【作者】 张阳；

【导师】 刘延琳；

【作者基本信息】 西北农林科技大学 ， 葡萄与葡萄酒学， 2019， 博士

【摘要】 葡萄酒中的萜类物质大多以结合态糖苷的形式存在,不易释放。β-葡萄糖苷酶(β-glucosidase,BGL)在糖苷水解过程中至关重要,然而在葡萄酒酿造过程中,其活性往往受葡萄酒发酵环境,即高浓度葡萄糖、乙醇及低pH等因素的影响,并且商业酶制剂的稳定性差、固定化酶的生产成本高等不利因素限制了BGL在葡萄酒酿造过程中的使用。近年来,酿酒酵母表面展示技术作为全细胞生物催化被认为是最有前景的方法之一,将酶展示到细胞表面,不仅可使其在活性和稳定性方面显著高于游离酶,而且具有不消耗固定化材料以及可重复利用等特点,为BGL在提升葡萄酒香气方面的应用提供了新的解决思路。本论文将来源于黑曲霉的BGL展示在酿酒酵母细胞表面,通过优化载体、启动子、锚定蛋白以及转录融合伴侣(translational fusion partner,TFP)等策略实现BGL的高效稳定展示,同时探索了酿酒酵母表面展示BGL的酶学特性及其在发酵环境下对香气糖苷的水解能力。主要研究内容及结果如下:(1)以绿色荧光蛋白(GFP)为靶蛋白,Sag1p、Sed1p、Cwp2p为锚定蛋白,利用无缝克隆技术将其插入到包含诱导型启动子GAL1的高拷贝载体pYES2/CT/α-Factor中,通过激光共聚焦显微镜观察GFP的位置。结果发现阳性对照菌株PBy-eG表达的GFP存在于细胞质中,而实验组菌株PBy-eGSA、PBy-eGSE、PBy-eGCW所表达的GFP具有向外分泌的趋势,同时在细胞周围也不存在游离形式的GFP,表明了酿酒酵母表面展示平台搭建成功。(2)分别将已构建的三种绿色荧光蛋白锚定载体上的启动子GAL1更换为GPD和SED1的启动子,绿色荧光蛋白基因(eGFP)替换为BGL基因(bgl1),成功构建出表面展示BGL的高拷贝质粒,并将其电转入宿主菌BY4741中进行表达。结果发现;六株重组菌所展示的酶活均随培养时间的延长而降低,其中GPD启动子驱动的阳性重组菌所展示的酶活均高于SED1启动子的;三种锚定蛋白(Sag1p、Sed1p、Cwp2p)中Sag1p表现最好;因此携带质粒GPD-HQM-Sag1的重组菌PBy-GBSa能够更为高效地展示BGL,且当培养到24 h,酶活达到最大值18.29±1.05 U/g。(3)构建了以HIS3为重组臂,URA3为筛选标记的酵母整合型载体Yip-loxp-URA3,分别将已构建的六种表面展示BGL表达盒插入到整合型载体中,成功构建了表面展示BGL的整合型质粒,通过实时定量PCR(Real time PCR)和酶活测定监测BGL在整个发酵过程中的转录水平和蛋白表达水平。结果发现,SED1启动子的转录水平要高于GPD启动子的;在蛋白表达水平,六株重组菌所展示的酶活均随培养时间的延长而逐渐升高,当培养至84 h时,酶活达到最大值;以GPD启动子驱动的阳性重组菌所展示的酶活高于SED1启动子的,且sed1p作为锚定蛋白时,所展示的BGL活性达到最高(25.22±0.81 U/g)。(4)将已构建的表面展示BGL酶活最高的阳性重组菌BY-GSE所用质粒Yip-GPD-BGL-Sed1中的MFalpha1 TFP替换为来源于酿酒酵母的23个不同的TFPs,考察TFPs对表面展示BGL活性的影响。结果显示,所有的TFPs都可以使BGL成功地展示在酵母表面,其中13-HSP150、17-SED1(195 aa)、23-CCW14和24-SED1(170 aa)的菌株所展示的BGL活性高于原始菌BY-GSE,且17-SED1(195 aa)重组菌展示的酶活最高(74.58±5.88 U/g)。(5)以商业酶制剂AR2000作为对照,考察了表面展示的BGL在不同条件下(葡萄糖、pH和乙醇)的稳定性及其在模拟葡萄汁发酵过程中对香气糖苷的水解能力。结果显示,当葡萄糖浓度低于5%时,游离态酶的活性被完全抑制,而表面展示BGL酶活的受抑制程度明显减弱,保留了12%左右的残留活性;在pH 3.0时,表面展示BGL的残留活性在50%左右,而游离态酶仅有15%左右,高出3倍以上;乙醇对表面展示BGL和商业酶制剂AR2000都具有抑制作用,且抑制趋势相似,在20%(v/v)乙醇浓度下,均保留了50%以上的残留活性;利用酵母细胞表面展示BGL所释放的萜烯醇浓度是商业酶制剂AR2000(37.58±1.16μg/L)的2.1倍。更多还原

