CN101948779B - Preparation and use of solid microbial enzyme preparation for producing L-cysteine through enzymatic conversion - Google Patents
Preparation and use of solid microbial enzyme preparation for producing L-cysteine through enzymatic conversion Download PDFInfo
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Abstract
The invention provides preparation and use of solid microbial enzyme preparation for producing L-cysteine through enzymatic conversion. The preparation method is characterized in that toluene is taken as a penetrating agent to treat bacterial suspension to obtain a TS-1138 strain permeabilized cell and the enzyme activity of the TS-1138 strain permeabilized cell is as high as 229.9U/g, about 7 times that of the initial cell; in a freeze-dried permeabilized cell, sodium glutamate, glycerine and mannitol are adopted as compound freeze-drying protective agents, the highest enzyme activity of the residuals after freeze-drying is 66.7%, and after the residuals are stored at 4 DEG C for 3 months, the enzyme activity can be still over 90%; and in a spray-dried cell, sodium glutamate, Arabic gums or beef extracts are adopted as the protective agents, the highest enzyme activity of the residuals after spray-drying is 68.92%, the activity of the prepared spray-dried enzyme powder is 614.7U/g and the enzyme powder can be stably stored for 3 months after being sealed at 4 DEG C. The solid microbial enzyme preparation obtained by the invention has high activity, is beneficial to storage and can be used for industrial production for preparing L-cysteine through enzyme method.
Description
[technical field]
The present invention utilizes microbial solid zymin enzymatic conversion method to produce amino acid, belongs to biological technical field.The osmotic treated and the freezing or spraying drying that relate to microorganism cells, and transforming the application of producing in the L-halfcystine.
[background technology]
The L-halfcystine is a kind of important sulfur-containing amino acid, is widely used in industries such as medicine, food, makeup and feed.At present the production of China L-halfcystine is main with hair-hydrolyzation, yields poorly, energy consumption is high, produces large quantity of exhaust gas and spent acid, contaminate environment in the hydrolytic process.And utilize enzyme process to prepare advantages such as the L-halfcystine has the reaction conditions gentleness, specificity is strong, efficient is high, by product is few, environmentally friendly, to energy-saving and emission-reduction, the development low-carbon economy significant, correlation technique is monopolized by offshore company for a long time.
2003; People such as Liu Zhong (microbiology circular, 2003,30 (6): 16～21) separate a strain and have the DL-2-of conversion amino-Δ 2 thiazolines-4 carboxylic acid (DL-2-Amino-Δ 2thiazoline-4-Carboxylic Acid; DL-ATC) the pseudomonas TS-1138 bacterial strain of generation L-halfcystine ability; Through being accredited as pseudomonas putida (Pseudomonas putida) (University Of Tianjin's journal, 2007,40 (4): 421～426).
People such as Jin Yongjie (microbiology circular; 2004; 31 (6): the related enzyme systems that 68-72) pseudomonas TS-1138 bacterial strain is transformed DL-ATC has been carried out separation and purification and zymologic property research, purifying L-ATC lytic enzyme and L-SCC lytic enzyme, the relative molecular mass of two kinds of enzymes is respectively 37.5kD and 42.8kD; The optimum temperuture of enzymatic reaction is 35 ℃; Optimal reaction pH is respectively 7.0 and 8.0, the Km value be respectively 0.67 mmole/liter and 0.15 mmole/liter, maximum reaction velocity is respectively 0.39 * 10
-3Mmole/LPM and 0.42 * 10
-3Mmole/LPM.
People such as Yu Yangsheng (Biosci.Biotechnol.Biochem., 2006,70 (9): 2262～2267) genes involved of pseudomonas putida TS-1138 bacterial strain conversion DL-ATC is studied; Made up the genome random library of TS-1138 bacterial strain; (tsB tsC), and expresses in intestinal bacteria to be cloned into two new genes; The result who expression product is carried out Function Identification shows; TsB genes encoding L-ATC lytic enzyme, tsC genes encoding L-SCC hydroamidase, thereby at the path for transformation of the clear DL-ATC of molecular level Shanghai Stock Exchange.
People such as Yu Yangsheng find that further (Frontiers of Biology in China, 2007,2 (4): 391-396), also have the L-cysteine desulfhydrase in the pseudomonas TS-1138 strain cell simultaneously, this enzyme can be decomposed into pyruvic acid, H with the L-halfcystine
2S and NH
3Thereby, influence the accumulation of L-halfcystine.Can partly suppress the activity of L-cysteine desulfhydrase through in enzymatic reaction liquid, adding azanol or Urea,amino-, avoid the decomposition of product.
