Issatchenkia terricola XJ-2乙醛脱氢酶的纯化和性质及其基因的克隆与表达
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摘要
醛脱氢酶超家族(Aldehyde Dehydrogenase superfamily, E.C.1.2.1.x)是一类重要的氧化还原酶类,广泛存在于动物、植物和微生物中。醛脱氢酶(ALDH)具有相似的一级结构,依赖NAD(P)+为辅酶将脂肪族和/或芳香族的醛类氧化成相应的酸类,从而调整生物体中醛类的浓度水平。根据国际癌症研究机构(IARC)和大量的实验证据表明,乙醛具有诱变作用和致癌性,其致诱变浓度为40-1000μmol/L。由于醛脱氢酶超家族中的乙醛脱氢酶能有效降解乙醛,有必要对乙醛脱氢酶进行深入研究,以减轻或消除乙醛的危害。本论文主要对高活性的乙醛脱氢酶产生酵母的筛选、乙醛脱氢酶的分离纯化和性质,以及乙醛脱氢酶基因的克隆和异源高效表达进行了研究,旨在为从蛋白和分子水平研究乙醛脱氢酶的结构和催化机理等提供理论依据,并为乙醛脱氢酶的生产和应用奠定基础。主要研究结果分述如下：
1.从新疆马奶葡萄中筛选了高产乙醛脱氢酶的酵母菌株并对其进行了鉴定。
采用黄豆芽培养基初筛、摇瓶复筛的筛选策略,从南京果园土壤和新疆马奶葡萄中分离筛选到三株具有乙醛脱氢酶活性的酵母菌株,其中筛自马奶葡萄的酵母菌株XJ-2酶活最高,达6.28U/mL,比活力为2.28U/mg。建立顶空气相色谱法检测菌株XJ-2粗酶液反应体系中的乙醛和乙酸,直接证明了该粗酶液中具有能够将乙醛氧化成乙酸的乙醛脱氢酶。采用酵母26S rDNA D1/D2区域序列的通用引物对菌株XJ-2进行PCR扩增,获得554bp的片段。将该PCR产物在GenBank数据库中BLAST软件进行同源搜索比对,利用Clustal X1.83和MEGA4.0软件进行多重比较后绘制系统发育树,结果表明：酵母菌株XJ-2的26S rDNA D1/D2区域序列与陆生伊萨酵母(Issatchenkia terricola) MH509的26S rDNA D1/D2区域序列同源性达100%；在系统发育树中,菌株XJ-2与I. terricola在同一分支,亲缘关系最近。结合常规形态特征、生理生化特征,鉴定该菌株为陆生伊萨酵母(I. terricola)。
2.对I. terricola XJ-2所产的乙醛脱氢酶进行了分离纯化并研究了其酶学性质。
经硫酸铵分级沉淀、DEAE-sephacel阴离子交换层析和Bio-Gel HTP羟基磷灰石吸附层析,从菌株XJ-2的粗酶液中获得了电泳纯的乙醛脱氢酶,比活力达到了31.2U/mg,较粗酶液纯化了14.3倍,酶活回收率为14.7%。
对乙醛脱氢酶纯酶进行SDS-PAGE和非变性PAGE电泳以及HPLC分析,结果表明该酶为同源四聚体结构。乙醛脱氢酶的最适反应温度和pH分别为50℃和9.0,在温度和pH分别为20-40℃和6.0-9.0的范围内稳定。乙醛脱氢酶有着较广的底物作用范围,偏好作用于短链脂肪族醛类,特别是丁醛和乙醛；能以NAD+和NADP+为辅酶,NAD+最佳。对乙醛的Km、Vmax、kcat和kcat/Km分别为0.73mmol/L、35.71U/mg、32.73/s和45.14×103/(mol·s)；对NAD+的Km和Vmax分别为0.24mmol/L和42.19U/mg。K+和NH4+是乙醛脱氢酶的激活剂,金属离子Mg2+、Ca2+、Ba2+、Co2+、Ni2+、Mn2+和化学试剂PMSF在1mmol/L和5mmol/L的浓度下对乙醛脱氢酶酶活具有一定程度的抑制作用,并随着浓度的增加而增强；Ag+、Cu2+、Zn2+、Hg2+则完全抑制了酶活性。还原剂2-ME和DTT对酶活有很强的促进作用。
4.克隆了I. terricola XJ-2乙醛脱氢酶基因并进行序列分析。
设计CODEHOP简并引物进行PCR扩增获得了乙醛脱氢酶基因的保守区序列,利用SiteFinding-PCR和Self-formed adaptor PCR染色体步移技术扩增了保守区的旁邻序列。经序列分析和拼接获得了I. terricola XJ-2乙醛脱氢酶基因(ist-ALD)的开放阅读框,长度为1578bp。对该基因编码的蛋白(ist-ALDH)进行生物信息学分析可知,ist-ALDH由525个氨基酸组成,预测分子量为57.2kDa；与Pichia angusta的ALDH(AAA83769)氨基酸序列的相似性达到73%,亲缘关系最近；含有16个在ALDH超家族中高度保守的氨基酸残基和10个保守性较高的氨基酸基元序列；预测Glu293和Cys327是其的催化活性位点以及第三和第四基元序列之间的氨基酸序列(GFGKTIG)是辅酶NAD(P)+的特异性结合位点。将ist-ALDH的氨基酸序列提交至http://swissmodel.expasy.org蛋白质在线分析服务器,预测其亚基的三级结构模型。该模型含有三个ALDH亚基的特征结构域：催化结构域、辅酶结合结构域和寡聚化结构域。通过一系列氨基酸序列分析证明了ist-ALD基因编码的蛋白是ALDH超家族的新成员。
4.实现了I. terricola XJ-2的乙醛脱氢酶基因在大肠杆菌中的异源表达并研究了重组酶的性质。
构建了非融合表达载体pET-23a-ist-ALD)和融合表达载体pET-32a-ist-ALD,将其分别转化到E. coli BL21(DE3)中均获得了活性表达,其中E. coli BL21/pET-32a-ist-ALD表达的重组乙醛脱氢酶活力最高,达到44.23U/mL,比活力为10.95U/mmg,是天然酶比活力的4.8倍。
重组酶ist-ALDH经凝血酶酶切和Ni-NTA一步分离纯化,达到了电泳纯,纯化倍数为8.99倍,回收率为18.06%。通过SDS-PAGE和非变性PAGE电泳测定重组酶的分子量分别为57kDa和232kDa,表明该酶是同源四聚体。ist-ALDH的最适反应温度和pH分别为40℃和9.0,在温度和pH分别为20-37℃和7.0-9.0的范围内稳定。与天然酶的酶学性质比较可知,重组酶ist-ALDH的最适反应温度较天然酶低10℃,热稳定性稍差,但其它酶学性质则与天然酶的基本一致。
