【作者】 鲁健章；

【导师】 何国庆；

【作者基本信息】 浙江大学 ， 食品科学， 2014， 博士

【摘要】 啤酒酿造特别是纯生啤酒的酿造中,高分子物质β-葡聚糖、阿拉伯木聚糖、蛋白质等不仅增加了啤酒粘度还极易堵塞过滤装置,造成过滤困难,影响生产。啤酒酿造中,高分子化合物含量越高,助滤剂硅藻土的使用量就越大。全球啤酒行业每年消耗的硅藻土是惊人的,初步计算每年硅藻上的使用量在200万吨-300万吨之间,这不仅需要企业付出大量成本,也给环境带来大量的污染。因而,研究如何降低啤酒的粘度,改善啤酒的易滤性,已成为业界和相关科研工作者亟待解决的问题之一。现有的科学研究和生产实践已经证明,啤酒酿造工艺中添加β-1,3-1,4-葡聚糖酶和β-1,4-木聚糖酶可以降低啤酒中β-1,3-1,4-葡聚糖和阿拉伯木聚糖的含量,从而降低啤酒粘度,是改善啤酒易滤性的可行途径之一。本研究从改造发酵菌株出发,采用基因工程技术,构建表达β-1,3-1,4-葡聚糖酶和p-木聚糖酶的基因工程酵母,赋予重组酵母降解啤酒中葡聚糖和阿拉伯木聚糖的能力。尝试通过酿酒酵母在发酵过程中的作用改善啤酒过滤困难的问题。研究结果如下：第一,构建了组成型分泌表达β-1,3-1,4-葡聚糖酶(GluZ)和β-1,4-木聚糖酶(XylB)的酿酒酵母基因工程菌。通过对商品化酵母穿梭质粒YEplac181的改造,构建了组成型分泌表达葡聚糖酶和木聚糖酶的重组质粒载体,该载体的表达盒包含了克隆自酿酒酵母基因组的PGK1启动子、MFα1信号肽、ADH1终止子序列,以及来自PUG6质粒的遗传霉素G418抗性基因KanMX,构建了组成型启动了PGKl调控的酵母分泌表达载体YEplac181-PMAK。将全基因合成的β-1,4-木聚糖酶基因(XylB)和来自YEplac181-KPMBT质粒的β-1,3-1,4-葡聚糖酶基因(GluZ)克降到YEplac181-PMAK上,构建了分泌表达XylB的YEplac181-PMXAK质粒和分泌表达GluZ的YEplac181-PMGAK质粒。分别将重组质粒YEplac181-PMXAK和YEplac181-PMGAK转化S. cerevisiae WZ65,构建了分泌表达XylB和GluZ的酿酒酵母基因工程菌S. cerevisiae PMXAK和S. cerevisiae PMGAK;同时将重组质粒YEplac181-PMXAK和YEplac181-PMGAK共同转化S. cerevisiae WZ65,构建了共分泌表达XylB和GluZ的酿酒酵母基因工程菌S. cerevisiae PMG-XAK,经透明圈实验证实三株重组酵母均可以分泌表达有活性的重组酶XylB和GluZ。摇瓶培养60h测得三株重组酵母的发酵液中酶活力分别为：S.cerevisiae PMXAK产XylB酶活为45.4U/mL; S. cerevisiae PMGAK产GluZ酶活为17.9U/mL; S. cerevisiae PMG-XAK共表达XylB和GluZ的酶活分别为21.7U/mL和8.7U/mL。第二,构建了组成型展示表达XylB和GluZ的酿酒酵母基因工程菌。以YEplac181-PMAK为出发质粒,分别克隆了XylB、GluZ以及凝集素C端320个aa的锚定序列,构建了展示表达XylB和GluZ的表达载体YEplac181-PMXAAK和YEplac181-PMGAAK。分别将重组质粒YEplac181-PMXAAK和YEplac181-PMGAAK转化S. cerevisiae WZ65,构建了展示表达XylB和GluZ的酿酒酵母基因工程菌S. cerevisiae PMXAAK和S. cerevisiae PMGAAK;同时将重组质粒YEplac181-PMXAAK和YEplac181-PMGAAK转化S. cerevisiae WZ65,构建了共展示表达XylB和GluZ的酿酒酵母基因工程菌S. cerevisiae PMG-XAAK,经透明圈实验证实在三株重组酵母中两种酶均为有活性的展示表达。