People such as Yu Yangsheng and Liu Chunqin has carried out reporting (biotechnology communication, 2006,17 (6): 911-913 to the conversion condition of the synthetic L-halfcystine of TS-1138 strain cell conversion DL-ATC respectively; The microbiology circular, 2008,35 (1): 45-49); Its righttest enzymatic reaction condition is: 42 ℃ of temperature of reaction, and reaction system pH 8.0,2.5 hours reaction times; Concentration of substrate 6 grams per liters, concentration of cell suspension 250 grams per liters, azanol concentration 1 mol in the reaction system.Under the righttest enzymatic reaction condition, the molar yield that is converted into the L-halfcystine by DL-ATC is 96.8%.
In addition, Chinese patent (application number: 200710056754.9) reported a kind of method of producing cystine by microorganism transformation.This invention is under the situation in enzyme source with pseudomonas putida TS-1138 cell, is suppressor factor with the azanol, and enzymatic conversion method substrate A TC obtains the L-halfcystine aqueous solution, and described halfcystine is changed into the method for Gelucystine through further mode of oxidizing.This method directly utilizes somatic cells to be enzyme source catalytic substrate, and transformation efficiency is more than 90%.
In above-mentioned conversion reaction research; All use the culturing cell of TS-1138 bacterial strain to be the enzyme source, enzyme source cell consumption big (250 grams per liter), the product growing amount is few relatively; And the enzyme source cell need be at present with existing system; Can't prolonged preservation because of poor stability, cause using inconvenience, influenced should technology industrialization.
[summary of the invention]:
Existing microbial enzyme method transforms resting cell (biotechnology communication, 2006,17 (6): 911-913 that the enzyme source of using in the report of producing the L-halfcystine is the TS-1138 bacterial strain; The microbiology circular, 2008,35 (1): 45-49; Chinese patent 200710056754.9), the enzyme source cell consumption of catalyzed reaction is big, and the product growing amount is few, and the enzyme source cell need be at present with existing system, can't prolonged preservation because of poor stability, cause using inconvenience.The present invention seeks to addressing the above problem, provide a kind of enzymatic conversion method to produce the preparation and the application of the microbial solid zymin of L-halfcystine.Specifically be the catalytic activity that improves the TS-1138 strain cell through the cell permeability processing, through the permeability cell is carried out lyophilize or spraying drying, obtain zymin with long preservation period simultaneously, for the production of L-cysteine by using enzyme is laid a good foundation.
The osmotic treated cell technology is meant under the prerequisite of not destroying the cell complete structure, changes the permeability of cell walls and cytolemma, thereby makes low molecular weight substance can free in and out the technology of cell.The cell that obtains by this method is commonly referred to the permeability cell.And the drying of microorganism cells is adopted vacuum lyophilization and spray drying technology usually.Vacuum lyophilization is called for short freeze-drying, is the drying and dehydrating technology that Refrigeration Technique combines with vacuum technique.The freeze-drying prods water cut is low, is difficult for oxidizedly, helps transportation, sale and prolonged preservation.Spraying drying be a kind ofly be widely used, economic, easy dry materials method; Spraying drying directly through atomization style form droplet and with process that the hot gas medium contacts in evaporating solvent; Thereby the generation dried powder, because moisture evaporation and heated time are short, can be through red-tape operati condition and the suitable protective material of adding; Prevent the sex change of active substance to greatest extent, thereby prepare stable powder granular products.To the problem of the easy inactivation of enzyme in vacuum lyophilization or the spray-drying process, select suitable protective material and spray-dired condition most important.
The present invention utilizes the Premeabilisation of cells treatment technology not destroying under the integrally-built prerequisite of pseudomonas putida TS-1138 strain cell, has changed the permeability of cell walls and cytolemma, has improved the mass-transfer efficiency of substrate and product.
The preparation method that enzymatic conversion method provided by the invention is produced the microbial solid zymin of L-halfcystine is: adopt pseudomonas putida TS-1138 somatic cells; With toluene is permeate agent; Osmotic treated obtains the permeability cell; Adopt lyophilize or spraying drying to obtain permeability cell dry powder, promptly can be used for the microbial solid zymin that enzymatic conversion method is produced the L-halfcystine.
