrate esterase families (CE1 to CE7, CE12, and CE16), of which CE7 enzymes possess a unique and narrow specificity for acetylated substrates. All structurally characterized members of this family are moderately to highly thermostable. The crystal structure of a novel, mesophilic CE7 AcXE (Axe1), from a soil metagenome, provides a basis for comparisons with thermostable CE7 enzymes. Using error-prone PCR and site-directed mutagenesis, we enhanced both the stability and activity of the mesophilic AcXE. With comparative structural analyses, we have also identified possible thermal stability determinants. These are valuable for understanding the thermal stability of enzymes within this family and as a guide for future protein engineering of CE7 and other α/β hydrolase enzymes.

Xylanolytic Extremozymes Retrieved From Environmental Metagenomes: Characteristics, Genetic Engineering, and Applications

“…The acetyl xylan esterases (AXEs) (EC 3.1.1.6) cleave the acetyl group from acetyl-xylose moieties and contribute to the complete degradation of the complex xylans. Recently, a few studies have uncovered a handful of AXEs using metagenomic approaches (Adesioye et al, 2018;Brito et al, 2018; Table 2). A metagenomic AXE (Axe1NaM1) was identified by using a hot desert hypolith metagenomic DNA sequence data set (Adesioye et al, 2018).…”

“…Recently, a few studies have uncovered a handful of AXEs using metagenomic approaches (Adesioye et al, 2018;Brito et al, 2018; Table 2). A metagenomic AXE (Axe1NaM1) was identified by using a hot desert hypolith metagenomic DNA sequence data set (Adesioye et al, 2018). On characterization, it was detected as a mesophilic (Topt.…”

Characterization of an alkaline esterase from an enriched metagenomic library derived from an oil-spill area

“…Specific activity of the purified enzyme was 366.7 U/mg protein. The specific activity of Est7S was in the upper range among family VII esterases such as Est-XG2 (17.71 U/mg protein) [17], EstDL30 (161 U/mg protein) [19], and an esterase NaM1 (488.9 U/mg protein) from a desert metagenome [29]. Compared to others, Est7S was in the middle range; an esterase Est2K (family VIII, 17.1 U/mg protein) from a compost metagenome [10], Lip7- [11], EstN7 of B. cohnii strain N1 (family IV, 336.89 U/mg protein) [30], EstSP (family IV, 745.14 U/mg protein) from a soil metagenome [31], and Est7K (family VIII, 790.2 U/mg protein) from the compost metagenome [32].…”

Recent Development of Extremophilic Bacteria and Their Application in Biorefinery

“…In addition, other thermo-resistance factors, including structural compactness, oligomerization, glycosylation, and hydrophobic interactions between subunits, are crucial for stability (Chakravorty et al, 2017). Synthetic biology, including site-directed mutagenesis and directed evolution of enzymes, has been used to improve the thermal stability of target proteins/enzymes, which is essential for their application in bioenergy industry (Adesioye et al, 2018).…”