5.对重组大肠杆菌高效表达乙醛脱氢酶的条件进行了优化并对重组大肠杆菌细胞转化清除乙醛的进行了研究。
通过单因素试验选择TB5培养基作为重组菌E. coliBL21(DE3)pET32a-ist-ALD的表达培养基,并确定了诱导剂乳糖的诱导时机为OD600=3.0-3.2、添加量为140μg/mL。通过Plackett-Burman试验和中心组合设计试验快速筛选出了影响重组大肠杆菌表达乙醛脱氢酶的关键因子并对其实现了条件优化：当诱导时间、装液量和接种量分别为20.68h、126.75mL和3%时,获得重组乙醛脱氢酶的酶活为496.65U/mL,较未优化前的44.23U/mL提高了12.5倍。
以最佳表达条件下培养的重组大肠杆菌为催化剂,通过对反应pH、反应温度、表面活性剂、底物浓度和NAD+浓度等因素对重组菌细胞转化清除乙醛的影响进行考察和分析,获得了细胞反应体系的最佳组成为：0.1mol/LGlycine-NaOH缓冲体系(pH9.5)、1mmol/L乙醛、2mmol/L NAD+、10mmol/L2-巯基乙醇,0.1mol/L KCl以及经0.075%(v/v)Triton X-100处理的湿菌体(1mg/mL)。当该体系于40℃反应15min,乙醛转化率为98%,细胞平均转化力为3.92mmol/g cells/h。
Aldehyde Dehydrogenase superfamily (E.C.1.2.1.x) comprises a group of very important oxidoreductases, widely distributing in animals, plants and microorganisms. ALDHs have the similar primary structure and can catalyze the NAD(P)+-dependent oxidation of aliphatic and/or aromatic aldehydes to their corresponding acids for the adjustment of the concentration of aldehydes in organisms. Acetaldehyde is a known mutagen and carcinogen according to the International Agency for Research on Cancer (IARC) and numerous experimental evidences, the mutagenic concentration of which is40-1000μmol/L. One of the most effective way for acetaldehyde elimination is its oxidation to acetate by acetaldehyde dehydrogenase, a member of ALDH superfamily. Thus, it is of interest to study the acetaldehyde dehydrogenase that has potential as an enzymatic product for detoxification of exogenous and endogenous acetaldehyde. The thesis presents the investigations on the screening of a high-activity acetaldehyde dehydrogenase producing yeast strain, purification and characterization of the enzyme, cloning and high-yield heterologous expression of the acetaldehyde dehydrogenase gene. The aim of the study is providing a theoretical support on the research of acetaldehyde dehydrogenase structure and catalysis mechanism. Furthermore, it lays a foundation for production and application of the enzyme in the future. The main results of this study are as follows:
1. A yeast strain was isolated from grapes which could produce high-activity acetaldehyde dehydrogenase and identified.
Soil and Manai grapes samples were collected from an orchard in Nanjing, Jiangsu province and Xinjiang province, respectively. Three yeast strains showing acetaldehyde dehydrogenase activity were isolated through prescreening of being cultivated in the soybean sprout media and then enzyme detection of being fermented in the malt media. The strain XJ-2collected from Manai grapes was selected for its ability to produce the highest