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Protein engineering of GH10 family xylanases for gaining a resistance to cereal proteinaceous inhibitors

Denisenko, Yury A., Gusakov, Alexander V., Rozhkova, Aleksandra M., Zorov, Ivan N., Bashirova, Anna V., Matys, Veronika Yu, Nemashkalov, Vitaly A., Sinitsyn, Arkady P.
Biocatalysis and agricultural biotechnology 2019 v.17 pp. 690-695
Penicillium, Talaromyces verruculosus, Trichoderma reesei, active sites, amino acids, auxotrophs, boiling, breadmaking, feeds, fungi, genes, protein engineering, proteins, rye, rye flour, viscosity, xylanases
Proteinaceous inhibitors from cereals reduce the efficiency of xylanase applications as a component of animal feeds, in bread making and other technologies based on processing of cereal grains. The inhibitors of XIP-type inhibit most of fungal xylanases from the GH10 and GH11 families of glycoside hydrolases, while they have no affect on bacterial xylanases. In order to gain a resistance to XIP-type proteins, an insertion of 5 amino acid residues was introduced into the peptide loop forming the active site cleft of two GH10 xylanases (PcXylA from Penicillium canescens and TrXyn3 from Trichoderma reesei) using a protein engineering technique. Genes encoding the wild-type and engineered forms of PcXylA and TrXyn3 were cloned and expressed in Penicillium verruculosum B1-537 and P. canescens RN3-11-7 auxotrophic recipient strains, respectively. The secreted recombinant xylanases were purified for characterization. Using a method based on measuring the enzyme effect on viscosity of the native extract of rye flour and that subjected to boiling in order to inactivate its inhibitory proteins, it was shown that the 5 aa insertion leads to relieving the inhibition of PcXylA and TrXyn3 by the proteinaceous inhibitors from rye.