摇瓶培养60h测得三株重组酵母的发酵液中XylB和GluZ的活力分别为：S. cerevisiae PMXAAK产XylB酶活为8.9U/mL; S. cerevisiae PMGAAK产GluZ酶活为4.1U/mL; S. cerevisiae PMG-XAAK共表达XylB和GluZ的酶活分别为4.2U/mL和2.3U/mL。第三,构建了诱导型展示表达XylB和GluZ的酿酒酵母基因工程菌。以YEplac181-PMGAAK和YEplac181-PMXAAK为出发质粒,克隆了源自酵母基因组的GAL1启动子,构建了诱导型启动子GALI控制的展示表达载体YEplac181-PMGAAK和YEplac181-PMXAAK。分别将重组质粒YEplac181-GMXAAK和YEplac181-GMGAAK转化S. cerevisiaeWZ65,构建了展示表达XylB和GluZ的酿酒酵母基因工程菌S. cerevisiae GMXAAK和S. cerevisiae GMGAAK;同时将重组质粒YEplac181-GMXAAK和YEplac181-GMGAAK转化S. cerevisiae WZ65,构建了共展示表达XylB和GluZ的酿酒酵母基因工和菌S. cerevisiae GMG-XAAK,经透明圈实验证实三株重组酵母均可以展示表达有活性的重组酶XylB和GluZ。摇瓶培养60h测得三株重组酵母的发酵液中酶活力分别为：S. cerevisiae GMXAAK产XylB酶活为15.6U/mL; S. cerevisiae GMGAAK产GluZ酶活为7.3U/mL; S. cerevisiae GMG-XAAK共表达XylB和GluZ酶活公别为7.6U/mL和3.4U/mL。第四,本文对重组酵母分泌表达和展示表达的XylB和GluZ的酶学性质进行了研究。结果表明：(1)重组酶XylB具有专一的水解β-1,4-木糖糖苷键活力,GluZ具有专一的水解β-1,3-1,4-葡葡糖糖苷键活力。(2)重组酵母菌株S. cerevisiae PMXAK、S. cerevisiae PMXAAK和S. cerevisiae GMXAAK所产XylB的最适反应温度均为50℃;重组酵母菌株S. cerevisiae PMGAAK和S. cerevisiae GMGAAK展示表达的GluZ最适反应温度是50℃;S. cerevisiae PMGAK分泌表达的GluZ最适反应温度是40℃。上述6种重组酶在10℃-50℃时都具有较高的的热稳定性,在最适反应湿度下保温1h后仍可保持70%以上的活力(3)重组酵母菌株S. cerevisiae PMXAK、S. cerevisiae PMXAAK和S. cerevisiae GMXAAK所产XylB最适反应pH为5.0;重组菌株S. cerevisiae PMGAK、S. cerevisiae PMGAAK和S. cerevisiae GMGAAK所产GluZ最适反应pH为6.0。上述6种重组酶在偏酸性环境中(pH3.0和pH4.0)时具有较好的稳定性。第五,本文对重组酵母的发酵性能和发酵中降低啤酒发酵液粘度的效果进行了研究。结果表明(1)重组菌与出发菌株相比较,生长性能略有下降,但是对啤酒的的表观发酵度和相实发酵度影响不大。(2)重组菌精酶液具有较好的降解麦汁葡聚糖和木聚糖的能力,麦汁粘度随葡聚糖或木聚糖含量的降低而下降。(3)三株共表达葡聚糖酶和木聚糖酶的重组菌在主发酵过程中都能不同程度的降低麦汁的粘度,其中分泌表达菌株降解能力最好,诱导型展示表达菌株降解能力最差。第六,为考察酿酒酵母基因工程菌株的生产性能进行了实验室小规模酿酒试验。重组菌株酿造的啤酒口味纯正,各项理化指标均达到啤酒国家标准。重组酿酒酵母在啤酒主发发酵和后发酵中使麦汁中的β-葡聚糖和阿拉伯木聚糖含量降低了60%-70%,与对照组S. cerevisiaeWZ65相比,啤酒粘度下降27%以上,麦汁中β-葡聚糖从312mg/L降至106mg/L和121mg/L,达到了管敦仪等人提出的啤酒在膜过滤除菌前β-葡聚糖含量应低于150mg/L的要求。更多还原

【Abstract】 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.更多还原