Biochemical Characterization and Mutational Analysis of a Lactone Hydrolase from Phialophora americana

Abstract

The mycotoxin zearalenone (ZEN) is a secondary metabolite produced mainly by Fusarium species. ZEN poses health hazards both for humans and animals, as a major contaminant in the food and feed industries. Currently, there is no effective technique for degrading ZEN during industrial processes. In this study, we isolated and biochemically characterized a novel lactone hydrolase, ZHD607, isolated from Phialophora americana, cloned, and exogenously expressed in Pichia pastoris. ZHD607 was characterized as a mesophilic lactone hydrolase having a neutral pH and showing optimal activity at 35 °C and pH 8.0. Two mutants, ZHDM1 and I160Y, generated from ZHD607 based on structure and sequence alignment analyses, exhibited 2.9-and 3.4-fold higher activity towards ZEN than did ZHD607. Molecular dynamics simulation revealed diverse mechanisms driving this improved catalytic activity. These findings enrich our knowledge about ZHD enzyme family and represent an important step toward industrialization of ZEN-detoxifying lactone hydrolases.

Engineering a Trypsin-Resistant Thermophilic α-Galactosidase to Enhance Pepsin Resistance, Acidic Tolerance, Catalytic Performance, and Potential in the Food and Feed Industry

“…[30][31][32]54 In this study, the thermostable Gal27B and Aga27A were resistant to trypsin, and the thermostable Gal27A was resistant to trypsin and pepsin (Figure S2 and Table 3). 34,36,38 Gal27B had 80.3, 39.3, and ≤12.0% sequence identity to Aga27A, Gal27A, and previously identified pepsin-resistant α-galactosidases, respectively, and showed a higher temperature optimum (75 °C), superior thermostability (50% activity at 70 °C for 15 min), better resistance to trypsin (99% activity after incubation with trypsin at 37 °C for 2 h), and greater specific activity (477 U/mg) (Table 3). Further, the thermostable Gal27B-A23 and Gly156Arg/Pro/His variants conferred pepsin resistance to Gal27B (Figure 2B−D, Figure 3B,C, Figure 4A,C,D, and Table 2).…”

“…20 The activities of Gal27B and its variants on raffinose and stachyose are much higher than those of many fungal αgalactosidases, such as Aga-BC7050 (23 and 14 U/mg), Gal27A (16 and 4.3 U/mg), GalC (5.4 and 3.2 U/mg), and PCGI (3.3 and 1.8 U/mg, respectively). 5,36,54,60 α-Galactosidases with high specific activity and excellent stability can economically and efficiently eliminate raffinose and stachyose in soybean products and increase the nutritional value.…”

“…The improvement of protein thermostability by mutating some residues or regions has been utilized to construct various thermostable enzymes. , The loop 4 replacement of Gal27B with residues 177–199 of loop 4 in Gal27A may improve the thermostability by enhancing the structural stability . Introducing the pyrrole ring of Gly156Pro may increase the thermostability by increasing its conformational rigidity .…”

“…KJ739690 and KF640698, respectively) and the synthesized DNA fragments containing the loop 4 region of the Gal27B-A23 variant (Table S1) as the templates, the variants were constructed by overlap extension PCR. 33,35,36 The primers used in this study are listed in Table S1. The loop 4 region in Gal27B was replaced with the corresponding residues 177−199 in Gal27A to construct the loop 4 replacement variant Gal27B-A23.…”

Kinetics, Thermodynamics and Mechanism of Enzymatic Degradation of Zearalenone in Degummed Corn Oil

“…In 2002, Takahashi Ando et al successfully expressed the lactone hydrolase encoding gene ZHD101 from Clonostachys rosea IFO7063 in Escherichia coli [ 14 ]. Since then, ZHD101 has gradually become a research hotspot and has been successfully expressed in a variety of hosts including Escherichia coli [ 14 ], Pichia pastoris [ 15 ], Saccharomyces cerevisiae [ 16 ] and Lactobacillus reuteri [ 17 ]. To obtain ZEN lactone hydrolase with better enzymology properties, some other microbial lactone hydrolases, such as ZHD607 [ 15 ] from Phylophora americana and Neurospora crassa ZENC [ 18 ], zhd518 [ 19 ] from Rhinocladiella machinenziei , CbZHD [ 20 ] from Cladophialophora bantiana and ZHD607 [ 15 ] from Phylophora americana , have been gradually excavated and studied.…”