Construction, Optimization And Characterization Of Bifunctional β-Glucanase-Xylanase And β-Glucanase-Phytase Fusions

To enhance the exploitation of anti-nutritional resources such asβ-glucan,xylan and phytate rich in cereals,β-glucanase(Glu),xylanase(Xyl)and phytase(Phy)that are separately produced are often mixed into compound formulations for use in feedstuff.The costs of such formulations could be largely reduced if bi- or multi-functional enzymes based on protein engineering were used to substitute the uni-functional enzymes.This study began from the construction ofβ-glucanase-xylanase andβ-glucanase-phytase fusions with no peptide linker,followed by inserting proper linkers between the two moieties of each fusion to improve expression of chimeric genes.Several bifunctionalβ-glucanase-xylanase fusions with increased catalytic efficiency for both moieties were obtained and some of theβ-glucanase-phytase fusions displaying enzymatic activities of both Glu and Phy were also achieved.Detailed results are summarized as follows.Construction ofβ-glucanase-xylanase andβ-glueanase-phytase fusions with no linker.The linker-free chimeric genes ofβ-glucanase-xylanase andβ-glucanase-phytase, named gx and gp,were constructed using the method of splicing-by-overlap extension(SOE) and expressed successfully in Escherichia coli BL21,respectively.The resultant fusion enzyme gx exhibited bothβ-glucanase and xylanase activities.Compared to parental enzymes,the catalytic efficiency of the gx Glu moiety(K_cat/K_m=6492)was 4.15 fold of that of the parent Glu(K_cat/K_m=1563),whereas the catalytic efficiency of the Xyl moiety was equivalent to 69%of the parental efficiency.Except for the less thermostability of the Glu domain,all enzymatic characteristics of both gx moieties in relation to temperature and pH were similar to those of the parental enzymes.However,the fusion gp displayed no activities of bothβ-glucanase and phytase.The results suggest that the interaction between the two moieties of a concerned fusion lead to not necessarily negative effect on their performance.It is likely to construct a bifunctional fusion with improved both catalytic efficiencies of both moieties by inserting a proper linker between the two moieties at a reasonable distance.The lack of a spacer between the two domains of the gp fusion could contribute to the complete loss of both activities ofβ-glucanase and phytase.Construction ofβ-glucanase-xylanase fusion genes by linker optimization.The flexible peptides(GGGGS)_n(n≤3),theα-helical peptides(EAAAK)_n(n＜3)and two other peptides were used as linkers to construct bifunctional fusions of Glu and Xyl for improved catalytic efficiencies of both moieties.Twelve chimeric genes ofβ-glucanase-xylanase were generated and,of those,five included the linkers(S3,S3-1,S3-2,S3-3 and S3-4)encoding the nucleotide sequence of(GGGGS)₃.Eight of the 12 genes were successfully expressed in BL21,generating the bifunctional fusion enzymes Glu-S3-Xyl,Glu-S2-Xyl,Glu-S1-Xyl, Glu-S-Xyl,Glu-H-Xyl,Glu-α3-Xyl,Glu-α2-Xyl and Glu-α1-Xyl.However,the chimeric genes with the linkers S3-1,S3-2,S3-3 and S3-4 were not successfully expressed in BL21, being excluded for further analysis.The nucleotide sequence encoding(GGGGS)₃ is a GC-rich sequence which may form stable mRNA secondary structure in the fusions and depress the translation of the intact chimeric genes.The decrease of the translation efficiency was likely attributed to the effect of codon pair utilization bias since the compatibility of adjacent aminoacyl-tRNA isoacceptors at sites A and P of a translated ribosome may affect the decoding accuracy and speed of mRNA.Thus,caution should be taken when the nucleotide sequence of a concerned linker peptide was designed.Impact of optimized linkers on the characteristics ofβ-glucanase-xylanase fusion. The eight fusion enzymes were purified sequentially using the methods of ammonium sulfate fractionation,precipitation via acid buffer(pH 5.0),and cation exchange chromatography.All the characterized fusions displayed catalytic efficiencies of the Glu (K_cat/K_m=4748-6658)and Xyl(K_cat/K_m=1073-1875)moieties equivalent to 304-426%and 82-143%of the parental enzymes,respectively.The peptide linker S2 resulted in the best fusion Glu-S2-Xyl,whose catalytic efficiency had a net increase of 326%for the Glu and of 43%for the Xyl.The two moieties of Glu-α3-Xyl with the linkerα3 also showed net increases of 262 and 31%for the catalytic efficiencies of both moieties,respectively.The enhanced bifunctional activities of theβ-glucanase-xylanase fusions imply that beneficial interaction may be attained by the optimized peptide linkers to separate the two moieties at a reasonable distance.Different types of peptides with flexibility or rigidity could result in similar efficiency.The length changes of a selected peptide could also affect overall performance of both moieties.It is thus necessary to optimize linkers for a fusion protein.Construction ofβ-glucanase-phytase genes by linker optimization.The flexible peptides(GGGGS)_n(n=2 or 3)and theα-helical peptides(EAAAK)_n(n=1 or 2)were used as linkers to construct the fusion genes encodingβ-glucanase and phytase.The four chimeric genes were expressed well in BL21,yielding the fusion proteins Glu-S2-Phy, Glu-pS3-Phy,Glu-α1-Phy and Glu-α2-Phy,respectively.The latter three were evidently bifunctional whereas the fusion Glu-S2-Phy displayed the activity ofβ-glucanase only.With the amino acid numbers of each linker in consideration,the folding structures of the selected flexible and rigid peptides in each fusion seem to be different.The results indicate that the optimized peptide linkers may relate to the structures and sizes of the two moieties for fusion due to the facts of the bifunctional Glu-S2-Xyl and the unifunctional Glu-S2-Phy.Conclusively,three bifunctionalβ-glucanase-xylanase fusions with enhanced catalytic efficiency of both Glu and Xyl were obtained by optimizing the peptide linkers to separate the two moieties at a reasonable distance for beneficial interaction.In combination with the achieved three bifunctionalβ-glucanase-phytase fusions,the structures and sizes of two given moieties in fusion proteins were,to some extent,determined by the optimization of peptide linkers.The results highlight the potential for developing commercial bi- or multi-functional enzymes in feed industry by means of protein engineering.