activity of6.28U/mL, with the specific activity of2.28U/mg. The enzyme activity was validated by oxidizing acetaldehyde to acetate with NAD+as coenzyme based on the headspace gas chromatography analysis. The26S rDNA D1/D2gene sequence of the yeast strain XJ-2was amplified by PCR, and a554-bp sequence was obtained. A homology search was performed on Genbank database, and a neighbor-joining phylogenetic tree was constructed using a ClustalX1.83program and a MEGA4program. The results indicated that the26S rDNA D1/D2gene sequence of strain XJ-2revealed the highest homology (100%) with that of Issatchenkia terricola MH509. In the phylogenetic tree, strain XJ-2and/terricola were in the same branch showing the closest kinship. In addition to the morphological, physiological, and biochemical characteristics of strain XJ-2, the isolate was identified as I. terricola.
2. The acetaldehyde dehydrogenase was purified from I. terricola strain XJ-2and its characterization was studied.
Acetaldehyde dehydrogenase was purified to electrophoretically homogeneity by combination of purification steps including ultrasonic cell disruption, ammonium sulfate fraction precipitation, DEAE-sephacel ion-exchange chromatography and Bio-Gel HTP hydroxyapatite adsorption chromatography. The specific activity of31.2U/mg of the purified protein was14.3-fold from the enzymatic extracts with a yield of14.7%.
By analysis of the molecular weight of acetaldehyde dehydrogenase through SDS-PAGE, native PAGE and HPLC, the enzyme existed as a homotetramer. The optimum temperature and pH of acetaldehyde dehydrogenase was50℃and9.0, respectively. The enzyme appeared to be stable in the range of20-40℃and pH6.0-9.0, respectively. It exhibited a broad substrate specificity for various aliphatic and aromatic aldehydes and preferred the former ones, especially butylaldehyde and acetaldehyde. Comparing with NADP+, NAD+was the preferred coenzyme. Kinetic study indicated that the acetaldehyde dehydrogenase had a Km value of0.73mmol/L, a Vmax value of35.71U/mg, a kcat value of32.73/s and a kcat/Km value of45.14×103/(mol·s) on acetaldehyde, respectively, while a Km value of0.24mmol/L and a Vmax value of42.19U/mg on NAD+, respectively. The enzyme activity was enhanced in the presence of K+, NHH+4,2-mercaptoethanol or DTT, while partially inhibited by several metal ions and chemicals at concentration of1mmol/L and5mmol/L, such as Mg2+, Ca2+, Ba2+, Co2+, Ni2+, Mn2+and PMSF. The higher concentration of the metal ions or chemicals was, the stronger inhibition showed. The activity was absolutely inhibited by Ag+, Cu2+,Zn2+and Hg2+.
