Improving Phytase And Xylanase Enzyme Properties Via Computational Guided Protein Engineering

Phytase and xylanase have been widely applied owing to their broad applications in industries,including feed manufacture,paper and pulp processing,environmental protection,etc.Among the above industries,phytase and xylanase play an important role in the feed manufacture for these enzyme products can provide with a sustainable development and environmental protection.In order to further reduce the production cost and improve the application performance of enzyme.Based on computational simulation and site-directed mutagensis,we modified phytase and xylanase with better enzymology characteristics.Firstly,structural comparison and B-factor normalization guided us the mutated points.Secondly,with the help of site-directed mutagenesis,we constructed,expressed the mutants,followed by determination of enzymatic characteristics.Finally,molecular mechanisms of the improved characteristics were explored by molecular dynamics(MD)simulations.Characteristics of the modified phytase and xylanase products were listed as follows:1.Aspergillus niger phyA phytase(phy-N)has high activity and poor thermostability.In contrast,phytase from Aspergillus fumigatus(phy-F)owns perfect thermostability;however,its enzyme activity is relatively low.An advantagous phytase can be constructed if we combine the thermostability of phy-F and enzyme activity of phy-N together.(1)We first superimposed the crystal structures of phy-N and phy-F,and calculated the main-chain deviation of the corresponding atoms.Three segments(Glu35-Ser42,Gly163-Arg168,Arg248-Ser254)were discovered with pronounced mainchain deviation?4 ?.Thus,we substituted 18 amino acids in the above three segments in phy-N to corresponding ones in phy-F,and named the mutant phy-N-18.Enzymology characteristics revealed that: recombinant phytase(phy-N-18)and wild type phytase(phy-N)owned the same temperature optimum(50°C),enzyme activity of both phytases decreased rapidly when temperature higher than 55°C,and they both belong to mosophilic enzyme;pH optimum of phy-N-18 is 6.0,larger than that of phy-N(5.5),and the pH stability of phy-N-18 is better than that of phy-N;moreover,phy-N-18 has higher thermostability than phy-N,after treated at 70°C,80°C and 90°C for 1h,phy-N-18 displayed higher temperature stability,for its has higher residual activity of 20.3%,19.8% and 3.4% than phy-N in the three temperatural conditions;besides,phy-N-18 was compared to 10 phytase products saled in market,its thermostability is middle among all provided products,while still better than phy-N.In this study,we improved the thermostability of phy-N,and proved that the 18 mutated points play an important role in phytase heat stability.(2)Although phy-N-18 with 18 mutated points has a higher themostability than phy-N,it is not sure which residues in the mutated points play the key role.Taking advantages of MD simulations,we explored the heat resistant mechanism of phy-N-18.After comparing and scanning the interactions formed with the 18 substituted sites in phy-N-18 and phy-N at 50°C and 70°C,we discovered 9 residues in phy-N-18 formed more hydrogen bonds than the corresponding ones in phy-N.Accordingly,we focused on these 9 residues and performed the second generation of mutagenesis.After site-directed mutagenesis,we expressed and purified the second phytase mutant,phy-N-9,and its enzymatic characteristics are as follows:phy-N-9 has the same temperatural adaptability as phy-N and phy-N-18,and its pH adatabilty is the same as that of phy-N-18;temperature stability tests revealed that phy-N-9 is the most thermostable among three phytases: relative residual enzyme activity of phy-N-9 is 14.1%,17.2% and 25.9% higher than that of phy-N-18 after treating at 70°C,80°C and 90°C for 1h,and 34.4%,37.0% and 29.3% higher than that of phy-N;compared to 10 phytase products saled in market,thermostability of phy-N-9 is in the leading level;however,the catalytic efficiency of phy-N-9 and phy-N-18 decreased 60.0% and 68.0% compared to that of phy-N,this phenomenon needs further investigation.2.Xynlanase from Neocallimastix patriciarum(XynCDBFV)is a thermostable xylanase widely used in industries.Due to the harsh and extreme conditions during biotechnological treatments,xylanases with advatageous properties such as excellent thermostability are highly demanded.Xylanase from Nonomuraea flexuosa(NFX)is a well known thermosphilic xylanase.We firstly normalized and compared the B-factors from crystal structures of XynCDBFV and NFX,discovered 207-NGGA-210 at the C-terminus of XynCDBFV with pronounced flexibility.Multiple sequence alighment revealed for the first time that sequences 207-SSGS-210 are conserved in both bacterial and fungal xylanases.Thus,207-SSGS-210 may contribute to the thermostability of xylanase,and we performed site-directed mutagenesis on XynCDBFV by substituting 207-NGGA-210 to 207-SSGS-210,and conducted a triple mutant Xyn-MUT(N207S,G208 S,A210S).We expressed and purified the mutant,Xyn-MUT,and its enzymatic characteristics are as follows:Temperature stability tests revealed that Xyn-MUT has improved thermostability: relative residual activities of Xyn-MUT had 5.8%,14.2% and 7.5% higher retention than that of XynCDBFV after treating at 70°C,80°C and 90°C for 1h;catalytic efficiency of Xyn-MUT were larger than that of XynCDBFV(0.6-fold).In order to explore the heat resistant mechanisms,MD simulations were conducted.It is discovered that S210 and S208 in Xyn-MUT has higher probability in forming hydrogen bonds than A210 and G208 in XynCDBFV.Moreover,secondary structure propensity analysis revealed that 207-SSGS-210 in Xyn-MUT own higher propensity in forming ?-sheet than 207-NGGA-210 of XynCDBFV.To our knowldge,this is the first report for glycose hydrolase familly 11 recombinant with improved thermostability based on C-terminus replacement.The successfully recombined mutant provides new insights in engineering a thermostable enzyme.In conclusion,taking advantage of the computational-guided site-directed mutagenesis,we obtained a recombinant phytase(phy-N-9)with improved thermostability(~30.0%)and pH adaptability,and we also obtained a recombinant xylanase(Xyn-MUT)with improved thermostability(~10.0%)and catalytic efficiency(0.6-fold higher than wild type).The resulting recombinants will become attractive candidates in industrial applications.