Characterization of Two VAO-Type Flavoprotein Oxidases from Myceliophthora thermophila.

The VAO flavoprotein family consists mostly of oxidoreductases harboring a covalently linked flavin cofactor. The linkage can be either monocovalent at position 8 with a histidine or tyrosine or bicovalent at position 8 with a histidine and at position 6 with a cysteine. Bicovalently bound flavoproteins show a preference for bulkier substrates such as oligosaccharides or secondary metabolites. The genome of the thermophilic fungus Myceliophthora thermophila C1 was found to be rich in genes encoding putative covalent VAO-type flavoproteins. Enzymes from this fungus have the advantage of being rather thermostable and homologous overexpression in M. thermophila C1 is feasible. Recently we discovered a new and VAO-type carbohydrate oxidase from this fungus: xylooligosaccharide oxidase. In this study, two other putative VAO-type oxidases, protein sequence XP_003663615 (MtVAO615) and XP_003665713 (MtVAO713), were expressed in M. thermophila C1, purified and characterized. Enzyme MtVAO615 was found to contain a bicovalently bound FAD, while enzyme MtVAO713 contained a monocovalent histidyl-bound FAD. The crystal structures of both proteins were obtained which revealed atypical active site architectures. It could be experimentally verified that both proteins, when reduced, rapidly react with molecular oxygen, a hallmark of flavoprotein oxidases. A large panel of alcohols, including carbohydrates, steroids and secondary alcohols were tested as potential substrates. For enzyme MtVAO713 low oxidase activity was discovered towards ricinoleic acid.

Discovery of a Xylooligosaccharide Oxidase from Myceliophthora thermophila C1.

By inspection of the predicted proteome of the fungus Myceliophthora thermophila C1 for vanillyl-alcohol oxidase (VAO)-type flavoprotein oxidases, a putative oligosaccharide oxidase was identified. By homologous expression and subsequent purification, the respective protein could be obtained. The protein was found to contain a bicovalently bound FAD cofactor. By screening a large number of carbohydrates, several mono- and oligosaccharides could be identified as substrates. The enzyme exhibits a strong substrate preference toward xylooligosaccharides; hence it is named xylooligosaccharide oxidase (XylO). Chemical analyses of the product formed upon oxidation of xylobiose revealed that the oxidation occurs at C1, yielding xylobionate as product. By elucidation of several XylO crystal structures (in complex with a substrate mimic, xylose, and xylobiose), the residues that tune the unique substrate specificity and regioselectivity could be identified. The discovery of this novel oligosaccharide oxidase reveals that the VAO-type flavoprotein family harbors oxidases tuned for specific oligosaccharides. The unique substrate profile of XylO hints at a role in the degradation of xylan-derived oligosaccharides by the fungus M. thermophila C1.

Expanding the substrate scope of chitooligosaccharide oxidase from Fusarium graminearum by structure-inspired mutagenesis.

Chitooligosaccharide oxidase from Fusarium graminearum (ChitO) oxidizes N-acetyl-D-glucosamine (GlcNAc) and its oligomers with high efficiency at the C1-hydroxyl moiety while it shows poor or no activity with other carbohydrates. By sequence and structural comparison with other known carbohydrate oxidases (glucooligosaccharide oxidase from Acremonium strictum and lactose oxidase from Microdochium nivale) eleven mutants were designed to redirect the catalytic scope of ChitO for improved oxidation of lactose, cellobiose and maltose. The catalytic properties of the most interesting mutants were further improved by combining single mutations. This has resulted in the creation of a set of ChitO variants that display totally different substrate tolerances. One ChitO variant shows a dramatic improvement in catalytic efficiency towards oxidation of glucose, cellobiose, lactose, and maltose. We also describe a ChitO variant with the highest catalytic efficiency in GlcNAc oxidation so far reported in the literature.

A fast, sensitive and easy colorimetric assay for chitinase and cellulase activity detection.

BACKGROUND: Most of the current colorimetric methods for detection of chitinase or cellulase activities on the insoluble natural polymers chitin and cellulose depend on a chemical redox reaction. The reaction involves the reducing ends of the hydrolytic products. The Schales' procedure and the 3,5-dinitrosalicylic acid (DNS) method are two examples that are commonly used. However, these methods lack sensitivity and present practical difficulties of usage in high-throughput screening assays as they require boiling or heating steps for color development. RESULTS: We report a novel method for colorimetric detection of chitinase and cellulase activity. The assay is based on the use of two oxidases: wild-type chito-oligosaccharide oxidase, ChitO, and a mutant thereof, ChitO-Q268R. ChitO was used for chitinase, while ChitO-Q268R was used for cellulase activity detection. These oxidases release hydrogen peroxide upon the oxidation of chitinase- or cellulase-produced hydrolytic products. The hydrogen peroxide produced can be monitored using a second enzyme, horseradish peroxidase (HRP), and a chromogenic peroxidase substrate. The developed ChitO-based assay can detect chitinase activity as low as 10 µU within 15 minutes of assay time. Similarly, cellulase activity can be detected in the range of 6 to 375 mU. A linear response was observed when applying the ChitO-based assay for detecting individual chito-oligosaccharides and cello-oligosaccharides. The detection limits for these compounds ranged from 5 to 25 µM. In contrast to the other commonly used methods, the Schales' procedure and the DNS method, no boiling or heating is needed in the ChitO-based assays. The method was also evaluated for detecting hydrolytic activity on biomass-derived substrates, that is, wheat straw as a source of cellulose and shrimp shells as a source of chitin. CONCLUSION: The ChitO-based assay has clear advantages for the detection of chitinase and cellulase activity over the conventional Schales' procedure and DNS method. The detection limit is lower and there is no requirement for harsh conditions for the development of the signal. The assay also involves fewer and easier handling steps. There is no need for boiling to develop the color and results are available within 15 minutes. These aforementioned features render this newly developed assay method highly suitable for applications in biorefinery-related research.