Wherein, the concentration range of permeate agent toluene is at 0.1% to 5% (v/v), and optimum concn is 2%, and the TR of osmotic treated is 15-35 ℃, and optimum temps is 25 ℃, and the treatment time is 15-90 minute, and Best Times is 30 minutes.Under best infiltration condition, the enzyme work of gained permeability cell can reach the 229.9U/ gram, is about 7 times of contrast (resting cell).The TS-1138 somatic cells is after permeability is handled, and the optimal reactive temperature that transforms DL-ATC generation L-halfcystine is 35 ℃, and is identical with the optimal reactive temperature of L-ATC lytic enzyme and L-SCC hydroamidase.The optimal reaction pH of permeability cell is 8.0.
Obtain among the preparation technology of permeability cell dry powder in lyophilize; The present invention adopts Plackett-Burman design method and response surface analysis method to obtain a kind of compound lyophilized vaccine of optimization; Compound lyophilized vaccine is the mixture of Sodium Glutamate, glycerine and N.F,USP MANNITOL; Concentration range is respectively: Sodium Glutamate 5-15 grams per liter, glycerine 10-50 milliliter/liter and N.F,USP MANNITOL 10-50 grams per liter, wherein optimum concn is Sodium Glutamate 11.21 grams per liters, 32 milliliters/liter of glycerine and N.F,USP MANNITOL 35 grams per liters.Under top condition, make the remaining enzyme work after the freeze-drying of permeability cell bring up to 66.7% by 12.5%.Freeze-drying permeability cell room temperature storage 3 months still keeps the activity more than 90%, and 4 ℃ of storages of resting cell enzyme work in 7 days is lost in more than 50%.Through experiment of single factor and response surface analysis experiment; Optimal cell concentration, concentration of substrate, reaction times have been confirmed; Obtained the influence rule of this three factor to freeze-drying permeability cell transformation reaction, having set up is the quadratic response face regression model of response value with the transformation efficiency, has optimized reaction process: FD concentration 35 grams per liters; Concentration of substrate 15 grams per liters, 2.5 hours reaction times.
Adopting spraying drying to obtain among the preparation technology of permeability cell dry powder, the present invention adopts orthogonal design that spray art is optimized, and investigates that inlet temperature, atomization speed, charging bacterium are dense, protective material content is to the influence of enzyme activity yield.Confirmed spray condition: protective material is Sodium Glutamate, gum arabic or beef extract, and concentration range is the 5-20 grams per liter, and inlet temperature is 100-140 ℃, and the charging bacterium is dense to be the 100-200 grams per liter, atomization speed 0.2-0.6 liter/hour.Wherein top condition is: protective material is Sodium Glutamate, gum arabic or the beef extract of 10 grams per liters; 120 ℃ of inlet temperatures; The charging bacterium is dense to be 200 grams per liters, and 0.2 liter/hour of atomization speed can obtain yield and be up to 68.92% permeability cellular enzymes powder under top condition.The spray powder enzyme work that makes reaches the 614.7U/ gram, preserves 3 months in the room temperature sealing, still keeps the activity more than 90%.
The solid zymin of the inventive method preparation is produced the application in the L-halfcystine in enzymatic conversion method; With DL-2-amino-Δ 2 thiazolines-4 carboxylic acid (DL-2-Amino-Δ 2thiazoline-4-Carboxylic Acid; ATC) be substrate; With permeability cell dry powder is that the solid zymin is the enzyme source, but the substrate A TC of 718 to 741.7 milligrams of every gram zymin catalysis generates the L-halfcystine of 595.1 to 614.7 grams per hour; The lyophozyme powder is respectively 35 ℃ or 40 ℃ with the temperature of reaction of the dried enzyme powder of spray, the best pH8.0 of reaction system, and reaction Best Times 2.5 hours, the initial optimum concn of substrate A TC is 4 grams per liters, optimum concn 1 mmole of azanol/liter.
Compare with the transformation efficiency of the per hour interior 31.4 milligrams L-halfcystine of resting cell; The spray powder stability in storage that makes is high, conversion performance is good; Unit time per hour in every gram solid zymin can transform 718 to 741.7 milligrams DL-ATC, need the wet resting cell of 4 grams approximately and prepare 1 gram solid zymin, after therefore being equivalent to the wet resting cell of every gram and processing the solid zymin; Can transform 179.5 to 185.4 milligrams DL-ATC in the unit time in per hour; Generate 148.8 to 153.7 milligrams L-halfcystine, efficient improves greatly, therefore has good actual application to be worth.