3. The acetaldehyde dehydrogenase gene of I. terricola strain XJ-2was cloned and the sequence analysis was performed.
The conserved gene fragment was amplified by PCR using the CODEHOP primers. The flanking regions of the conserved gene fragment were amplified by SiteFinding-PCR and Self-formed adaptor PCR. By sequence analysis and assembling, the ORF of acetaldehyde dehydrogenase gene of I. terricola strain XJ-2(ist-ALD) was obtained with1578bp in length. The bioinformatics analysis of the predicted protein of ist-ALD revealed that the protein contained525amino acids with a predicted molecular weight of57.2kDa. Ist-ALDH showed the highest identity (73%) to ALDH from Pichia angusta. Sixteen amino acid residues that are found in more than95%of ALDH proteins were all critically conserved in the predicted protein, while ten most conserved motifs in ALDHs were also existed. Two presumed active-site residues, GIU293and CyS327, known to be catalytically essential amino acids and a glycine motif involved in NAD(P)+binding (GFGKITG) were identified in the protein. The amino acid sequence of ist-ALDH was submitted to http://swissmodel.expasy.org to predict the structure of one subunit. The characteristic domains of the subunit of ALDH, which are the catalysis domain, cofactor-binding domain and the oligomerization domain, respectively, were found in the tertiary structure model. A series of amino acid analysis demonstrated that ist-ALDH was a new member of ALDH superfamily.
4. The acetaldehyde dehydrogenase gene of I. terricola strain XJ-2was heterologous expressed in E. coli and the characterization of the recombinant enzyme was studied.
Two recombinant expression vectors, pET-23a-ist-ALD and pET-32a-ist-ALD were constructed, and transformed to E. coli BL21(DE3), respectively. The two recombinants were able to express acetaldehyde dehydrogenase. The recombinant E. coli harboring pET-32a-ist-ALD yield a higher activity of44.23U/mL, the specific activity of which reached10.95U/mg,4.8-fold than that of native enzyme.
The His6-tagged ist-ALDH in the cell-free extract was purified by Ni-NTA agarose column and appeared one band on a SDS-PAGE gel and a native PAGE gel with a calculated molecular weight of57kDa and232kDa, respectively, indicating that the purified ist-ALDH presented as a homotetramer. Ist-ALDH exhibited the optimal activity at40℃and pH9.0, respectively. The recombinant enzyme was stable in the range of20-37℃and pH7.0-9.0, respectively. By comparing with the native acetaldehyde dehydrogenase, the optimal temperature of the recombinant enzyme was10℃less than that of native one. Moreover, the thermal stability of ist-ALDH was worse than that of the native enzyme, while other characterics of the two enzymes were comparable.
5. Culture conditions of the recombinant E. coli for high-yield acetaldehyde dehydrogenase expression were optimized and the clearance of acetaldehyde transformed by the recombinant E. coli was studied.
Based on the results of single factor experiments, TB5medium was selected as the expression medium of recombinant E. coli BL21(DE3)/pET32a-wist-ALD. The recombinant ist-ALDH was induced to express when OD600reached3.0-3.2with140μg/mL of lactose added. The key factors affecting the expression of recombinant ist-ALDH were screened by the Plackett-Burman experiments and optimized using the central composite design experiments. The predicted optimum culture conditions for maximum expression of recombinant ist-ALDH were found to be comprised of20.68h post-induction,126.75mL medium volume, and3%(v/v) inoculum content with an experimental acetaldehyde dehydrogenase activity of496.65U/mL, resulting in12.5-fold increment after optimization.
