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1,4-β-Endoglucanase is one of the most important biocatalysts in modern industries. Here, a glycoside hydrolase (GH) family 45 endoglucanase from thermophilic fungus Theilavia terrestris (TtCel45A) was expressed in Pichia pastoris . The recombinant protein shows optimal activity at 60 °C, pH 4–5. The enzyme exhibits extraordinary thermostability that more than 80% activity was detected after heating at 80 °C for 2.5 h. The high resolution crystal structures of apo-form enzyme and that in complex with cellobiose and cellotetraose were solved to 1.36–1.58 Å. The protein folds into two overall regions: one is a six-stranded β-barrel, and the other one consists of several extended loops. Between the two regions lies the substrate-binding channel, which is an open cleft spanning across the protein surface. A continuous substrate-binding cleft from subsite −4 to +3 were clearly identified in the complex structures. Notably, the flexible V–VI loop ( 113 Gly- 114 Gly- 115 Asp- 116 Leu- 117 Gly- 118 Ser) is found to open in the presence of −1 sugar, with D115 and L116 swung away to yield a space to accommodate the catalytic acid D122 and the 2,5 B boat conformation of −1 sugar during transition state. Collectively, we characterized the enzyme properties of P. pastoris -expressed TtCel45A and solved high-resolution crystal structures of the enzyme. These results are of great interests in industrial applications and provide new insights into the fundamental understanding of enzyme catalytic mechanism of GH45 endoglucanases. [ABSTRACT FROM AUTHOR]

Cellulose is the most abundant renewable biomass on earth, and its decomposition has proven to be very useful in a wide variety of industries. Endo-1,4-β-D-glucanase (EC 3.2.1.4; endoglucanase), which can catalyze the random hydrolysis of β-1,4-glycosidic bonds to cleave cellulose into smaller fragments, is a key cellulolytic enzyme. An endoglucanase isolated from Aspergillus aculeatus F-50 (FI-CMCase) that was classified into glycoside hydrolase family 12 has been found to be effectively expressed in the industrial strain Pichia pastoris. Here, recombinant FI-CMCase was crystallized. Crystals belonging to the orthorhombic space group C2221, with unit-cell parameters a = 74.2, b = 75.1, c = 188.4 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 1.6 Å resolution. Initial phase determination by molecular replacement clearly shows that the crystal contains two protein molecules in the asymmetric unit. Further model building and structure refinement are in progress. [ABSTRACT FROM AUTHOR]

A thermophilic glycoside hydrolase family 16 (GH16) β-1,3-1,4-glucanase from Clostridium thermocellum (CtLic16A) holds great potentials in industrial applications due to its high specific activity and outstanding thermostability. In order to understand its molecular machinery, the crystal structure of CtLic16A was determined to 1.95 Å resolution. The enzyme folds into a classic GH16 β-jellyroll architecture which consists of two β-sheets atop each other, with the substrate-binding cleft lying on the concave side of the inner β-sheet. Two Bis–Tris propane molecules were found in the positive and negative substrate binding sites. Structural analysis suggests that the major differences between the CtLic16A and other GH16 β-1,3-1,4-glucanase structures occur at the protein exterior. Furthermore, the high catalytic efficacy and thermal profile of the CtLic16A are preserved in the enzyme produced in Pichia pastoris , encouraging its further commercial applications. [ABSTRACT FROM AUTHOR]

1,3-1,4-β- d-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-β- d-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ∼30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity. [ABSTRACT FROM AUTHOR]

Ochratoxin A (OTA) is among the most prevalent mycotoxins detected in agroproducts, posing serious threats to human and livestock health. Using enzymes to conduct OTA detoxification is an appealing potential strategy. The recently identified amidohydrolase from Stenotrophomonas acidaminiphila , termed ADH3, is the most efficient OTA-detoxifying enzyme reported thus far and can hydrolyze OTA to nontoxic ochratoxin α (OTα) and L- β -phenylalanine (Phe). To elucidate the catalytic mechanism of ADH3, we solved the single-particle cryo-electron microscopy (cryo-EM) structures of apo-form, Phe- and OTA-bound ADH3 to an overall resolution of 2.5–2.7 Å. The role of OTA-binding residues was investigated by structural, mutagenesis and biochemical analyses. We also rationally engineered ADH3 and obtained variant S88E, whose catalytic activity was elevated by 3.7-fold. Structural analysis of variant S88E indicates that the E88 side chain provides additional hydrogen bond interactions to the OTα moiety. Furthermore, the OTA-hydrolytic activity of variant S88E expressed in Pichia pastoris is comparable to that of Escherichia coli -expressed enzyme, revealing the feasibility of employing the industrial yeast strain to produce ADH3 and its variants for further applications. These results unveil a wealth of information about the catalytic mechanism of ADH3-mediated OTA degradation and provide a blueprint for rational engineering of high-efficiency OTA-detoxifying machineries. [Display omitted] • Structure of a highly effective ochratoxin A-detoxifying enzyme ADH3 was reported. • The mechanism of action of ADH3 was revealed by complex structure of ochratoxin A. • Rational design yielded variants showing up to 4-fold higher catalytic activity. • ADH3 and the highly active variant produced by Pichia pastoris is fully functional. [ABSTRACT FROM AUTHOR]

