CN102417897B - Process for preparing aldehyde dehydrogenase (ALDH) by enzyme hydrolysis method - Google Patents
Process for preparing aldehyde dehydrogenase (ALDH) by enzyme hydrolysis method Download PDFInfo
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Abstract
The invention relates to a process for preparing aldehyde dehydrogenase (ALDH) by an enzyme hydrolysis method. The process is characterized by comprising the following steps: (1) preparing buffer solution by evenly mixing CBP (chitin-binding protein) solution with alpha-amylase liquid in a volume ratio of 5:(1-2); and placing beer yeast and the buffer solution in a reaction tank, and then evenly stirring; (2) adjusting the pH value to 7-8 while stirring, and stirring for reacting at the temperature of 42-50 DEG C for 22-28 hours to obtain enzymatic beer yeast hydrolyzate; (3) carrying out centrifugation and ultrafiltration concentration on the enzymatic beer yeast hydrolyzate to obtain concentrated solution; and (4) drying the concentrated solution to obtain ALDH powder. According to the invention, raw materials are simple to obtain, used preparation equipment is simple, and the process is simple to operate and lasts for a short time.
Description
[technical field]
The present invention relates to a kind of technique for preparing acetaldehyde dehydrogenase with enzymolysis process.
[background technology]
Acetaldehyde dehydrogenase (aldehyde dehydrogenase, EC 1.2.1.n, english abbreviation ALDH) and ethanol dehydrogenase (alcohol dehydrogenase, EC 1.1.1.1, english abbreviation ADH) the common ethanol dehydrogenase that forms is to be responsible for the ethanol decomposition metabolism in catalytic body in human body.The product acetaldehyde dehydrogenation CH of ALDH catalysis ADH wherein
3CHO generates the acetic acid CH3COOH of toxicological harmless, reaction needed nadide (NAD
+) or coenzyme II (NADP
+) participate in:
ALDH mainly contains two kinds: tenuigenin isozyme (ALDH
1) and Mitochondria Isoenzyme (ALDH
2).The former is lower than the latter to the avidity of acetaldehyde.Being both isozyme, is one of most important enzyme in the alcohol metabolism approach.Therefore, ALDH is widely used in medication, plays an important role in the clinical diagnosis of the diseases such as it is poisoning at Alcoholic, alcoholic liver disease, upper digestive tract cancer and cancer of the stomach and treatment.In recent years, certain application and development is also arranged on food, healthcare products.
[summary of the invention]
The purpose of this invention is to provide a kind of technique for preparing acetaldehyde dehydrogenase with enzymolysis process, preparation is simple, quick.
Above-mentioned purpose is achieved through the following technical solutions:
A kind of technique for preparing acetaldehyde dehydrogenase with enzymolysis process is characterized in that, comprises the following steps:
1) buffered soln: with CBP liquid (CBP: radish chitin-binding protein) and α-amylase liquid be 5 by volume: 1-2 mixes, and gets buffered soln; Get cereuisiae fermentum and described buffered soln is put in retort, stir;
2) under agitation adjust pH is to 7-8, and stirring reaction 22h-28h under 42-50 ℃ of condition gets enzymolysis cereuisiae fermentum liquid;
3) above-mentioned enzymolysis cereuisiae fermentum liquid through centrifugal, ultrafiltration and concentration, gets concentrated solution;
4) above-mentioned concentrated solution drying gets acetaldehyde dehydrogenase enzyme powder.
By such scheme as can be known, the present invention carries out enzymolysis to obtain enzymolysis cereuisiae fermentum liquid to cereuisiae fermentum by the condition of setting by CBP liquid and α-amylase liquid, again through centrifugal, ultrafiltration and concentration and dry acetaldehyde dehydrogenase enzyme powder, wherein, centrifugal, ultrafiltration and concentration and dry these steps are all the routine techniques means; Raw material of the present invention obtains simply, and Preparation equipment is simple, process operation is simple and the time is not long.