The recombinant E. coli was prepared under the optimized condition, and served as a catalyst for the clearance of acetaldehyde. By studying the effect of reaction pH, temperature, surfactants, concentration of acetaldehyde and NAD+on the whole cell biotransformation, the optimum reaction system was as follows:0.1mol/L Glycine-NaOH buffer (pH9.5),1mmol/L acetaldehyde,2mmol/L NAD+,10mmol/L2-ME,0.1mol/L KC1, and1mg/mL wet cells that were treated by0.075%(v/v) Triton X-100. When the reaction was performed at40℃for15min,98%of acetaldehyde could be removed with a total cell average transformation of3.92mmol/g cells/h.
1.从新疆马奶葡萄中筛选了高产乙醛脱氢酶的酵母菌株并对其进行了鉴定。
采用黄豆芽培养基初筛、摇瓶复筛的筛选策略,从南京果园土壤和新疆马奶葡萄中分离筛选到三株具有乙醛脱氢酶活性的酵母菌株,其中筛自马奶葡萄的酵母菌株XJ-2酶活最高,达6.28U/mL,比活力为2.28U/mg。建立顶空气相色谱法检测菌株XJ-2粗酶液反应体系中的乙醛和乙酸,直接证明了该粗酶液中具有能够将乙醛氧化成乙酸的乙醛脱氢酶。采用酵母26S rDNA D1/D2区域序列的通用引物对菌株XJ-2进行PCR扩增,获得554bp的片段。将该PCR产物在GenBank数据库中BLAST软件进行同源搜索比对,利用Clustal X1.83和MEGA4.0软件进行多重比较后绘制系统发育树,结果表明：酵母菌株XJ-2的26S rDNA D1/D2区域序列与陆生伊萨酵母(Issatchenkia terricola) MH509的26S rDNA D1/D2区域序列同源性达100%；在系统发育树中,菌株XJ-2与I. terricola在同一分支,亲缘关系最近。结合常规形态特征、生理生化特征,鉴定该菌株为陆生伊萨酵母(I. terricola)。
2.对I. terricola XJ-2所产的乙醛脱氢酶进行了分离纯化并研究了其酶学性质。
经硫酸铵分级沉淀、DEAE-sephacel阴离子交换层析和Bio-Gel HTP羟基磷灰石吸附层析,从菌株XJ-2的粗酶液中获得了电泳纯的乙醛脱氢酶,比活力达到了31.2U/mg,较粗酶液纯化了14.3倍,酶活回收率为14.7%。
对乙醛脱氢酶纯酶进行SDS-PAGE和非变性PAGE电泳以及HPLC分析,结果表明该酶为同源四聚体结构。乙醛脱氢酶的最适反应温度和pH分别为50℃和9.0,在温度和pH分别为20-40℃和6.0-9.0的范围内稳定。乙醛脱氢酶有着较广的底物作用范围,偏好作用于短链脂肪族醛类,特别是丁醛和乙醛；能以NAD+和NADP+为辅酶,NAD+最佳。对乙醛的Km、Vmax、kcat和kcat/Km分别为0.73mmol/L、35.71U/mg、32.73/s和45.14×103/(mol·s)；对NAD+的Km和Vmax分别为0.24mmol/L和42.19U/mg。K+和NH4+是乙醛脱氢酶的激活剂,金属离子Mg2+、Ca2+、Ba2+、Co2+、Ni2+、Mn2+和化学试剂PMSF在1mmol/L和5mmol/L的浓度下对乙醛脱氢酶酶活具有一定程度的抑制作用,并随着浓度的增加而增强；Ag+、Cu2+、Zn2+、Hg2+则完全抑制了酶活性。还原剂2-ME和DTT对酶活有很强的促进作用。
4.克隆了I. terricola XJ-2乙醛脱氢酶基因并进行序列分析。
设计CODEHOP简并引物进行PCR扩增获得了乙醛脱氢酶基因的保守区序列,利用SiteFinding-PCR和Self-formed adaptor PCR染色体步移技术扩增了保守区的旁邻序列。经序列分析和拼接获得了I. terricola XJ-2乙醛脱氢酶基因(ist-ALD)的开放阅读框,长度为1578bp。对该基因编码的蛋白(ist-ALDH)进行生物信息学分析可知,ist-ALDH由525个氨基酸组成,预测分子量为57.2kDa；与Pichia angusta的ALDH(AAA83769)氨基酸序列的相似性达到73%,亲缘关系最近；含有16个在ALDH超家族中高度保守的氨基酸残基和10个保守性较高的氨基酸基元序列；预测Glu293和Cys327是其的催化活性位点以及第三和第四基元序列之间的氨基酸序列(GFGKTIG)是辅酶NAD(P)+的特异性结合位点。将ist-ALDH的氨基酸序列提交至http://swissmodel.expasy.org蛋白质在线分析服务器,预测其亚基的三级结构模型。该模型含有三个ALDH亚基的特征结构域：催化结构域、辅酶结合结构域和寡聚化结构域。通过一系列氨基酸序列分析证明了ist-ALD基因编码的蛋白是ALDH超家族的新成员。
4.实现了I. terricola XJ-2的乙醛脱氢酶基因在大肠杆菌中的异源表达并研究了重组酶的性质。
