Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

Abstract

a b s t r a c tA xylanase produced by Thermomyces lanuginosus 195 by solid state fermentation (SSF) was purified 9.3-fold from a crude koji extract, with a 7.6% final yield. The purified xylanase (with an estimated mass of 22 kDa by SDS-PAGE) retained 18% relative activity when treated for 10 min at 100• C and approximately 90% relative activity when incubated at pH values ranging from 6 to 10. Xylanase activity in the purified preparation was significantly enhanced following treatment with manganese and potassium chlorides (p < 0.05) but significantly reduced by calcium, cobalt and iron (p < 0.05). The purified enzyme was also shown to be exclusively xylanolytic. The gene encoding xylanase activity from T. lanuginosus 195 was functionally expressed by Pichia pastoris. MALDI-ToF mass spectrometry and zymography were employed to confirm functional recombinant expression. Maximum xylanase titres were achieved following 120 h induction of the recombinant culture, yielding 26.8 U/mL. Achieving functional protein expression facilitates future efforts to optimise the cultivation conditions for heterologous xylanase production.

A novel highly thermostable xylanase stimulated by Ca2+ from Thermotoga thermarum: cloning, expression and characterization

“…The K m value of the thermostable xylanase from Thermomyces lanuginosus , reported 7.3 mg mL -1 and 60.2 mg mL -1 for soluble and insoluble oat spelt xylan respectively, however, was higher than that of Xyn10A xylanase. Furthermore, the Xyn10A was stable at 85°C for 2h, while Thermomyces lanuginosus xylanase was only stable at 60°C for 1 h [32]. Therefore, it can be concluded that Xyn10A exhibits better enzymatic properties for bioconversion of xylan at even higher temperature.…”

Optimization of Xylanase Production byThermomyces lanuginosusin Solid State Fermentation

“…13) A single xylanase from T. lanuginosus 195 has been purified 9.3-fold from a crude koji extract, with a specific activity of 6,182 U/mg of protein. 53) Following cultivation by SSF, xylanase from T. lanuginosus 195 exhibited optimal activity at 80 C (data not shown). Furthermore, the crude xylanase preparation was shown be exclusively xylanolytic, with stability demonstrated over a wide pH range (pH 2-10) and at elevated temperatures (30% RA at 100 C).…”

“…Furthermore, the crude xylanase preparation was shown be exclusively xylanolytic, with stability demonstrated over a wide pH range (pH 2-10) and at elevated temperatures (30% RA at 100 C). 53) Such characteristics indicate that the xylanase preparation from T. lanuginosus 195 can be used in the treatment of paper pulp, without the need for purification or excessive downstream processing.…”

Optimized expression, purification and characterization of a family 11 xylanase (AuXyn11A) from Aspergillus usamii E001 in Pichia pastoris

“…), confirming its purity. The specific activity of purified AuXyn11A towards birchwood xylan was 22 714 U mg −1 , which is significantly higher than that of purified Thermomyces lanuginosus 195 xylanase (6182 U mg −1 ) and A. niger xylanase (717.5 U mg −1 ) …”