Performance Improvement Of The Acidotolerant And Thermophilic Xylanases From Fungi

Xylanase is the most important hemicellulase and has been widely used in many fields.However,its application in different industries requires xylanases different enzymatic properties.It is of vital importance to mine xylanase resources for favorable candidates and modify them to meet the industrial purposes.Thermotolerant Talaromyces leycettanus JCM12802,acidophilic Bispora sp.MEY-1 and lignocellulose-degrading Gloeophyllum trabeum CBS 900.73 were selected to obtain the xylanase-encoding genes.These xylanases were then characterized and modified for better performance by the aid of computer-assisted design.Six xylanases of glycoside hydrolase(GH)family 10 and one xylanase of GH11 were functionally characterized,and four of them,TlXyn10A,TlXyn11B,Xyl10E and GtXyn10,showed excellent properties.TlXyn10A showed maximum activity at pH 4.5 and 80?,and retained 81.7%,74.8%and 58.2%activity after incubation at 85?,90? and 95? for 1 h.TlXyn11B with pH and temperature optima at 3.5 and 65? had a specific activity of up to 8300±32 U/mg.Xyl10E was optimally active at pH 4.0 and 95 ?,and may represent the most thermophilic fungal xylanase known so far.And GtXyn10 had pH and temperature optima of 4.5 and 75? and showed capacity to degrade xylan and barley ?-glucan.These four xylanases demonstrated great potentials for industrial applications.The highly active TlXyn11B was unstable under 70? and above.Thus TlXyn11B was subjected to thermostability improvement.It has been reported that the N-terminal sequences of GH11 xylanases play an important role in thermostability,and saturated mutagenesis was used to optimize the N-terminal sequence.After two rounds of mutagenesis,five mutants(S3F,S3F/D35V,S3F/D35Q,S3F/D35I and S3F/D35M)with comparable activities showed improved thermostability with increased Tm values of 2.3-7.8?.Molecular dynamics simulation analysis revealed that the N-termini of TlXyn11B and mutants S3F and S3F/D35I formed different amino acid interaction networks,and S3F/D35I demonstrated more interactions with a typical sandwich structure(Ser45-Phe3-Thr47/Ile35)to stabilize the N-terminal local conformation.When using beechwood xylan as the substrate,TlXyn11B,S3F,S3F/D35V and S3F/D35I mainly released xylobiose,xylotriose and xylotetraose as the hydrolysis products,and thus have potentials for the production of probiotic xylo-oligosaccharides.TlXyn10A and Xyl10E with excellent thermostability were improved for catalytic performance.The Xyl10C with excellent stability and catalytic performance was selected as the template.The non-conserved residues in the catalytic pocket of TlXyn10A were replaced by the corresponding ones of Xyl10C.Three mutants,TlXyn10A_P,TlXyn10A_N and TlXyn10A_C,were then constructed and characterized.TlXyn10A_P showed increased Km and Vmax values;as results,the specific activity was promoted,but the catalytic efficiency was not significantly elevated.Molecular dynamics simulation analysis suggested that the mutation of Gly149 on the loop 4 of TlXyn10A_P to Asp may introduce a hydrogen bond with the substrate,and substitution of Phe232 with Glu decreased the substrate affinity.The effect of loop 4 on catalytic efficiency was further explored in Xyl10E,in which sites 160 and 161 in Xyl10E corresponded to the residues of site 148 and 149 of TlXyn10A.When mutated Ala160 and Ala161 to Asp or Glu,great differences were detected.Mutants Ala160Asp or Ala160Glu showed decreased Km values while A161D or A161E showed increases in the Vmax values.Molecular dynamics simulation analysis demonstrated the Glu160 might form a hydrogen bond with Tyr204,which contributed to the increased substrate affinity,while Asp161 was capable of forming a hydrogen bond with the substrate to assist substrate binding or accelerate the products release.The improved performance of TlXyn10A and Xyl10E verified the roles of loop 4 in thecatalytic performance of xylanases.This study enriched the xylanase resources with industrial application potentials and investigated the molecular mechanisms of thermostability and catalytisis,and therefore is of significance and importance in the basic researches and improvements of industrial enzymes.