构建了非融合表达载体pET-23a-ist-ALD)和融合表达载体pET-32a-ist-ALD,将其分别转化到E. coli BL21(DE3)中均获得了活性表达,其中E. coli BL21/pET-32a-ist-ALD表达的重组乙醛脱氢酶活力最高,达到44.23U/mL,比活力为10.95U/mmg,是天然酶比活力的4.8倍。
重组酶ist-ALDH经凝血酶酶切和Ni-NTA一步分离纯化,达到了电泳纯,纯化倍数为8.99倍,回收率为18.06%。通过SDS-PAGE和非变性PAGE电泳测定重组酶的分子量分别为57kDa和232kDa,表明该酶是同源四聚体。ist-ALDH的最适反应温度和pH分别为40℃和9.0,在温度和pH分别为20-37℃和7.0-9.0的范围内稳定。与天然酶的酶学性质比较可知,重组酶ist-ALDH的最适反应温度较天然酶低10℃,热稳定性稍差,但其它酶学性质则与天然酶的基本一致。
5.对重组大肠杆菌高效表达乙醛脱氢酶的条件进行了优化并对重组大肠杆菌细胞转化清除乙醛的进行了研究。
通过单因素试验选择TB5培养基作为重组菌E. coliBL21(DE3)pET32a-ist-ALD的表达培养基,并确定了诱导剂乳糖的诱导时机为OD600=3.0-3.2、添加量为140μg/mL。通过Plackett-Burman试验和中心组合设计试验快速筛选出了影响重组大肠杆菌表达乙醛脱氢酶的关键因子并对其实现了条件优化：当诱导时间、装液量和接种量分别为20.68h、126.75mL和3%时,获得重组乙醛脱氢酶的酶活为496.65U/mL,较未优化前的44.23U/mL提高了12.5倍。
以最佳表达条件下培养的重组大肠杆菌为催化剂,通过对反应pH、反应温度、表面活性剂、底物浓度和NAD+浓度等因素对重组菌细胞转化清除乙醛的影响进行考察和分析,获得了细胞反应体系的最佳组成为：0.1mol/LGlycine-NaOH缓冲体系(pH9.5)、1mmol/L乙醛、2mmol/L NAD+、10mmol/L2-巯基乙醇,0.1mol/L KCl以及经0.075%(v/v)Triton X-100处理的湿菌体(1mg/mL)。当该体系于40℃反应15min,乙醛转化率为98%,细胞平均转化力为3.92mmol/g cells/h。
Aldehyde Dehydrogenase superfamily (E.C.1.2.1.x) comprises a group of very important oxidoreductases, widely distributing in animals, plants and microorganisms. ALDHs have the similar primary structure and can catalyze the NAD(P)+-dependent oxidation of aliphatic and/or aromatic aldehydes to their corresponding acids for the adjustment of the concentration of aldehydes in organisms. Acetaldehyde is a known mutagen and carcinogen according to the International Agency for Research on Cancer (IARC) and numerous experimental evidences, the mutagenic concentration of which is40-1000μmol/L. One of the most effective way for acetaldehyde elimination is its oxidation to acetate by acetaldehyde dehydrogenase, a member of ALDH superfamily. Thus, it is of interest to study the acetaldehyde dehydrogenase that has potential as an enzymatic product for detoxification of exogenous and endogenous acetaldehyde. The thesis presents the investigations on the screening of a high-activity acetaldehyde dehydrogenase producing yeast strain, purification and characterization of the enzyme, cloning and high-yield heterologous expression of the acetaldehyde dehydrogenase gene. The aim of the study is providing a theoretical support on the research of acetaldehyde dehydrogenase structure and catalysis mechanism. Furthermore, it lays a foundation for production and application of the enzyme in the future. The main results of this study are as follows:
