The Studies On The Enzymatic Poperties Of Acetaldehyde Dehydrogenase In Angel Yeast

Aldehyde dehydrogenase?ALDH?is one of the key enzymes to decompose acetaldehyde in human body.ALDH can catalyze many aliphatic and aromatic aldehydes and react to produce corresponding acids to reduce the toxicity of aldehydes in human body.At present,ALDH is mainly isolated from animal and plant cells.It is difficult to produce large-scale ALDH because of its high cost and low yield.With the increasing demand of the market,if we can find a high-content alternative to ALDH and get aldehyde dehydrogenase easily and quickly,it becomes the focus of research.In nature,microorganisms have the characteristics of easy cultivation and fast cycle,thus providing great prospects for obtaining ALDH.?1?ALDH was extracted by cellulase and saline-alkali mixed wall-breaking method.Through single factor test,it was found that when the wall-breaking temperature was 70?,the wall-breaking time was 4.5 h,the ratio of material to liquid?yeast concentration?was20%,and NaCl/NaOH was 1:3,the wall-breaking rate of yeast ALDH was higher,and the enzyme activity was also higher,which was 2.85 U/mL.On the basis of single factor experiment,response surface analysis was carried out,and the optimum extraction conditions were obtained as follows:breaking temperature 74?,breaking time 5 h,material-liquid ratio 22%,NaCl/NaOH=1:3,breaking rate of yeast ALDH was 96.20%,enzyme activity was2.99 U/mL.From P value,the significant degree of influencing ALDH wall-breaking rate and enzyme activity was concentration>time>temperature>NaCl/NaOH.?2?After primary purification,the activity of ALDH was the highest when the saturation of ammonium sulfate was 75%.On this basis,ALDH was purified by aqueous two-phase method.Through single factor test,it was found that when the relative molecular weight of polyethylene glycol?PEG?was 2000,the pH was 8,the concentration of NaCl was 1%,and the concentration of NAD⁺was 0.15 mol/L,the purification multiple and recovery of yeast ALDH were 3.1 and 43%,respectively.On the basis of single factor experiment and orthogonal test,the optimal combination of two-phase extraction was obtained:PEG2000,pH8,NaCl concentration 1%,NAD⁺concentration 0.15 mol/L.The results of range analysis showed that the significant degree of influence on purification multiple and recovery of ALDH was PEG2000>pH>NAD⁺>NaCl.?3?The ALDH of yeast was searched in EMBL database.The results showed that the enzyme consisted of four subunits?Two alphas consist of 501 bases.Two betas consist of 500bases?.The active center of the enzyme was Cys-302 of alpha subunit and the sequence near the active center?Cys-302?was highly conserved.The molecular weight of the purified ALDH of yeast was further determined by SDS-PAGE.The electrophoretic bands showed that the molecular weight of the purified ALDH was two,respectively.54 kD,49 kD,combined with EMBL database search results,the molecular weight of ALDH is 206 kD.?4?The factors affecting ALDH activity of yeast were studied.It was found that the enzyme activity was the highest at 25?and pH 7.Na⁺,Ca²⁺and thiourea had inhibitory effects on the enzyme,and the concentration of thiourea was positively correlated with the inhibitory effect.When the concentration of Zn²⁺is lower than 25 mmol/L,it activates the enzyme weakly,and when the concentration of Zn²⁺is between 25 mmol/L and 125 mmol/L,it inhibits the activity of the enzyme;K⁺,Mg²⁺,DTT and EDTA can activate the enzyme,among which K⁺and Mg²⁺can increase the activity of the enzyme weakly;DTT and EDTA can significantly increase the activity of the enzyme,especially DTT.At 75 mmol/L,the activity of ALDH was increased.Ascorbic acid had no effect on the activity of ALDH.?5?Yeast ALDH needs limited hydrolysis modification because of its high molecular weight,which is not conducive to human absorption.Firstly,the amino acid sequence of yeast ALDH was downloaded from NCBI database.Peptide Cutter analysis was carried out by ExPasy software.It was found that there were relatively many arginine?Arg?and lysine?Lyr?sites in the enzyme.The binding site of the enzyme was Cys-302,and the highly conserved sequence near it was Gln-Gly-Gln-Cys.Yeast ALDH can be hydrolyzed into 54 fragments of alpha-peptide chain without destroying the active center.Then the hydrolysis conditions were discussed by single factor experiment.It was found that the hydrolysis effect was better when the temperature was 39?,the reaction time was 7 h,the pH was 7,and the concentration of trypsin was 30 mg/mL.The enzyme activity was 0.89 U/mL at this time.Further orthogonal analysis showed that the order of significant factors was trypsin concentration>pH>temperature>time.?6?In order to improve the enzyme activity of yeast ALDH in human body,sodium alginate-glutaraldehyde-charcoal immobilization experiments were carried out.Single factor experiments showed that sodium alginate concentration was 3%,pickled dialdehyde 1%,cross-linking for 2 h and immobilization for 2.5 h.Response surface analysis showed that the optimum conditions were sodium alginate 3%,glutaraldehyde concentration 1%,and cross-linking time for 2 h.The immobilization time was 2.4 h.The activity of yeast ALDH was 3.34U/mL.From P value,the order of significant factors was:immobilization time>sodium alginate concentration>crosslinking time>glutaraldehyde concentration.The immobilized yeast ALDH can maintain the enzyme activity under the action of trypsin and improve the enzyme activity in alkaline environment.In summary,the optimum technological conditions of yeast ALDH were explored through wall breaking,purification and identification,enzyme activity,hydrolysis modification and immobilization.