【作者】 张强；

【导师】 何国庆；

【作者基本信息】 浙江大学 ， 食品科学， 2008， 博士

【摘要】 β-葡聚糖在啤酒生产中会造成过滤困难以及成品啤酒的非生物性混浊等问题,一定程度上影响啤酒的质量和保质期。啤酒泡沫,特别是纯生啤酒的泡沫问题会受到蛋白酶A（PrA）很大程度的影响。现代啤酒工业中通过生产过程中添加微生物来源的β-葡聚糖酶制剂以及PrA的抑制剂等手段来降低它们带来的消极影响,但这无疑使啤酒生产变得复杂而且提高了成本。因此,本研究采用同源重组技术以枯草芽孢杆菌的β-葡聚糖酶基因替换酿酒酵母染色体组中的一个PEP4等位基因,构建能够降解β-葡聚糖而PrA低表达的重组酿酒酵母。结果表明,构建的重组菌株能有效的分泌β-葡聚糖酶同时降低了PrA的表达,在删除了进行基因操作时引入的抗性标记基因后,构建完成了适合于啤酒工业特别是纯生啤酒酿造的重组酿酒酵母菌株。同时对重组酿酒酵母菌株的生理和发酵性能进行了研究。主要研究结果如下:从酿酒酵母基因组中PCR扩增PGK1启动子、MFα1信号肽以及ADH1终止子序列,构建了在酿酒酵母的启动子、分泌序列、终止子作用下的枯草芽孢杆菌bg1S基因（编码β-1,3-1,4-葡聚糖酶）在酿酒酵母中的表达单元。并以KanMX的kan^r为筛选标记,以质粒Yeplac181为出发质粒构建了酿酒酵母的表达载体。表达载体中包含的bg1S表达单元转入工业酿酒酵母菌株WZ65得到了表达,并得到了有效分泌。用DNS法对重组酵母菌生长过程中分泌到培养介质中的β-葡聚糖酶活性进行了测定,结果显示在60h出现最大值,然后下降,在接下来的60h内基本保持稳定。在前人研究的基础上对β-葡聚糖酶活性测定方法进行改进,用刚果红法可以更好的对本文构建的重组酿酒酵母菌株分泌的β-葡聚糖酶酶活进行测定。对重组酶的酶学性质进行了研究,并与枯草芽孢杆菌中表达的野生型β-葡聚糖酶的性质进行了对比。研究发现,本试验构建的重组酵母表达的重组β-葡聚糖酶酶学性质发生了改变。40℃热处理20min造成重组酶活很大损失,残留的酶活只有30℃处理的63.4%;70℃的热处理与40℃处理相比酶活性只降低了17.5%,保持了45.9%的最初酶活,重组酶在高温下的稳定性增强。与野生酶相比,重组酶的最适pH值为5.0,而野生酶在6.0-7.0之间;相比野生酶在pH6.0-7.0有较高的稳定性,重组酶在pH5.0-6.0之间有最大的稳定性。由于酿酒酵母中表达载体不稳定,为了使β-葡聚糖酶在重组酵母菌株更稳定的表达而且降低蛋白酶A对啤酒泡沫阳性蛋白的降解作用,在前期试验证明了构建的bg1S表达单元正确的基础上,用bg1S基因表达单元替换了出发酵母菌株WZ65染色体上的一个PEP4等位基因,构建了PEP4等位基因杂合的重组酿酒酵母菌株SC-βG1、SC-βG2,其基因型为:PEP4/pep4∷KanMX-bg1S。β-1,3-1,4-葡聚糖酶的编码基因在进行替换后得到了有效的表达和分泌,菌株SC-βG1、SC-βG2培养过程中最高酶活性出现在培养56-64h时,分别为28.1U和20.2U,与用表达载体表达时的出现最高酶活性的时间基本一致。重组菌对数期的生长速度与出发菌株相比略慢,但稳定期的细胞浓度基本一致。而且稳定期以后的生长变化趋势基本一致。考虑到菌株应用的安全性,利用Cre/loxP系统成功的删除了在构建产β-1,3-1,4-葡聚糖酶的酵母菌株SC-βG1时整合在染色体上的KanMX,而且利用实验中使用的包含Cre/loxP系统的质粒pSH-Z的不稳定性,通过传代使质粒丢失而筛选到2株无抗性标记的重组酵母菌株SC-βG1A、SC-βG1B,整合到重组菌株基因组中的bg1S基因表达单元没受到影响。对最终获得的无抗性标记的菌株SC-βG1A、SC-βG1B分泌的β-1,3-1,4-葡聚糖酶酶活进行了测定,结果显示删除Kan^r的菌株保留了原来菌株的β-1,3-1,4-葡聚糖酶活性,分别为32.3U和29.1U。对最终获得的重组菌株在实验室进行了继代培养并对其遗传稳定性、生长性能、发酵力、凝聚性、热死灭温度等几个重要的生理指标进行了测试,并对发酵液进行了蛋白酶A活性,β-葡聚糖酶活性的测定和研究。结果表明重组菌株在麦芽汁发酵过程中能够产生较高活性的β-葡聚糖酶。被替换掉一个等位PEP4基因的酿酒酵母菌株的蛋白酶A的活性与出发菌株WZ65相比降低了30-40%。重组菌SC-βG1A、SC-βG1B中bg1S表达单元在传代过程中遗传稳定,不发生丢失。与对照菌株相比,重组菌初期生长变缓,但稳定期细胞浓度基本相同,稳定期以后的浓度变化一致;热死灭温度降低2℃;转化菌遗传性能稳定,与对照菌生长性能、发酵力、凝聚性均基本相似。我们选用Saccharomyces cerevisiae SC-βG1A进行了小试,对其啤酒酿造过程中的生长发酵性能以及成品的口味风味特性等方面进行了测试;对该菌株酿制啤酒的多项指标包括浓度、浊度、色度、苦味值、pH、α-N、总酸、酒精度、发酵度、外观糖度、双乙酰、泡持性、CO₂、口味、风味等也进行了测试。试验结果表明,重组菌酿制的啤酒酯香味突出、苦味较重,其它与现行干啤相近;各种理化指标均达到国家标准优级;啤酒泡沫的泡持性从202s增加到274s。更多还原

