Heterologous Expression And Function Study On Xylanases And Acetyl Xylan Esterases From Thermobifida Halotolerans YIM 90462~T

Xylan is a very abundant renewable carbon source in nature,and it’s effectively used in food,paper,bioenergy,animal feed and agricultural solid waste treatment,which can not only reduce environmental pollution,but also bring huge economic benefits.The endo-1,4-beta-xylanases and acetyl xylan esterase in the xylan degrading enzyme system are the key enzymes for effective utilization of xylan,because they can act on the beta-1,4-glucoside bond of the main chain and the O-acetyl group of the side chain,respectively.However,environmental conditions,such as strong acid,strong base,high temperature and high salt in industry,will deactivate enzymes and limit their application.Therefore,it is of great significance to explore xylanase and acetyl xylan esterase with good stability to reply the above extreme environmental conditions.Thermobifida halotolerans YIM 90462^T is an Actinomyces,which was isolated from the Hejing salt mine in Yunnan Province and grew in the flatters of 20℃–50℃,p H 6.0–9.0 and 0%–10%（w/v）Na Cl.In this study,two potential xylanase genes and one potential acetyl xylan asterase（AXEs）gene were obtained by T.halotolerans YIM 90462^T genomic annotations,they were named th Xyn10B,th Xyn10C and th AXE.The protein sequences encoded by the above three genes,is compared with the homologous sequences in Swiss Prot database.The results showed that the three proteins and the sequences in the database have the similarity of less than 50%,which has research significance and potential application value.In this paper,the heterologous expression,enzymological properties,substrate specificity and substrate action mechanism of ThXyn10B,ThXyn10C and ThAXE were studied respectively.The main research results were as follows:1.Enzymatic characteristics,substrate specificity and substrate action mechanism of ThXyn10B and ThXyn10C.（1）The optimal p H of ThXyn10B and ThXyn10C were 7.0 and 8.5,respectively,and both maintained more than 70%activity in the range of p H 4.0–11.0 for 1 h.The optimum temperatures of ThXyn10B and ThXyn10C were 55℃and 50℃,respectively,and the half-lives of both were greater than 1 h at 80℃.The enzyme activity is not affected after tolerance in 2.5%–15%（w/v）Na Cl solution for 1 h.10m M Co²⁺and 1%（v/v）β-mercaptoethanol increased the catalytic activity of ThXyn10C by 1.29 times and 0.97 times,respectively.Anhydrous ethanol,acetonitrile,DMSO,n-butanol,Triton 100,Tween-80,methanol and Tween-20 can increase the enzyme activity of ThXyn10C to 119%～158%.（2）The optimum substrate for ThXyn10B and ThXyn10C was bagasse xylan,and the enzyme activity was 2.67 U/mg and 1.68 U/mg,respectively.The enzyme activity of bagasse xylan was 3.6,3.0 times that of beechwood xylan and 5.0,7.3times that of oat-spelt xylan.（3）The molecular docking results of ThXyn10B and ThXyn10C with xylotriose showed that amino acid residues Lys73,His106,Gln113,His234 and Glu263 may be involved in the binding and catalytic process of ThXyn10B with the xylotriose.Amino acid residues Asn47,Lys50,His83,Asn131,Gln207,His209,Glu237 and Trp277 may be involved in the binding and catalytic process of ThXyn10C with the xylotriose.After each amino acid was mutated into Ala,the substrate kinetics was determined.The results showed that Glu155 was the key amino acid involved in substrate binding and catalysis of ThXyn10B.Glu237,Asn47,Lys50,His83 and Gln207 are the key amino acids involved in substrate binding of ThXyn10C.2.Enzymatic characteristics,substrate specificity and substrate action mechanism of ThAXE.（1）The optimal p H and temperature of ThAXE were 8.5 and 50℃,respectively.The activity of ThAXE remained over 70%after incubation in p H 6.0–10.0 buffer for1 h,and over 60%after tolerance at 90℃for 50 min.After tolerance to 25%（w/v）Na Cl buffer for 1 h,ThAXE also retained 58%enzyme activity.1 m M of K⁺,Na⁺,Mg⁺,Li⁺,Fe³⁺,Fe²⁺,and Al³⁺increased ThAXE activity to 110%to 141%.（2）The activity of ThAXE against p-nitrophenol acetate p-NPC₂was 2533.1U/mg,the K_m value was 1.0±0.10 m M,and the K_catvalue was 2339.8 s^-1.ThAXE has no hydrolytic activity against acetylated xylan,but has hydrolytic activity for7-Aminocephalosporanic Acid（7-ACA）.7-ACA was deacetylated by ThAXE to produce deacetylated 7-aminocephalosporanic acid（D-7-ACA）with activity of 7.4U/mg,K_m of 18.5±2.10 m M and K_catof 46.70 s^-1.（3）Ser186,Asp272 and His301 were the key amino acids involved in the catalysis of 7-ACA,and Arg208 and Asp308 were the key amino acids involved in the binding of 7-ACA.Ser186,Asp272 and His301 are the key amino acids involved in p-NPC₂ binding and catalysis,and Gln187 is also the key amino acids involved in p-NPC₂binding.In summary,the enzymatic characteristics of xylanase ThXyn10B,ThXyn10C and acetyl xylan esterase ThAXE from T.halotolerans YIM 90462^T were identified.The ThXyn10B and ThXyn10C have good stability under acidic（p H 3.0–5.0）,alkaline（p H 8.0–12.0）,high temperature（70–80℃）and high salt（20%–25%Na Cl）conditions.They can act on a variety of xylans to produce xyoligosaccharides,and have good industrial application potential.Acetyl xylan esterase ThAXE has deacetylation activity on 7-ACA,which provides a potential choice for the industrial synthesis of novelβ-lactam antibiotics.Through homology modeling and molecular docking,the binding and catalytic mechanisms of xylanase ThXyn10B and ThXyn10C with substrates xylotriose,and the binding and catalytic mechanisms of acetyl xylan esterase ThAXE with substrates p-NPC₂ and 7-ACA were preliminatively studied.This study provided a theoretical basis for subsequent molecular modification and application of ThXyn10B,ThXyn10C and ThAXE.