Deoxynivalenol (DON) and its acetylated derivatives such as 3-acetyldeoxynivalenol (3A-DON) and 15-acetyldeoxynivalenol (15A-DON) are notorious mycotoxins in Fusarium contaminated cereals, which pose a great threat to human and livestock health. The specialized glyoxalase I from Gossypium hirsutum (SPG) can lower the toxicity of 3A-DON by conducting isomerization to transfer C8 carbonyl to C7 and double bond from C9-C10 to C8-C9. Here we report that the substrate–flexible SPG can also recognize 15A-DON and DON, probably following the same isomerization mechanism as that for 3A-DON. The crystallographic, mutagenesis, and biochemical analyses revealed that SPG provides a hydrophobic pocket to accommodate the substrate and residue E167 might serve as the catalytic base. A variant SPGY62A that was constructed based on structure–based protein engineering exhibited elevated catalytic activity towards DON, 3A-DON, and 15A-DON by >70%. Furthermore, variant SPGY62A was successfully expressed in Pichia pastoris , whose catalytic activity was also compared to that produced in Escherichia coli. These results provide a blueprint for further protein engineering of SPG and reveal the potential applications of the enzyme in detoxifying DON, 3A-DON and 15A-DON. • A specialized glyoxalase SPG can recognize DON, 3A-DON and 15A-DON. • The crystal structure of SPG was determined to high resolution. • Docking and mutagenesis clarified the substrate binding and catalytic mechanism. • Mutant SPGY62A with catalytic activity increased by 77% was rationally designed. • The variant SPGY62A exhibited potentials in expressed in Pichia pastoris. [ABSTRACT FROM AUTHOR]

[Display omitted] • Developing enzyme-mediated PET degradation is an important task. • Using Pichia pastoris to produce PET degrading enzyme is a promising strategy. • The enzyme production level reaches commercialization standard. • Crude fermentation product shows PET degradation capacity without purification. • The enzyme production cost of the reported strain is estimated to ∼ 116 USD/ton PET. Using enzymes to decompose poly(ethylene terephthalate) (PET) is an attractive strategy to the sustainable utilization of PET, and an effective production platform of PET degrading enzyme is a prerequisite to achieve this goal. Here, we exploited the industrial yeast strain Pichia pastoris to produce a potent PET hydrolase termed FAST-PETase, whose performance was further elevated by removing two N -linked glycosylations through molecular engineering. The expression of the yielded variant, FAST-PETase-212/277, was elevated by antibiotics selection and chaperon co-expression to exceed 3 g/L in a 30-L fermenter. Notably, the crude fermentation product can be directly applied to decompose PET without purification. More than 95 % postconsumer PET can be achieved by 0.5 mg enzyme g PET -1 in 24 h in a 10-L reaction system in a reactor. These results demonstrate the economic viability of producing PET hydrolytic enzyme with modern fermentation facilities for large scale PET decomposition. [ABSTRACT FROM AUTHOR]

Eukaryotic 1,4-β-endoglucanases (EC 3.2.1.4) have shown great potentials in many commercial applications because they effectively catalyze hydrolysis of cellulose, the main component of the plant cell wall. Here we expressed a glycoside hydrolase family (GH) 5 1,4-β-endoglucanase from Aspergillus niger (AnCel5A) in Pichia pastoris , which exhibits outstanding pH and heat stability. In order to further investigate the molecular mechanism of AnCel5A, apo-form and cellotetraose (CTT) complex enzyme crystal structures were solved to high resolution. AnCel5A folds into a typical (β/α) 8 -TIM barrel architecture, resembling other GH5 members. In the substrate binding cavity, CTT is found to bind to −4 – −1 subsites, and several polyethylene glycol molecules are found in positive subsites. In addition, several unique N -glycosylation motifs that may contribute to protein higher stability were observed from crystal structures. These results are of great importance for understanding the molecular mechanism of AnCel5A, and also provide guidance for further applications of the enzyme. [ABSTRACT FROM AUTHOR]