Enhancement of thermostability of GH10 xylanase E Penicillium canescens directed by ΔΔG calculations and structure analysis.

Hydrolytic enzymes are highly demanded in the industry. Thermostability is an important property of enzymes that affects the economic costs of the industrial processes. The rational design of GH10 xylanase E (XylE) Penicillium canescens for the thermostability improvement was directed by ΔΔG calculations and structure analysis. Amino acid substitutions with stabilizing values of ΔΔG and providing an increase in side-chain volume of buried residues were performed experimentally. From the six designed substitutions, four substitutions appeared to be stabilizing, one - destabilizing, and one - neutral. For the improved XylE variants, values of Tm were increased by 1.1-3.1 °C, and times of half-life at 70 °C were increased in 1.3-1.7-times. Three of the four stabilizing substitutions were located in the N- or the C-terminus region. This highlights the importance of N- and C-terminus for the thermostability of GH10 xylanases and also enzymes with (ß/α)8 TIM barrel type of structure. The criteria of stabilizing values of ΔΔG and increased side-chain volume of buried residues for selection of substitutions may be applied in the rational design for thermostability improvement.

Heterologous Expression of Thermogutta terrifontis Endo-Xanthanase in Penicillium verruculosum, Isolation and Primary Characterization of the Enzyme.

Heterologous endo-xanthanase (EX) from the thermophilic planktomycete Thermogutta terrifontis strain was obtained using Penicillium verruculosum 537 (ΔniaD) expression system with the cellobiohydrolase 1 gene promoter. Homogeneous EX with a molecular weight of 23.7 kDa (pI 6.5) was isolated using liquid chromatography methods. This xanthan degrading enzyme also possesses the enzymatic activity towards CM-cellulose, ß-glucan, curdlan, lichenan, laminarin, galactomannan, xyloglucan but not towards p-nitrophenyl derivatives of ß-D-glucose, mannose and cellobiose. The temperature and pH optima of EX were 55°C and 4.0, respectively; the enzyme exhibited 90% of its maximum activity in the temperature range 50-60°C and pH 3-5.

Site-directed mutagenesis of GH10 xylanase A from Penicillium canescens for determining factors affecting the enzyme thermostability.

In order to investigate factors affecting the thermostability of GH10 xylanase A from Penicillium canescens (PcXylA) and to obtain its more stable variant, the wild-type (wt) enzyme and its mutant forms, carrying single amino acid substitutions, were cloned and expressed in Penicillium verruculosum B1-537 (niaD-) auxotrophic strain under the control of the cbh1 gene promoter. The recombinant PcXylA-wt and I6V, I6L, L18F, N77D, Y125R, H191R, S246P, A293P mutants were successfully expressed and purified for characterization. The mutations did not affect the enzyme specific activity against xylan from wheat as well as its pH-optimum of activity. One mutant (L18F) displayed a higher thermostability relative to the wild-type enzyme; its half-life time at 50-60°C was 2-2.5-fold longer than that for the PcXylA-wt, and the melting temperature was 60.0 and 56.1°C, respectively. Most of other mutations led to decrease in the enzyme thermostability. This study, together with data of other researchers, suggests that multiple mutations should be introduced into GH10 xylanases in order to dramatically improve their stability.