Advantage of the present invention and beneficial effect: the present invention utilizes the Premeabilisation of cells treatment technology not destroying under the integrally-built prerequisite of pseudomonas putida TS-1138 strain cell; Changed the permeability of cell walls and cytolemma; Improved the mass-transfer efficiency of substrate and product, the enzyme work of prepared permeability cell is up to 7 times of original resting cell.Simultaneously; Adopt lyophilize or spraying drying; The permeability cell preparation that is difficult for preserving is become the solid zymin that stability is high, the conversion vigor is good, compare under the optimal conversion processing condition with the primary resting cell, the reaction efficiency of cell has approximately improved 6 times; Can be used for efficient production L-halfcystine, so the present invention has good industrialization value in the field of industrialized production of L-halfcystine.
[description of drawings]
Fig. 1 is the influence of toluene consumption to permeability cellular enzymes vigor.
Fig. 2 is the influence of osmotic treated temperature and time to permeability cellular enzymes vigor.
Fig. 3 is the influence of temperature to enzymic activity.
Fig. 4 is the influence of temperature to enzyme stability.
Fig. 5 is the influence of pH value to enzymic activity.
[embodiment]
The preparation method that enzymatic conversion method provided by the invention is produced the microbial solid zymin of L-halfcystine is: adopt pseudomonas putida TS-1138 somatic cells; With toluene is permeate agent; Osmotic treated obtains the permeability cell; Adopt lyophilize or spraying drying to obtain permeability cell dry powder, promptly enzymatic conversion method is produced the microbial solid zymin of L-halfcystine.Wherein, the actual conditions that the preparation method relates to confirms through following embodiment, the microbial solid zymin the concrete operations of the production application of L-halfcystine can with reference to Chinese patent (application number: 200710056754.9):
The preparation of embodiment 1 TS1138 somatic cells and enzyme are lived and are analyzed
Picking one ring TS-1138 bacterial classification inserts (glucose 20 grams, ATC 3 grams, steeping water 5 grams, urea 3 grams, NaCl 1.5 grams, MnSO the liquid seed culture medium from flat board
4H
2O 0.1 gram, K
2HPO
43 grams, MgSO
47H
2O 0.5 gram, FeSO
47H
2O 0.01 gram is settled to 1 liter, and pH 7.5), 28 ℃, 200 are changeed and cultivated 16 hours, then with 1% inoculum size transfer (glucose 20 grams, ATC 4 grams, steeping water 1 gram, urea 3 grams, NaCl 1.5 grams, MnSO in producing the enzyme substratum
4H
2O 0.1 gram, K
2HPO
43 grams, MgSO
47H
2O 0.5 gram, FeSO
47H
2O 0.01 gram is settled to 1 liter, pH7.5); Change for 28 ℃, 200 and cultivated 16 hours, the gained fermented liquid 6,000 left the heart 10 minutes down in 4 ℃; Collect thalline, with 50 mmoles/liter phosphoric acid buffer (pH 8.0) suspend centrifugally again after the washing, the collection thalline is subsequent use as resting cell.
With 50 mmoles/liter phosphoric acid buffer (pH 8.0) prepare certain density cell suspension; Join with 50 mmoles/liter the DL-ATC solution of 2 times of volume 6 grams per liters of phosphoric acid buffer (pH 8.0) preparation in; Behind the mixing; In 35 ℃ of insulations 10 minutes, the L-cysteine content that adopts the rapid test of acid ninhydrin method to generate then.With the L-halfcystine is standard substance drawing standard curve.Enzyme activity unit is defined as under above-mentioned reaction conditions, and the enzyme amount that per hour transforms the required enzyme source cell of 1 milligram of L-halfcystine of DL-ATC generation is 1 enzyme activity unit (U).Enzyme work is represented with the contained enzyme activity unit of every gram cell (U/ gram).
The selection of embodiment 2 osmotic agent
With somatic cells with 50 mmoles/liter phosphoric acid buffer (pH 8.0) add different osmotic agent (seeing table 1) respectively after being made into 10% bacteria suspension; 30 ℃ vibrated 30 minutes; 4 ℃ 6,000 left the heart 10 minutes down, collect thalline; Again with 50 mmoles/liter phosphoric acid buffer (pH 8.0) washing back centrifugal, the permeability cell.Press the enzyme of embodiment 1 method detection permeability cell and live, to confirm best osmotic agent.
The different osmotic agent of table 1 are handled the enzyme of back gained permeability cell and are lived
The TS-1138 somatic cells is after the permeate agent osmotic treated; The ability that transforms DL-ATC generation L-halfcystine is seen table 2; Wherein 2% toluene is superior to other permeate agent to the osmotic effect of TS-1138 somatic cells, and the enzymic activity of gained permeability cell is more than 7 times of permeation cell (resting cell) not.The not collaborative enhancement of increase that toluene and EDTA or ethanol are lived to TS-1138 somatic cells enzyme.