【Abstract】 It is well known that most of the terpenoids in wine exist in the form of bound glycosides,which are not easily released.β-glucosidase(BGL)is essential in the hydrolysis of glycosides,while its activity is always inhibited by the factors such as high glucose,ethanol and low pH during the winemaking process.Moreover,the disadvantages of poor stability of commercial enzyme preparations and high production costs of immobilized enzymes limit the use of BGL in the winemaking process.In recent years,cell surface-display technique of Saccharomyces cerevisiae,as as a whole-cell biocatalyst,has been considered as one of the most promising methods.By displaying the enzyme on the cell surface,the activity and stability of enzyme could be significantly enhanced,and it does not need to consume the immobilized material,which makes it reusable,providing a new solution for the application of BGL in improving the wine aroma.In our study,BGL derived from Aspergillus niger was displayed on the surface of Saccharomyces cerevisiae cells,and different vectors,promoters,anchoring proteins and translational fusion partners(TFPs)were used to obtain an efficient and stable display of BGL.In addition,the enzymatic properties and the capacity of surface-displayed BGL in hydrolyzing aroma glycosides during fermentation were investigated.The main research contents and results are as follows:(1)The green fluorescent protein(GFP)was used as the target protein,and was fused with 3 anchoring proteins,including Sag1 p,Sed1p and Cwp2 p,which were inserted into the vector pYES2/CT/α-Factor containing a inducible promoter Gal1 using In-fusion technology.Then,the location of GFP was observed using laser confocal microscopy.The results showed that GFP expressed by the positive control strain PBy-eG was present in the cytoplasm,while in the experimental strains like PBy-eGSA、PBy-eGSE、PBy-eGCW,GFP had a tendency to be secreted to the outside of the cell,meanwhile no free GFP was detected,confirming that the Saccharomyces cerevisiae surface display platform has been successfully constructed.(2)The promoter GAL1 in the three GFP-anchoring vectors constructed in chapter 2 was replaced with the promoters of GPD and SED1,respectively,and the gfp gene was replaced by β-glucosidase gene(Bgl1),thus the high-copy plasmids displaying BGL on the surface were successfully constructed and then electroporated into the host strain BY4741 for expression.Results showed that the enzyme activities displayed by the six recombinant strains all decreased with the prolongation of the culture time,among which the enzyme activities of the positive recombinant strain driven by the GPD promoter were higher than that of the SED1 promoter.As for the three anchoring proteins(Sag1p,Sed1 p,Cwp2p),Sag1 p performed best.Therefore,the recombinant strain PBy-GBSa carrying the plasmid GPD-HQM-Sag1 was able to display BGL more efficiently.When it was cultured for 24 h,the enzyme activity reached a maximum of 18.29±1.05 U/g.(3)The yeast integrative plasmids Yip-loxp-URA3 with HIS3 as the recombination arm and URA3 as the selection marker was constructed,and then the six surface-display BGL cassettes in chapter 3 were inserted into the integrative plasmids,thus the surface-displayed BGL integrative plasmids were successfully constructed.qRT-PCR and enzyme activity assay were performed to monitor the BGL expression at both transcription and protein levels during the fermentation process.It was found that the transcription level of the SED1 promoter was higher than that of the GPD promoter,while at the protein expression level,the enzyme activities displayed by the six recombinant strains all gradually increased with the prolongation of the culture time,which reached the maximum at 84 h.The enzyme activities of the positive recombinant strains driven by GPD promoter were higher than that of the SED1 promoter,which reached the maximum(25.22±0.81 U/g)when sed1 p was used as anchoring protein.(4)The MFalpha1 TFP in the plasmid Yip-GPD-BGL-Sed1 of the positive recombinant strain BY-GSE in chapter 4 was replaced with 23 different TFPs derived from Saccharomyces cerevisiae,and effects of TFPs on the BGL activities were investigated.The results showed that all the strains with different TFPs could successfully display BGL on the yeast cell surface,among which strains 13-HSP150、17-SED1(195 aa)、23-CCW14 and 24-SED1(170 aa)had higher BGL activities than the original strain BY-GSE,and activity of BGL in the strain with 17-SED1(195 aa)(74.58±5.88 U/g)was 3 times higher than that of the control.(5)The stability of surface-displayed BGL under different conditions(different concentrations of glucose,ethanol and pH)and its capacity of hydrolyzing glycosidic bonds during the fermentation process were investigated,using commercial enzyme preparation AR2000 as control.It was found that,the surface-display BGL was less inhibited by glucose less than 5%,and still had 12% residual activity under 5% glucose,while the free enzyme was completely inhibited.At pH 3.0,the residual activity of surface-displayed BGL was about 50%,which was more than three times higher than that of free enzyme(only about 15%).Ethanol had an inhibitory effect on the surface-displayed BGL and the commercial enzyme preparation AR2000,and the inhibition trend was similar.At 20%(v/v)ethanol concentration,more than 50% residual activity was retained.The concentration of terpenols released by the surface-displayed BGL was 2.1 times that of the commercial enzyme preparation AR2000(37.58±1.16 μg/L).更多还原