【Abstract】 Beta-glucans could reduce the extract yields in the brewhouse,affect filtration performance and participate in haze formation in wort and beer.Commercialβ-glucanase was added into the wort and beer during the fermentation to eliminate the impact ofβ-glucan.Yeast proteinase A （PrA）,a proteolytic enzyme found in the vacuole of Saccharomyces cerevisiae,can be excreted into beer under stress or by yeast autolysis and is detrimental to beer foam stability.PrA inhibitor was used in brewhouse to reduce the negative impact of PrA on foam active-protein.To resolve the problems caused byβ-glucan and PrA in beer production,we constructed the recombinant S.cerevisiae strain with one of the PEP4 gene was replaced by bg1S gene from B.subtilis.Activelyβ-glucanase secreting by the recombinant strains was detected and the PrA was lower than the control strain.The fermentation property of the recombinants was test and show near to the control.Some of the process and results showed as follow:The bg1S gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtilis mutant ZJF-1A5 was cloned.The bg1S expression cassette including PGK1 promoter,bg1S gene fused to MFα1 signal peptide sequence,and ADH1 terminator,with G418-resistence as the selection marker,was constructed in the shuttle vector Yeplac-181.A notableβ-glucanase activity was detected in the culture with DNS method when the recombinants,obtained after the expression vector Yeplac-181 was transformed into the industrial S.cerevisiae strain WZ65,were incubated.The maximum activity was detected after 60h of incubation in the YEPD yeast culture.With the modified Congo-red method the enzyme Properties of theβ-glucanase secreted by the recombinant yeast was tested and showed a variation contrast to the initial enzyme of the B.subtilis.There is an obvious change in the optimal temperature and heat-stability of the recombinant enzyme contrast to the native enzyme.Changing in temperature lead to a significant reduction of the active enzyme in a relatively low temperature,below 40℃.There was a small impact to the reduction of the activity by raising the temperature when it over 40℃,The optimal pH of the recombinant enzyme was lower to 5.0 contrasts to 6.0-7.0 of the native.To enhance the expression stability of theβ-glucanase by the recombinants,the bg1S expression cassette was integrated on the S.cerevisiae chromosome after replacing one of the PEP4 gene allele and resulting recombinant SC-βG1 and SC-βG2 with the gene type,PEP4/pep4∷KanMX-bg1S.The maximumβ-glucanase activity of SC-βG1 and SC-βG2 was between 56-64h of the incubation course,almost the same time point mentioned above.The recombinant strains, SC-βG1 and SC-βG2,exibit an unobvious feeble in growth capability but had the same ultimate cell concentration to the control.To make a considering safely yeast strain of the recombinant we have constructed,the KanMX,working as the resistance marker,was deleted under the action of Cre/loxP system constructed in the plasmid pSH-Z.Two strains with the bglS cassette preserved and the plasmids pSH-Z missing were screened during the subculture,were named SC-βG1A and SC-βG1B respectively.Experiment result showed that the recombinant strains SC-βG1A and SC-βG1B remained theβ-glucanase activity.The physiological characteristics of SC-βG1A and SC-βG1B were tested including genetic stability,growing performance,fermenting ability,flocculation ability,heat-resistance temperature, etc.The activity of proteinase A andβ-glucanase was also measured.It is shown there is a lower proteinase A activity,contract to the very initial strain WZ65,and notableβ-glucanase activity in the ultimate recombinant strains SC-βG1A and SC-βG1B.The genetic performance was stable and the fermenting ability,flocculation ability of these recombinants was similar to the control strain except for an unobvious slowly starting growth and a slight lower heat-resistance temperature.Saccharomyces cerevisiae SC-βG1A was chosed to brewing beer in a 250L fermentation tank. The groth performance and fermenting property were tested and physic-chemical indexes were measured including concentration,turbidness,colourity,bitterness value,pH,α-N,total acidity, alcohol by volume and by weight,attenuation degree,apparent sugar concentration,bi-acetyl, head retention,CO₂ and flavor substances were also detected.The results indicated that the flavour of the beer brewing with the recombinant sacchramosasice cerevisiae strain similar to the current dry beer.Every of the physic-chemical indexe was reached superior grade according to the national standard,especially the head retention time was enhangced from 202s to 274s.Further more investigation was needed to test the brewing performance of the next generations of the recombinant Saccharomyces cerevisiae SC-βG1A.更多还原