Adopt the toluene pair cell of different concns to carry out osmotic treated respectively, the result is as shown in Figure 1.30 minutes effects of toluene osmotic treated of interpolation 2% are better.
The influence that embodiment 3 osmotic treated temperature and times are lived to enzyme
Measure the optimal temperature scope and the treatment time of infiltration respectively with identical biomass, the result is as shown in Figure 2, is contrast with the enzyme work of permeation cell not, and living at the 20-35 ℃ of enzyme of handling the permeability cell that obtained in 15-90 minute all increases more than 2 times.But, in lesser temps and short period scope, (handled 15～60 minutes), along with the enzyme of the prolongation gained permeability cell of penetration time is lived and increased as 20～25 ℃; When penetration time continuation growth, enzyme is lived and is descended on the contrary, possibly be the cause (like 20 ℃ of processing 90 minutes) that the part intracellular enzyme is revealed.Under the higher situation of temperature, after 15 minutes, enzyme is lived and is just begun to reduce like 35 ℃ of processing, and the thermostability that this enzyme is described is not fine.In addition, though 90 minutes effects of 20 ℃ of infiltrations are best, the enzyme work of gained permeability cell can reach the 165U/ gram, compares enzyme with 25 ℃ of infiltrations 30 minutes (enzyme is lived and is the 160U/ gram) and lives and only improve 3% approximately, penetration time that but need 3 times.Because the prolongation of low temperature osmotic and penetration time all can increase energy consumption, therefore take all factors into consideration, for the bacteria suspension of 100 grams per liters, in 30 minutes the bests of 25 ℃ of following osmotic treated.
The preparation of embodiment 4 TS-1138 bacterial strain permeability cell suspensions
With the TS-1138 strain cell with 10 mmoles/liter phosphoric acid buffer (pH 8.0) preparation 100 grams per liter bacteria suspensions after add the toluene of 0.1-5% (v/v), 20-35 ℃ osmotic treated 15-90 minute, then in 4 ℃ down 6; 000 left the heart 10 minutes, abandoning supernatant, and the permeability cell suspends with identical phosphoric acid buffer and washs 2 times; 4 ℃, 6,000 left the heart 10 minutes; Collect the permeability cell, add an amount of above-mentioned phosphoric acid buffer, it is subsequent use to obtain the permeability cell suspension.
The screening of embodiment 5 lyophilized vaccines
Choose sucrose, trehalose, skim-milk, sorbyl alcohol, N.F,USP MANNITOL, vitamins C, Sodium Glutamate and 8 kinds of protective materials of glycerine respectively as investigating object; Utilize the Plackett-Burman experimental design; Lyophilized vaccine to being applicable to TS-1138 bacterial strain permeability cell screens, and experimental design and result see table 2.Can find out from table 2, not add after the freeze-drying of protectant permeability cell only remaining 12.5% enzyme and live, add protective material after, the enzyme of freeze-drying permeability cell is lived all has raising in various degree, reaches as high as 61.2% at the scope of experiment endoenzyme protection ratio of living.
Table 2Plackett-Burman experimental design and response value (N=12)
Utilize Design-Expert software that the Plackett-Burman experimental result is carried out statistical study, the result sees table 3.Wherein, the influence that when certain factor is from low-level " 1 " to high level " 1 " variation in degree of the influence finger print type model response value is caused, negative value representes that response value is had negative effect, the shadow loudness value shows the response value influence big more more greatly.The per-cent of each factor sum of squares summation in the sum of squares of certain factor and the model in the model contribution rate finger print type, its value size shows the size of this factor to the response value influence.
Can find out by table 3; Active protection in freeze-drying process has remarkable negatively influencing to vitamins C to the permeability cell; Be not suitable as the lyophilized vaccine of TS1138 bacterial strain permeability cell, the protection that other 7 kinds of additives are lived to enzyme all has positive-effect, and wherein Sodium Glutamate, glycerine and N.F,USP MANNITOL are main affecting factors; The contribution rate of model all more than 10%, can be used for further research.
