Co-expression Of β-1,3-1,4-glucanase And β-1,4-xylanase In Saccharomyces Verevisiae And Investigation Of The Performance And Application

In beer brewing industry, especially for draft beer, macromolecular compounds such as β-glucan, araboxylan, protein are not only to increases the viscosity of beer but also to block filter set, causing filter difficulties and affecting production. Macromolecular compounds can also cause non-biological stability of beer in storage. The global beer industry consumes about2-3million tons diatomite a year, which bringing lots of environment pollution. And during the filter process of beer, the higher content of macromolecular compound, the more diatomite consumed. Therefore, studying how to reduce the viscosity of beer to make it filtrating easier has become one of the problems which need to be solved in beer industry. Now scientific research and production practice has proved that adding β-1,3-1,4-glucanase and β-1,4-xylanase in brewing process can reduce the content of0-1,3-1,4-glucan and araboxylan and decrease the viscosity of beer. So it is one of the feasible ways to make beer filtration easier.This study aims to resolve the beer filtration problem from modifying fermentation strain with genetic engineering technology. In this study several recombinant saccharomyces cerevisiae strains were constructed which can express β-1,3-1,4-glucanase and β-1,4-xylanase and be able to degrade the β-1,3-1,4-glucan and raboxylan. In this way, through fermentation the viscosity of beer can be decreased and the filtration problem can be solved without extra enzyme added. The results are as follows.First, the recombinant yeast with the ability of constitutive secretion expression of β-1,4-xylanase (Xy1B) and β-1,3-1,4-glucanase (GluZ) were constructed. Through the modification of commercialization yeast shuttle plasmid YEplac181, recombinant plasmids were constructed. The expression plasmid YEplac181-PMAK contains PGK1promoter, MFal signal peptide, ADH1terminator which from S. cerevisiae genome, and G418resistance gene KanMX which from PUG6plasmid. Xy1B from gene synthesis and GluZ from plasmid YEplac181-KPMBT were cloned to construct plasmid YEplac181-PMXAK and YEplac181-PMGAK. Then, the recombinant plasmids YEplac181-PMXAK and YEplac181-PMGAK were transformed into S. cerevisiae WZ65. Recombinant yeast strains S. cerevisiae PMXAK and S. cerevisiae PMGAK were created with the ability of secretory expression Xy1B and GluZ respectively. Furthermore, the recombinant plasmids YEplac181-PMXAK and YEplac181-PMGAK were transformed into S. cerevisiae WZ65together to construct a recombinant yeast named as S. cerevisiae PMG-XAK which can produce Xy1B and GluZ through transparent circle experiment confirmed. The maximum enzyme activities were reached after60h shaking flask cultivation. The activity of GluZ of S. cerevisiae PMGAK and S. cerevisiae PMG-XAK is45.4U/mL and21.7U/mL and the activity of XylB of S. cerevisiae PMXAK and S. cerevisiae PMG-XAK is17.9U/mL and8.7U/mL.Second, the recombinant yeasts with the ability of constitutive surface display expression of XylB and GluZ were constructed. The a-agglutinin gene containing the3’half of the region encoding320amino acids and a238-bp flanking region and XylB were cloned to plasmid YEplac181-PMAK to create a surface display plasmid YEplac181-PMXAAK. Based on YEplac181-PMXAAK plasmid, the GluZ was cloned to replace Xy1B to construct another display plasmid YEplac181-PMGAAK. The320C-terminal amino acids of α-agglutinin were used as an anchor to link the enzyme to cell surface of yeast. Then, the recombinant plasmids YEplac181-PMXAAK and YEplac181-PMGAAK were transformed into S. cerevisiae WZ65respectively to construct recombinant S. cerevisiae PMXAAK and S. cerevisiae PMGAAK with the ability of displaying expression XylB and GluZ. Furthermore, the recombinant plasmids YEplac181-PMXAK and YEplac181-PMGAK were transformed into S. cerevisiae WZ65to construct a new recombinant S. cerevisiae PMG-XAAK which can produce XylB and GluZ together through transparent circle experiment. The maximum enzyme activities were reached after60h shaking flask cultivation. The GluZ of S. cerevisiae PMGAAK and S. cerevisiae PMG-XAAK is4.1U/mL and2.3U/mL and the XylB of S. cerevisiae PMXAAK and S. cerevisiae PMG-XAAK is8.9U/mL and4.2U/mL.Third, the recombinant yeasts with the ability of induced surface display expression of Xy1B and GluZ were constructed. The GAL1promoter gene was cloned to plasmid YEplac181-PMGAAK and YEplac181-PMXAAK to instead of the PGK1promoter. New plasmids were transformed into S. cerevisiae WZ65respectively to construct S. cerevisiae GMXAAK and S. cerevisiae GMGAAK with the ability of displaying expression Xy1B and GluZ. Furthermore, the recombinant plasmids were transformed into S. cerevisiae WZ65together to construct S. cerevisiae GMG-XAAK which can produce XylB and GluZ together through transparent circle experiment confirmed. The maximum enzyme activities were reached after60h shaking flask cultivation. The GluZ of S. cerevisiae GMGAAK and S. cerevisiae GMG-XAAK is7.6U/mL and3.4U/mL and the Xy1B of S. cerevisiae PMXAK and S. cerevisiae PMG-XAK is15.6U/mL and7.3U/mL.Fourth, in this paper, the properties of XylB and GluZ were investigated which were produced by recombinant yeasts with secretory expression and cell surface display expression. The results are as follows. Xy1B and GluZ are specific hydrolase to hydrolyze β-1,3-1,4-glucan and β-1,4-xylan respectively. The optimum temperature of the recombinant enzymes is50℃, except the GluZ produced by S. cerevisiae PMGAK which is40℃. The recombinant enzymes are stable at10℃to50℃. The optimum pH of recombinant XylB and GluZ is5.0and6.0respectively. All types of the recombinant enzymes are stable under acidic condition around pH3.Fifth, the performance of fermentation and degradation of beer viscosity in beer were studied. The results are as follows. Comparing with the original strain, the growth performance of recombinant yeasts reduces slightly, but both apparent fermentation degree and real fermentation degree of original and recombinant strains are very close to one another. The0-1,3-1,4-glucan, araboxylan and wort viscosity were reduced through treatment with the fermentation broth or yeasts cell which containing Xy1B and GluZ. All three recombinant yeasts which can produce Xy1B and GluZ can lower beer viscosity during fermentation. The result showed that the secretory expression strain was most effective and the induced surface display strain effect is worst.