Further scheme is, described step 1) α-amylase liquid is made by following technique: mixing step 3) residue and step 4) residue get mixture, described mixture is added pH6.0-pH7.0,0.01mol/L phosphoric acid buffer, the quality g of mixture is 1: 2 with the ratio of the volume ml of phosphoric acid buffer, stir 0.5h, through centrifugal, ultrafiltration and concentration, get α-amylase liquid.This programme makes this preparation technology can be salvaged, and when just beginning to prepare acetaldehyde dehydrogenase, can first obtain α-amylase liquid from other place, just can utilize in process of production afterwards corresponding residue α-amylase Producer in next life liquid.
Further scheme is, step 2) in described cereuisiae fermentum for discarded beer yeast slurry, namely produce the cereuisiae fermentum that filters out after beer; These discarded beer yeast slurries can be provided by brew-house, can waste reclaimation, and low-cost low consumption environment protection.
Further scheme is, step 1) in, the quality of described cereuisiae fermentum (g) is 1: 1 with the ratio of the volume (ml) of buffered soln.
Further scheme is, described step 3) specifically comprise:
301) above-mentioned enzymolysis cereuisiae fermentum liquid suction whizzer, through the centrifugal collection supernatant liquor of 1200-1500r/min normal temperature, the residue press filtration regathers supernatant liquor, merges twice supernatant liquor, and residue keeps standby;
302) above-mentioned merging supernatant liquor suction whizzer, through the centrifugal collection supernatant liquor of 14000-17000r/min normal temperature, residue keeps standby;
303) with step 302) supernatant liquor of gained is through ultrafiltration and concentration, gets concentrated solution.
Further scheme is, described step 4) be specially, above-mentioned concentrated solution changes lyophilize in freeze drier over to, or dry with the cold acetone washing dehydration below-15 ℃, gets the acetaldehyde dehydrogenase powder.
[description of drawings]
Fig. 1 is preparation technology's schema of the present invention;
Fig. 2 is the equipment flowsheet that adopts in preparation technology of the present invention;
The schematic diagram of temperature on the impact of enzyme activity when Fig. 3 is enzymolysis;
Fig. 4 is that the enzymolysis the reaction time is on the schematic diagram of the impact of enzyme activity;
The schematic diagram of pH value on the impact of enzyme activity when Fig. 5 is enzymolysis;
The schematic diagram of the matter liquor ratio of cereuisiae fermentum and buffered soln on the impact of enzyme activity when Fig. 6 is enzymolysis.
[embodiment]
In conjunction with Fig. 1, Fig. 2 and table 1, the present invention includes following steps:
1) (discarded beer yeast slurry is to produce the cereuisiae fermentum that filters out after beer to get the discarded beer yeast slurry of 100kg, provided by Qingdao Beer Brewery (Zhuhai), can certainly be with discarded beer yeast slurry or other cereuisiae fermentum of other companies) put in retort, add 100L buffered soln, stir, this 100L buffered soln comprises that the enzyme activity of 75L is that the CBP liquid (preferred range of the enzyme activity of CBP liquid is 500-1000U/mL) of 800U/mL and the enzyme activity of 25L are the α-amylase liquid (preferred range of the enzyme activity of α-amylase liquid is 1.17-1.36U/mL) of 1.17U/mL, wherein, CBP liquid can be by being prepared in the technical scheme that on January 30th, 2011 was 201110033110.4 at the number of patent application of China application, name is put down in writing in being called the patent of invention of " method of the standby radish chitin-binding protein of the poly-legal system of a kind of macromolecular cold-condensation " by the applicant.
2) adjust pH to 8 under agitation, insulation reaction 25h under low whipping speed 100r/min, the condition of 45 ℃ gets enzymolysis cereuisiae fermentum liquid;
3) above-mentioned enzymolysis cereuisiae fermentum liquid suction whizzer first uses low-speed centrifugal (1500r/min, normal temperature) to collect supernatant liquor, and press filtration once regathers supernatant liquor to residue through pressure filter again, merges twice supernatant liquor, and residue keeps standby;
4) above-mentioned merging supernatant liquor again through tubular-bowl centrifuge centrifugal (15000r/min, normal temperature), is collected approximately 185L of supernatant liquor, and enzyme activity reaches 0.19U/ml, and residue keeps standby;
5) with step 4) supernatant liquor of gained pumps into ultrafilter, through ultrafiltration (filtration condition: collect molecular weight and be the material 10000 dalton more than) must concentrated solution about 30L, enzyme activity 0.62U/mL;
6) concentrated solution is changed in freeze drier over to lyophilize or with the cold acetone washing dehydration below-15 ℃ three times, get ALDH enzyme powder (being dry powder) 1.54kg, enzyme activity reaches 11U/g.