The impact effect of each factor of table 3Plackett-Burman experiment
The response surface method of embodiment 6 frozen-dried protective agent prescriptions is optimized
According to the Plackett-Burman experimental result; With Sodium Glutamate, glycerine and N.F,USP MANNITOL is independent variable(s); With enzyme protection ratio alive is response value; Box-Behnken design method with three factors, three levels is optimized the protective material prescription that filters out, and the value of empirical factor and level is seen table 4, and experimental design and experimental result are seen table 5.Can find out from table 5; The mixture of Sodium Glutamate, glycerine and N.F,USP MANNITOL is effective compound lyophilized vaccine; Its concentration range is respectively: Sodium Glutamate 5-15 grams per liter, glycerine 10-50 milliliter/liter and N.F,USP MANNITOL 10-50 grams per liter, the protection efficient that composite protectant is lived to enzyme is at least 49.1%.
Table 4Box-Behnken empirical factor and level
Table 5 Box-Behnken experimental design and result
After experimental data used the ANOVA program of Design-Expert software to carry out the quadratic regression match, the gained regression equation was:
Y＝67.17+2.45A+1.71B+3.19C+0.075AB-0.075AC
-1.10BC-4.98A
2-7.61B
2-5.21C
2
In the formula: Y represents response value, i.e. enzyme protection ratio alive; A, B, C are respectively Sodium Glutamate, glycerine and N.F,USP MANNITOL.
Utilize Design-Expert software to carry out variance analysis, the significance of checking regression model and each parameter, the result sees table 6.
The variance analysis of table 6 response surface
Annotate: a, Prob＞F value shows that less than 0.05 model or investigation factor have remarkably influenced; Prob＞F value influences highly significant less than 0.01 expression.
Visible by table 6, the Prob of regression equation＞F value is 0.0003, shows this model highly significant.The probability of a representation model predictor and not match of actual value is intended in the model mistake, and the Prob＞F value of model mistake plan item is 0.059 in the table, and difference is not remarkable, therefore can replace testing truly putting with this regression model and analyze experimental result.In addition, the coefficient R of model
2Value is 98.8%, greater than 90%, shows that the degree of fitting of regression equation is better.The degree of confidence of the variation coefficient (CV) reflection model, the degree of confidence of the low more model of CV value is high more, and the CV value of model is 1.93% in the table 6, explains that model equation can reflect real experimental value well.
Utilize the optimization program in the Design-Expert software to predict; Obtain to make enzyme protection ratio alive to reach peaked protective material proportioning: Sodium Glutamate 11.22 grams per liters; 31.85 milliliters/liter of glycerine; N.F,USP MANNITOL 35.88 grams per liters, the predictor of enzyme protection ratio alive is 68.014%, satisfactory degree is 0.45.Consider the convenience of actual preparation; The concentration of glycerine and N.F,USP MANNITOL is revised; Be that 32 milliliters/liter, mannitol concentration are that 35 grams per liters, enzyme live that to be not less than 68% be that limit value is predicted to protection ratio promptly, obtain the protective material prescriptions of 5 groups of optimizations, see table 7 with glycerol concentration.Therefrom choose first group and carry out confirmatory experiment, the enzyme protection ratio of living is 66.7% as a result, with the relative error of model predication value be 1.9%, explain according to the model of foundation to predict it is feasible in practice.
The protective material prescription that table 7 model is optimized
The protectant selection of embodiment 8 spraying dryings
In the bacteria suspension of permeability cell; The carbohydrate, polymer, polyvalent alcohol, protein, tensio-active agent, salt etc. that add 20 grams per liters respectively are as investigating object, and fully after the dissolving, preheating is 30 minutes in 50 ℃ of water-baths; With blank residual enzyme vigor is 100%, and relatively the relative surplus enzyme is lived.The result is as shown in table 8, and Sodium Glutamate, gum arabic and beef extract all have good protective action, can preferentially select Sodium Glutamate or gum arabic if consider cost.
Table 8 protective material is to the influence of temperature stability
The investigation of embodiment 9 spray drying treatment conditions
The permeability cell suspension of 100-200 grams per liter concentration is added a certain amount of Sodium Glutamate, gum arabic or beef extract as protective material; According to four factors, three horizontal quadrature contrived experiments; Under different inlet temperatures, atomization speed, spray; Calculate enzymatic activity recovery, thereby confirm the spray drying treatment condition, the result sees table 9, table 10 and table 11 respectively.
