Dataset Information

A Glycoside Hydrolase Family 62 A-L-Arabinofuranosidase from Trichoderma Reesei and Its Applicable Potential during Mashing.

ABSTRACT: Arabinoxylan is the second most abundant component in the endosperm cell wall of barley and it has been shown to have negative effects on the viscosity and filtration rate of wort and beer. In this study, a glycoside hydrolase (GH) family 62 ?-L-arabinofuranosidase (AFase), termed as TrAbf62A, was purified from the culture filtrate of Trichoderma reesei CICC 41495 by a combined chromatographic method. The preferred substrates of the purified TrAbf62A were soluble, highly substituted arabinoxylan oligosaccharides and polymers, similar to the type found in barley grain. TrAbf62A exhibited activity towards oligomeric and polymeric arabinoxylans, as well as colorimetric arabinose-based substrates, thus meeting the criteria to be classified as a type B AFase. TrAbf62A released mainly arabinose and xylose from soluble wheat arabinoxylan, thus indicating a dual lytic enzyme activity. Supplementation of TrAbf62A during mashing, with a loading of 12 mU/g malt, resulted in a 36.3% decrease in arabinoxylan polymer content, a 5.6% reduction in viscosity, and finally, a 22.1% increase in filtration rate. These results revealed that TrAbf62A has a high potential value in improving lautering performance during mashing.

SUBMITTER: Sun J

PROVIDER: S-EPMC7143738 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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A Glycoside Hydrolase Family 62 A-L-Arabinofuranosidase from <i>Trichoderma Reesei</i> and Its Applicable Potential during Mashing.

Sun Junyong J Xu Feng F Lu Jian J

Foods (Basel, Switzerland) 20200319 3

Arabinoxylan is the second most abundant component in the endosperm cell wall of barley and it has been shown to have negative effects on the viscosity and filtration rate of wort and beer. In this study, a glycoside hydrolase (GH) family 62 α-L-arabinofuranosidase (AFase), termed as TrAbf62A, was purified from the culture filtrate of <i>Trichoderma reesei</i> CICC 41495 by a combined chromatographic method. The preferred substrates of the purified TrAbf62A were soluble, highly substituted arabi ...[more]

PMID: 32204354

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Project description:A GH (glycoside hydrolase) family 54 alpha-L-arabinofuranosidase from Trichoderma koningii G-39 (termed Abf) was successfully expressed in Pichia pastoris and purified to near homogeneity by cation-exchange chromatography. To determine the amino acid residues essential for the catalytic activity of Abf, extensive mutagenesis of 24 conserved glutamate and aspartate residues was performed. Among the mutants, D221N, E223Q and D299N were found to decrease catalytic activity significantly. The kcat values of the D221N and D299N mutants were 7000- and 1300-fold lower respectively, than that of the wild-type Abf. E223Q was nearly inactive. These results are consistent with observations obtained from the Aspergillus kawachii alpha-L-arabinofuranosidase three-dimensional structure. This structure indicates that Asp221 of T. koningii Abf is significant for substrate binding and that Glu223 as well as Asp299 function as a nucleophile and a general acid/base catalyst for the enzymatic reaction respectively. The catalytic mechanism of wild-type Abf was further investigated by NMR spectroscopy and kinetic analysis. The results showed that Abf is a retaining enzyme. It catalyses the hydrolysis of various substrates via the formation of a common intermediate that is probably an arabinosyl-enzyme intermediate. A two-step, double-displacement mechanism involving first the formation, and then the breakdown, of an arabinosyl-enzyme intermediate was proposed. Based on the kcat values of a series of aryl-alpha-L-arabinofuranosides catalytically hydrolysed by wild-type Abf, a relatively small Brønsted constant, beta(lg)=-0.18, was obtained, suggesting that the rate-limiting step of the enzymatic reaction is the dearabinosylation step. Further kinetic studies with the D299G mutant revealed that the catalytic activity of this mutant depended largely on the pK(a) values (>6) of leaving phenols, with beta(lg)=-1.3, indicating that the rate-limiting step of the reaction becomes the arabinosylation step. This kinetic outcome supports the idea that Asp299 is the general acid/base residue. The pH activity profile of D299N provided further evidence strengthening this suggestion.

Project description:Arabinofuranose substitutions on xylan are known to interfere with enzymatic hydrolysis of this primary hemicellulose. In this work, two novel α-L-arabinofuranosidases (ABFs), TtABF51A from Thielavia terrestris and EpABF62C from Eupenicillium parvum, were characterized and functionally analyzed. From sequences analyses, TtABF51A and EpABF62C belong to glycoside hydrolase (GH) families 51 and 62, respectively. Recombinant TtABF51A showed high activity on 4-nitrophenyl-α-L-arabinofuranoside (83.39 U/mg), low-viscosity wheat arabinoxylan (WAX, 39.66 U/mg), high-viscosity rye arabinoxylan (RAX, 32.24 U/mg), and sugarbeet arabinan (25.69 U/mg), while EpABF62C preferred to degrade arabinoxylan. For EpABF62C, the rate of hydrolysis of RAX (94.10 U/mg) was 2.1 times that of WAX (45.46 U/mg). The optimal pH and reaction temperature for the two enzymes was between 4.0 and 4.5 and 65 °C, respectively. Calcium played an important role in the thermal stability of EpABF62C. TtABF51A and EpABF62C showed the highest thermal stabilities at pH 4.5 or 5.0, respectively. At their optimal pHs, TtABF51A and EpABF62C retained greater than 80% of their initial activities after incubation at 55 °C for 96 h or 144 h, respectively. 1H NMR analysis indicated that the two enzymes selectively removed arabinose linked to C-3 of mono-substituted xylose residues in WAX. Compared with the singular application of the GH10 xylanase EpXYN1 from E. parvum, co-digestions of WAX including TtABF51A and/or EpABF62C released 2.49, 3.38, and 4.81 times xylose or 3.38, 1.65, and 2.57 times of xylobiose, respectively. Meanwhile, the amount of arabinose released from WAX by TtABF51A with EpXYN1 was 2.11 times the amount with TtABF51A alone. KEY POINTS: • Two novel α-l-arabinofuranosidases (ABFs) displayed high thermal stability. • The thermal stability of GH62 family EpABF62C was dependent on calcium. • Buffer pH affects the thermal stability of the two ABFs. • Both ABFs enhance the hydrolysis of WAX by a GH10 xylanase.

Dataset Information

A Glycoside Hydrolase Family 62 A-L-Arabinofuranosidase from Trichoderma Reesei and Its Applicable Potential during Mashing.

Publications

A Glycoside Hydrolase Family 62 A-L-Arabinofuranosidase from <i>Trichoderma Reesei</i> and Its Applicable Potential during Mashing.

Similar Datasets

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