Related data in table 1 preparation process
| Step | Cumulative volume/L | Enzyme activity U/mL | Total activity/U | Activity recovery/% |
| Supernatant liquor | 185 | 0.19 | 35150 | 100 |
| Concentrated |
30 | 0.62 | 18600 | 52.9 |
| Dry powder (g) | 1540g | 11U/g | 16940 | 48.2 |
By above-mentioned table 1 as can be known, the discarded beer yeast slurry of 100kg must be done enzyme powder 1.54kg, and productive rate is 1.54%.
Determining of several technical parameters:
In conjunction with Fig. 3, the selection of temperature during enzymolysis: according to above-mentioned steps 1) configuration five parts of reaction solns (the discarded brewer yeast mud that adds buffered soln), then equal adjust pH to 7 under agitation, stirring velocity 50r/min, reacting 28h respectively at 40 ℃, 45 ℃, 50 ℃, 55 ℃, 65 ℃ temperature (namely requires except temperature condition, other term harmonizations), after testing, the ALDH enzyme activity is respectively 0.017U/mL, 0.101U/mL, 0.079U/mL, 0.0715U/mL, 0.0037U/mL.
in conjunction with Fig. 4, the selection of enzymolysis the reaction time: according to above-mentioned steps 1) configuration reaction soln (the discarded brewer yeast mud that namely adds buffered soln), adjust pH to 8 under agitation, low whipping speed 100r/min, insulation reaction under the condition of 50 ℃, then it is 0 hour in the reaction times respectively, 3 hours, 16 hours, 22 hours, 25 hours, 28 hours, 30 hours, the moment of 40 hours is detected enzymolysis cereuisiae fermentum liquid, the ALDH enzyme activity is respectively 0.041U/mL, 0.055U/mL, 0.112U/mL, 0.133U/mL, 0.161U/mL, 0.125U/mL, 0.134U/mL, 0.137U/mL.
In conjunction with Fig. 5, the selection of pH value during enzymolysis: according to above-mentioned steps 1) configuration five parts of reaction solns (the discarded beer yeast slurry that adds buffered soln), adjust pH most 5,6,7,8,9 under agitation respectively, then all under low whipping speed 120r/min, the condition of 45 ℃ insulation reaction 25h (namely require except pH value condition, other term harmonizations), after testing, the ALDH enzyme activity is respectively 0.105U/mL, 0.123U/mL, 0.168U/mL, 0.17U/mL, 0.113U/mL.
in conjunction with Fig. 6, the selection of the matter liquor ratio of cereuisiae fermentum and buffered soln during enzymolysis: according to above-mentioned steps 1) configure five parts of not reaction solns (the discarded beer yeast slurry that adds buffered soln) of homogeneity liquor ratio (ratio of the volume (ml) of the quality of cereuisiae fermentum (g) and buffered soln), the matter liquor ratio is respectively 0.33, 0.4, 0.5, 0.67, 1, 1.5, 2, equal adjust pHs to 7 under agitation, low whipping speed 80r/min, insulation reaction 22h under the condition of 42 ℃ (namely requires except matter liquor ratio condition, other term harmonizations), after testing, the ALDH enzyme activity is respectively 0.034U/mL, 0.055U/mL, 0.056U/mL, 0.095U/mL, 0.152U/mL, 0.053U/mL, 0.035U/mL.