Table 9 Sodium Glutamate is made protectant orthonormal design of experiments
Table 10 gum arabic is made protectant orthonormal design of experiments
| Factor | 1 | 2 | 3 | 4 | The result |
| Experiment | Protective material concentration | The charging bacterium is dense | Inlet temperature | Atomization speed | Enzyme activity reclaims |
| (grams per liter) | (grams per liter) | (℃) | (L/ hour) | (％) | |
| 1 | 2 | 200 | 140 | 0.6 | 50.25 |
| 2 | 2 | 150 | 120 | 0.4 | 48.10 |
| 3 | 2 | 100 | 100 | 0.2 | 51.02 |
| 4 | 1 | 200 | 120 | 0.4 | 65.40 |
| 5 | 1 | 150 | 100 | 0.2 | 51.21 |
| 6 | 1 | 100 | 140 | 0.6 | 45.06 |
| 7 | 0.5 | 200 | 100 | 0.2 | 48.9 |
| 8 | 0.5 | 150 | 140 | 0.6 | 47.4 |
| 9 | 0.5 | 100 | 120 | 0.4 | 45.11 |
Table 11 beef extract is made protectant orthonormal design of experiments
Visible from the result; Make protective material with Sodium Glutamate, gum arabic or beef extract; In inlet temperature is 100-140 ℃; The charging bacterium is dense to be the 100-200 grams per liter, atomization speed 0.2-0.6 liter/hour condition under the bacteria suspension of permeability cell is carried out spraying drying, enzymatic activity recovery all can reach more than 40%.Simultaneously through the orthonormal design of experiments interpretation of result; Take the condition of the convenient and instrument of actually operating into consideration; Preferred spraying drying condition is: protective material is Sodium Glutamate, gum arabic or the beef extract of 10 grams per liters; Inlet temperature is 120 ℃, and atomization speed is 0.2 liter/hour, and the charging bacterium is dense to be 200 grams per liters.Under this optimum condition, carry out confirmatory experiment, obtain actual enzymatic activity recovery and be up to 68.92%.
The investigation of the conversion condition of embodiment 10 solid zymins
Variation to a certain degree may take place in the permeability of TS 1138 strain cells film in lyophilize or spray-drying process; Cell is affected to the mass transfer and the heat transfer property of materials such as substrate DL-ATC, product L-halfcystine, and then influences the catalyzed reaction characteristic.Therefore, the enzymatic reaction character of the solid zymin of permeability cell is studied, and compared with the wet cell before and after the permeability.
1 temperature is to the influence of the conversion reaction of solid zymin
The solid zymin that freeze-drying or spray are done with 50 mmoles/liter phosphoric acid buffer (pH 8.0) suitably after the dilution, it is alive under differing temps, to measure enzyme, the enzyme work during with 35 ℃ is 100%.Can be found out that by Fig. 3 the righttest conversion reaction temperature of the resting cell of pass through changing is 40-45 ℃, the righttest conversion reaction temperature of freeze-drying permeability cell is 35 ℃, and is identical with the optimal reactive temperature of permeability cell before the freeze-drying.The righttest conversion reaction temperature that the permeability cell is done in spray is 40 ℃.
In order to investigate the influence of temperature to the stability of solid zymin, the zymin that freeze-drying or spray are done after being incubated 30 minutes respectively under the differing temps, ice bath cooling rapidly; Measuring remaining enzyme then lives; With initial enzyme work is 100%, and the result sees Fig. 4, can know; Solid zymin after freeze-drying or spray are done is compared with resting cell and permeability cell; Thermostability slightly descends, but the solid zymin is being incubated 30 minutes below 35 ℃, and the vigor that transforms DL-ATC generation L-halfcystine still can keep more than 95%.
The 2pH value is to the influence of the conversion reaction of solid zymin
The active optimum pH of permeability cell and solid zymin is investigated the result and is seen Fig. 5.The result shows that the optimal reaction pH of the solid zymin after permeability cell and freeze-drying thereof or spray are done is the same with the optimal reaction pH of resting cell, is pH8.0.
The investigation of 3 solid zymin transformation efficiencies
In order to investigate the transformation efficiency of solid zymin, respectively with resting cell, permeability cell, lyophozyme powder with to spray dried enzyme powder be the enzyme source, with DL-ATC substrate, under optimum reaction conditions, measure transformation efficiency, calculate enzyme and live, the result sees table 10.Visible from table, be the enzyme source with the resting cell, under its optimum reaction conditions, the wet resting cell of every gram can transform 37.9 milligrams of DL-ATC in 1 hour unit time, prepares 31.4 milligrams L-halfcystine, and the enzyme of cell is lived not high.