1. A yeast strain was isolated from grapes which could produce high-activity acetaldehyde dehydrogenase and identified.
Soil and Manai grapes samples were collected from an orchard in Nanjing, Jiangsu province and Xinjiang province, respectively. Three yeast strains showing acetaldehyde dehydrogenase activity were isolated through prescreening of being cultivated in the soybean sprout media and then enzyme detection of being fermented in the malt media. The strain XJ-2collected from Manai grapes was selected for its ability to produce the highest activity of6.28U/mL, with the specific activity of2.28U/mg. The enzyme activity was validated by oxidizing acetaldehyde to acetate with NAD+as coenzyme based on the headspace gas chromatography analysis. The26S rDNA D1/D2gene sequence of the yeast strain XJ-2was amplified by PCR, and a554-bp sequence was obtained. A homology search was performed on Genbank database, and a neighbor-joining phylogenetic tree was constructed using a ClustalX1.83program and a MEGA4program. The results indicated that the26S rDNA D1/D2gene sequence of strain XJ-2revealed the highest homology (100%) with that of Issatchenkia terricola MH509. In the phylogenetic tree, strain XJ-2and/terricola were in the same branch showing the closest kinship. In addition to the morphological, physiological, and biochemical characteristics of strain XJ-2, the isolate was identified as I. terricola.
2. The acetaldehyde dehydrogenase was purified from I. terricola strain XJ-2and its characterization was studied.
Acetaldehyde dehydrogenase was purified to electrophoretically homogeneity by combination of purification steps including ultrasonic cell disruption, ammonium sulfate fraction precipitation, DEAE-sephacel ion-exchange chromatography and Bio-Gel HTP hydroxyapatite adsorption chromatography. The specific activity of31.2U/mg of the purified protein was14.3-fold from the enzymatic extracts with a yield of14.7%.
By analysis of the molecular weight of acetaldehyde dehydrogenase through SDS-PAGE, native PAGE and HPLC, the enzyme existed as a homotetramer. The optimum temperature and pH of acetaldehyde dehydrogenase was50℃and9.0, respectively. The enzyme appeared to be stable in the range of20-40℃and pH6.0-9.0, respectively. It exhibited a broad substrate specificity for various aliphatic and aromatic aldehydes and preferred the former ones, especially butylaldehyde and acetaldehyde. Comparing with NADP+, NAD+was the preferred coenzyme. Kinetic study indicated that the acetaldehyde dehydrogenase had a Km value of0.73mmol/L, a Vmax value of35.71U/mg, a kcat value of32.73/s and a kcat/Km value of45.14×103/(mol·s) on acetaldehyde, respectively, while a Km value of0.24mmol/L and a Vmax value of42.19U/mg on NAD+, respectively. The enzyme activity was enhanced in the presence of K+, NHH+4,2-mercaptoethanol or DTT, while partially inhibited by several metal ions and chemicals at concentration of1mmol/L and5mmol/L, such as Mg2+, Ca2+, Ba2+, Co2+, Ni2+, Mn2+and PMSF. The higher concentration of the metal ions or chemicals was, the stronger inhibition showed. The activity was absolutely inhibited by Ag+, Cu2+,Zn2+and Hg2+.
