Purification, Characterization, Gene Cloning And Expression Of Acetaldehyde Dehydrogenase From Issatchenkia Terricola Strain XJ-2

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.