After resting cell processed the permeability cell, can effectively improve the reactive behavior and the reaction efficiency of cell.The wet permeability cell of every gram can transform 277.4 milligrams DL-ATC in 1 hour unit time.Further, with permeability cell freezing drying or spraying drying, every gram lyophozyme powder can transform 718 milligrams DL-ATC in 1 hour unit time, and every gram sprays dried enzyme powder can transform 741.7 milligrams in 1 hour unit time DL-ATC.Preparation 1 gram solid zymin needs the wet resting cell of 4 grams approximately; Therefore; After being equivalent to the wet resting cell of every gram and processing the solid zymin; In 1 hour unit time, can transform 179.5 to 185.4 milligrams DL-ATC, generate 148.8 to 153.7 milligrams L-halfcystine, the reaction efficiency of cell has approximately improved 6 times.
The transformation efficiency of several kinds of enzyme source cells of table 10 relatively
4 package stabilities
With freeze-drying or spray do permeability cell and untreated permeability cell respectively 4 ℃ with room temperature under airtight storage, the remaining enzyme of certain hour sampling and measuring is lived at interval, is 100% with initial enzyme work, the result sees table 11.
The investigation of table 11 package stability
The result shows, passes through the resting cell of changing, and places 1 day in room temperature, and enzyme is lived and descended closely half, places 3 days, and enzyme work is close to and completely loses; Stored 3 days at 4 ℃, enzyme is lived and is lost＞30%.Same, the permeability cell is poorer in the stability of room temperature and 4 ℃, and when storing for 4 ℃, all endoenzymes loss of living is more than 70%.So resting cell and permeability can only be at present with existing preparations.By contrast, the lyophozyme powder was stored 7 days room temperature and 4 ℃ with the dried enzyme powder of spray, and enzyme is lived loss all＜5%, stores 3 months at 4 ℃, and enzyme is lived and still can be kept more than 90%.This shows, process the solid zymin after the freeze-drying of permeability cell or spray are done, but the long period deposit, be convenient to practical application.
Claims (5)
1. enzymatic conversion method is produced the preparation method of the microbial solid zymin of L-halfcystine; It is characterized in that: adopt pseudomonas putida TS-1138 somatic cells; With toluene is permeate agent, and the volumetric concentration scope of toluene is 0.1% to 5%, and the TR of osmotic treated is 15-35 ℃; The time range of handling is 15-90 minute; Osmotic treated obtains the permeability cell, adopts lyophilize or spraying drying to obtain permeability cell dry powder, and promptly enzymatic conversion method is produced the microbial solid zymin of L-halfcystine; Wherein, Adopt lyophilize to obtain among the preparation technology of permeability cell dry powder; The compound lyophilized vaccine that uses is the mixture of Sodium Glutamate, glycerine and N.F,USP MANNITOL, concentration range be respectively Sodium Glutamate 5-15 grams per liter, glycerine 10-50 milliliter/liter and N.F,USP MANNITOL 10-50 grams per liter; Adopt spraying drying to obtain among the preparation technology of permeability cell dry powder; Protective material is Sodium Glutamate, gum arabic or beef extract, and concentration range is the 5-20 grams per liter, and inlet temperature is 100-140 ℃; The atomization speed scope be the 0.2-0.6 liter/hour, the dense scope of charging bacterium is the 100-200 grams per liter.
2. method according to claim 1, the optimal volume concentration that it is characterized in that toluene is 2%, and the optimum temps of osmotic treated is 25 ℃, and the Best Times of processing is 30 minutes.
3. method according to claim 1 is characterized in that: the optimum concn of Sodium Glutamate is 11.21 grams per liters, 32 milliliters/liter of glycerine and N.F,USP MANNITOL 35 grams per liters in the compound lyophilized vaccine.
4. method according to claim 1 is characterized in that: the Sodium Glutamate that best spraying drying condition is 10 grams per liters is as protective material, and inlet temperature is 120 ℃, and atomization speed is 0.2 liter/hour, and the charging bacterium is dense to be 200 grams per liters.
5. the solid zymin of the said method preparation of claim 1 is produced the application in the L-halfcystine in enzymatic conversion method, it is characterized in that: with DL-2-amino-Δ
2Thiazoline-4 carboxylic acid (DL-2-Amino-Δ
2Thiazoline-4-Carboxylic Acid) is substrate; Permeability cell dry powder with claim 1 preparation is that the solid zymin is the enzyme source; But this zymin enzymatic conversion method is produced the L-halfcystine, but the substrate of 718 to 741.7 milligrams of every gram zymin catalysis generates the L-halfcystine of 595.1 to 614.7 grams per hour; The temperature of reaction of lyophozyme powder is 35 ℃, and the temperature of reaction of spraying dried enzyme powder is 40 ℃, the best pH8.0 of reaction system, and reaction Best Times 2.5 hours, the initial optimum concn of substrate is 4 grams per liters, optimum concn 1 mmole of azanol/liter.
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