Abstract
As a feed additive, xylanase has been widely applied in the feed of monogastric animals, which contains multiple plant polysaccharides. However, during feed manufacture, the high pelleting temperatures challenge wild-type xylanases. The aim of this study was to improve the thermostability of Aspergillus sulphureus acidic xylanase. According to the predicted protein structure, a series of disulphide bridges and proline substitutions were created in the xylanase by PCR, and the mutants were expressed in Pichia pastoris. Enzyme properties were evaluated following chromatographic purification. All the recombinant enzymes showed optima at pH 3.0 and 50 °C or 55 °C and better resistance to some chemicals except for CuSO4. The specific activity of the xylanase was decreased by introduction of the mutations. Compared to the wild-type enzyme, a combined mutant, T53C-T142C/T46P, with a disulphide bond at 53–142 and a proline substitution at 46, showed a 22-fold increase of half-life at 60 °C. In a 10-L fermentor, the maximal xylanase activity of T53C-T142C/T46P reached 1,684 U/mL. It was suggested that the T53C-T142C/T46P mutant xylanase had excellent thermostability characteristics and could be a prospective additive in feed manufacture.
Highlights
Xylan, present in plant cell walls and middle lamella, is the most abundant natural cell wall polysaccharide next to cellulose[1]
An acidic β-1,4-xylanase gene xynA was cloned from Aspergillus sulphureus and constitutively expressed in Pichia pastoris[12, 13]
Two types of mutations were introduced into the wild-type A. sulphureus xylanase to improve its thermostability
Summary
Present in plant cell walls and middle lamella, is the most abundant natural cell wall polysaccharide next to cellulose[1]. Due to the technical requirements during processes such as feed pelleting and food baking, people need higher-thermal-capacity xylanase. Scientists have developed multiple means of improving the thermostability of xylanase. An earlier means was to clone hyper-thermophilic xylanase from a variety of micro-organism sources, such as Thermotoga maritima MSB86, Caldocellum saccharolyticum[7], Geobacillus sp. Later means were based on rational design and directed evolution methods to improve the properties of xylanases. Satyanarayana[10] enhanced the thermostability of xylanase (MxylM4) by substituting serine/ threonine with arginine residues by site-directed mutagenesis. Wang et al.[11] improved the thermal performance of Thermomyces lanuginosus GH11 xylanase by introducing a disulphide bridge, Q1C-Q24C, into the N-terminal region of the enzyme. We created xylanase mutants by introducing a disulphide bridge and proline residue substitutions to improve the enzyme’s thermostability
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