3. The acetaldehyde dehydrogenase gene of I. terricola strain XJ-2was cloned and the sequence analysis was performed.
The conserved gene fragment was amplified by PCR using the CODEHOP primers. The flanking regions of the conserved gene fragment were amplified by SiteFinding-PCR and Self-formed adaptor PCR. By sequence analysis and assembling, the ORF of acetaldehyde dehydrogenase gene of I. terricola strain XJ-2(ist-ALD) was obtained with1578bp in length. The bioinformatics analysis of the predicted protein of ist-ALD revealed that the protein contained525amino acids with a predicted molecular weight of57.2kDa. Ist-ALDH showed the highest identity (73%) to ALDH from Pichia angusta. Sixteen amino acid residues that are found in more than95%of ALDH proteins were all critically conserved in the predicted protein, while ten most conserved motifs in ALDHs were also existed. Two presumed active-site residues, GIU293and CyS327, known to be catalytically essential amino acids and a glycine motif involved in NAD(P)+binding (GFGKITG) were identified in the protein. The amino acid sequence of ist-ALDH was submitted to http://swissmodel.expasy.org to predict the structure of one subunit. The characteristic domains of the subunit of ALDH, which are the catalysis domain, cofactor-binding domain and the oligomerization domain, respectively, were found in the tertiary structure model. A series of amino acid analysis demonstrated that ist-ALDH was a new member of ALDH superfamily.
4. The acetaldehyde dehydrogenase gene of I. terricola strain XJ-2was heterologous expressed in E. coli and the characterization of the recombinant enzyme was studied.
Two recombinant expression vectors, pET-23a-ist-ALD and pET-32a-ist-ALD were constructed, and transformed to E. coli BL21(DE3), respectively. The two recombinants were able to express acetaldehyde dehydrogenase. The recombinant E. coli harboring pET-32a-ist-ALD yield a higher activity of44.23U/mL, the specific activity of which reached10.95U/mg,4.8-fold than that of native enzyme.
The His6-tagged ist-ALDH in the cell-free extract was purified by Ni-NTA agarose column and appeared one band on a SDS-PAGE gel and a native PAGE gel with a calculated molecular weight of57kDa and232kDa, respectively, indicating that the purified ist-ALDH presented as a homotetramer. Ist-ALDH exhibited the optimal activity at40℃and pH9.0, respectively. The recombinant enzyme was stable in the range of20-37℃and pH7.0-9.0, respectively. By comparing with the native acetaldehyde dehydrogenase, the optimal temperature of the recombinant enzyme was10℃less than that of native one. Moreover, the thermal stability of ist-ALDH was worse than that of the native enzyme, while other characterics of the two enzymes were comparable.
5. Culture conditions of the recombinant E. coli for high-yield acetaldehyde dehydrogenase expression were optimized and the clearance of acetaldehyde transformed by the recombinant E. coli was studied.
Based on the results of single factor experiments, TB5medium was selected as the expression medium of recombinant E. coli BL21(DE3)/pET32a-wist-ALD. The recombinant ist-ALDH was induced to express when OD600reached3.0-3.2with140μg/mL of lactose added. The key factors affecting the expression of recombinant ist-ALDH were screened by the Plackett-Burman experiments and optimized using the central composite design experiments. The predicted optimum culture conditions for maximum expression of recombinant ist-ALDH were found to be comprised of20.68h post-induction,126.75mL medium volume, and3%(v/v) inoculum content with an experimental acetaldehyde dehydrogenase activity of496.65U/mL, resulting in12.5-fold increment after optimization.
The recombinant E. coli was prepared under the optimized condition, and served as a catalyst for the clearance of acetaldehyde. By studying the effect of reaction pH, temperature, surfactants, concentration of acetaldehyde and NAD+on the whole cell biotransformation, the optimum reaction system was as follows:0.1mol/L Glycine-NaOH buffer (pH9.5),1mmol/L acetaldehyde,2mmol/L NAD+,10mmol/L2-ME,0.1mol/L KC1, and1mg/mL wet cells that were treated by0.075%(v/v) Triton X-100. When the reaction was performed at40℃for15min,98%of acetaldehyde could be removed with a total cell average transformation of3.92mmol/g cells/h.
引文
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