Therefore, make the enzyme activity of enzymolysis cereuisiae fermentum liquid higher, better condition is: the matter liquor ratio is that 1: 1, pH value are that 7-8, temperature are that 42-50 ℃, reaction times are 22-28h.
in process of production, we utilize step 3) residue and step 4) residue prepare α-amylase liquid: mixing step 3) residue and step 4) residue get mixture, this mixture is added pH6.0-pH7.0, 0.01mol/L phosphoric acid buffer, the quality of mixture (g) is 1: 2 with the ratio of the volume (ml) of phosphoric acid buffer, stir 0.5h, first centrifugal (1200-1500r/min, normal temperature) collect supernatant liquor, residue repeats once centrifugal (14000-17000r/min, normal temperature) collect supernatant liquor, merge supernatant liquor twice, concentrated through ultrafiltration (filtration condition: the collection molecular weight is the material more than 10000 dalton), get α-amylase liquid, after testing, enzyme activity reaches 1.17-1.36U/mL, be 63.3-76.8U/mg protein than vigor.
The vitality test technique of enzymolysis cereuisiae fermentum liquid, supernatant liquor, concentrated solution and enzyme powder: add pH8.0,0.05mol/L Tris-Hcl damping fluid 1.95mL in quartz colorimetric utensil, 0.015mol/L NAD
+(or NADP
+) solution (with above-mentioned Tris-Hcl damping fluid preparation) 0.2mL, 0.14mol/L 4MP (tetramethyl-pyrazoles, with above-mentioned Tris-Hcl damping fluid preparation) 0.25mL, 0.25mL enzyme liquid (diluent of enzymolysis cereuisiae fermentum liquid or supernatant liquor or concentrated solution or enzyme powder), mixing, after 25 ℃ of insulations are constant, add 45% (mass percentage concentration) acetaldehyde 0.25mL of same insulation.Contrast adds 0.25mL 0.1% (mass/volume) bovine serum albumin (with above-mentioned Tris-Hcl damping fluid preparation) to replace enzyme liquid.Measure photoabsorption (A under the 340nm wavelength
340) value, read number one time every 30s, read altogether 3min.With A
340To time (s) mapping, obtain per minute A
340Rising value (Δ A
340).Produce the enzyme amount of 1nmol NADH (reduction-state nadide) or NADPH (reduction-state is coenzyme II) as an enzyme activity unit (U) take per minute catalysis.Calculate according to following formula:
U/ml＝ΔA
340×2.9/(0.25×6.22)
U/g enzyme=U/mL * extension rate/enzyme quality
The vitality test technique of radish chitin-binding protein: take micrococcus lysodeikticus (Micrococcus lysodeikticus) as substrate.The a small amount of substrate of picking adds the phosphate buffered saline buffer of quantitative 1/15mol/L pH6.2 in mortar, grind broken wall, constantly add above-mentioned damping fluid, grind, make suspension under the 450nm wavelength, absorbance value reaches 0.6～0.9 photoabsorption unit, is substrate suspension; During enzyme assay, take above-mentioned phosphate buffered saline buffer as contrast, add substrate suspension 2.5ml in measuring cuvette under 25 ℃, add rapidly 0.1ml enzyme liquid (diluent of enzyme powder), stir evenly, after reading immediately absorbance value (A), read number one time every 30s, read altogether 3min.Obtain average light absorption value (Δ A).Descend the enzyme amount of 0.001 unit as 1 enzyme activity unit (U) take the absorbance value under the 450nm wavelength of per minute under these conditions, calculate enzyme activity unit according to following formula:
Enzyme activity unit (U/ml)=Δ A
450/ (0.001 * 3 * 0.1)
Enzyme activity unit (U/g)=U/mL * extension rate/enzyme quality (g)
Alpha-starch activity determination technique: get enzyme liquid, (the 1g Zulkovsky starch adds 0.01mol/L pH6.9 phosphoric acid buffer 100ml to 1% Zulkovsky starch solution, this phosphoric acid buffer includes 0.05mol/L NaCl) insulation is constant in 25 ℃ of water-baths respectively, respectively get 0.5ml, rapid mixing, in 25 ℃ of lower accurate response 3min, (1g 3 to add immediately the 1.0ml developer, add a small amount of distilled water in the 5-dinitrosalicylic acid, stir evenly, add the 20ml 2mol/L NaOH aqueous solution, make its dissolving, then add the 30.0g Rochelle salt, be settled to 100ml with distilled water after its dissolving) termination reaction.Put and heat 5min in boiling water bath, the cooling rear 4ml distilled water diluting of using.Measure light absorption value (A under the 540nm wavelength
540), and add in the 1ml developer the same insulation of the above-mentioned damping fluid of 1ml cooling again with the 4ml distilled water diluting as contrast.With A
540Be worth on the glucose typical curve, find reducing sugar content.To discharge the enzyme amount of 1 μ mol/L reducing sugar from Zulkovsky starch be 1 enzyme activity unit to per minute under these conditions.
Protein content determination: protein content determination is pressed Bradford (1976) technique, and take bovine serum albumin as standard protein.
The present invention is not limited to above-described embodiment, based on above-described embodiment, simple replacement that do not make creative work, should belong to the scope that the present invention discloses.
Claims (2)
1. a method for preparing acetaldehyde dehydrogenase with enzymolysis process, is characterized in that, comprises the following steps:
1) buffered soln: radish chitin-binding protein liquid and α-amylase liquid by volume for 5:1-2 mixes, are got buffered soln; Get cereuisiae fermentum and described buffered soln is put in retort, stir, the quality g of described cereuisiae fermentum is 1:1 with the ratio of the volume ml of buffered soln;
2) under agitation adjust pH is to 7-8, and stirring reaction 22h-28h under 42-50 ℃ of condition gets enzymolysis cereuisiae fermentum liquid;
3) above-mentioned enzymolysis cereuisiae fermentum liquid through centrifugal, ultrafiltration and concentration, gets concentrated solution;
4) above-mentioned concentrated solution changes lyophilize in freeze drier over to, or dry with the cold acetone washing dehydration below-15 ℃, gets acetaldehyde dehydrogenase enzyme powder;
Described step 3) specifically comprises:
301) above-mentioned enzymolysis cereuisiae fermentum liquid suction whizzer, through the centrifugal collection supernatant liquor of 1200-1500r/min normal temperature, the residue press filtration regathers supernatant liquor, merges twice supernatant liquor, and residue keeps standby;
302) above-mentioned merging supernatant liquor suction whizzer, through the centrifugal collection supernatant liquor of 14000-17000r/min normal temperature, residue keeps standby;
303) with step 302) supernatant liquor of gained is through ultrafiltration and concentration, gets concentrated solution.
2. the method for preparing acetaldehyde dehydrogenase with enzymolysis process according to claim 1, is characterized in that, the described cereuisiae fermentum in step 1) namely produces for discarded beer yeast slurry the cereuisiae fermentum that filters out after beer.
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Non-Patent Citations (7)
| Title |
|---|
| Recombinant industrial brewing yeast strains with ADH2 interruption using self-cloning GSH1+CUP1 cassette;Wang ZY et al.;《FEMS Yeast Res》;20090630;第9卷(第4期);第574-581页 * |
| Wang ZY et al..Recombinant industrial brewing yeast strains with ADH2 interruption using self-cloning GSH1+CUP1 cassette.《FEMS Yeast Res》.2009,第9卷(第4期),第574-581页. |
| 从废弃啤酒酵母提取乙醛脱氢酶的研究;毛跟年等;《中国酿造》;20101231(第12期);第38-40页 * |
| 吴桂英等.酿酒酵母产乙醛脱氢酶发酵条件的研究.《酿酒科技》.2009,(第5期),第26-28页. |
| 毛跟年等.从废弃啤酒酵母提取乙醛脱氢酶的研究.《中国酿造》.2010,(第12期),第38-40页. |
| 毛跟年等.从废弃啤酒酵母提取乙醛脱氢酶的研究.《陕西科技大学学报（自然科学版）》.2010,第28卷第33-36页. * |
| 酿酒酵母产乙醛脱氢酶发酵条件的研究;吴桂英等;《酿酒科技》;20090531(第5期);第26-28页 * |
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