CN112175930B - Immobilized lysine decarboxylase and preparation method thereof - Google Patents
Immobilized lysine decarboxylase and preparation method thereof Download PDFInfo
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Abstract
The invention relates to immobilized lysine decarboxylase and a preparation method thereof, wherein the immobilized lysine decarboxylase is obtained by immobilizing lysine decarboxylase and a carrier and then treating tannin. The immobilized lysine decarboxylase provided by the invention is subjected to tannin treatment, so that the stability of a combination structure of a polymer of the lysine decarboxylase and an immobilized carrier is greatly improved, the catalytic stability and the use efficiency of the immobilized lysine decarboxylase are obviously improved, the high-activity multi-batch repeated use of the immobilized lysine decarboxylase can be realized, and the raw material cost of an enzyme preparation and the production cost of 1, 5-pentanediamine prepared by a biological method are greatly reduced.
Description
Technical Field
The invention relates to the field of bioengineering, and particularly relates to immobilized lysine decarboxylase, a preparation method thereof and a method for producing pentamethylene diamine.
Background
The biocatalytic reaction has the characteristics of mild reaction conditions, high selectivity, high reaction efficiency and the like, so that the biocatalytic reaction is increasingly applied to the field of chemical synthesis. However, the stability of biological enzymes in vitro industrial environment is poor and the cost of enzyme preparation is too high, which greatly limits the practical application of biological catalysis in industrial field. The immobilized enzyme technology not only effectively solves the problem that the catalyst cannot be reused by binding (or combining) enzyme molecules on the water-insoluble carrier, but also can obviously improve the stability and catalytic properties (substrate affinity, stereoselectivity and the like) of the biological enzyme. At present, the research content of immobilized enzymes is wide, and the immobilized enzymes relate to synthesis and characterization of carriers, and influence of geometric parameters, surface activity, physicochemical properties and the like of the carriers on the aspects of enzyme immobilization capacity, enzyme activity recovery rate, stability and the like. At present, enzymes that have been successfully immobilized industrially and applied are penicillin acylase, glucose isomerase, and the like.
The preparation method of the immobilized enzyme mainly comprises an embedding method, a covalent bond method, an ion adsorption method, a crosslinking method and the like, wherein the covalent bond method is used for forming covalent bonds between certain groups of the zymoprotein and active functional groups in a carrier so as to realize the immobilization of the zymoprotein. The immobilized enzyme prepared by the covalent bond method has good stability and long half-life period because the enzyme protein is firmly combined with the carrier and cannot fall off from the carrier, can be repeatedly used for a long time, can simplify the extraction and the refining of products, reduces the cost of enzyme preparations, is favorable for industrial production, and becomes an enzyme immobilization method which is applied and researched more at present.
1, 5-diaminopentane (also referred to as 1, 5-pentanediamine, abbreviated as pentanediamine) is an important 5-carbon compound in the chemical industry, and is mainly used for producing polyamides, polyurethanes, and the like, and also can be used for producing important chemical raw materials such as isocyanates, pyridines, piperidines, and the like. To date, diamines have been produced industrially primarily chemically from petroleum-based raw materials via dicarboxylic acid intermediates or by chemical decarboxylation of amino acids. With the development of biotechnology, people can synthesize the pentanediamine by a biological method, and the pentanediamine is obtained mainly by catalyzing the decarboxylation of a substrate lysine by using a microorganism or exogenously over-expressed lysine decarboxylase.
Currently, in a process for producing pentamethylene diamine by catalyzing lysine decarboxylation by lysine decarboxylase produced by a biological method, lysine decarboxylase in a free state or cells containing lysine decarboxylase are generally used, or pentamethylene diamine is produced by fermentation by using a strain capable of producing lysine and lysine decarboxylase at the same time. In recent years, researchers have produced pentamethylene diamine by means of immobilized cells, and patent application JP2004298033A describes that a lysine decarboxylase-producing strain is embedded by carrageenan, then enzyme production is cultured, and the cultured immobilized microorganism is collected to catalyze lysine salt to produce pentamethylene diamine, wherein the substrate concentration of lysine hydrochloride is 246g/L, and the pentamethylene diamine is produced at a concentration of 40g/L and the molar conversion rate of lysine hydrochloride is about 30% after catalysis of immobilized cells for 150 hours. Jiangli and the like adopt 3 wt% of calcium alginate to fix cells containing lysine decarboxylase, the stability of the fixed cells is poor, the enzyme activity is obviously reduced when the 2 nd catalysis is carried out, and the enzyme activity is reduced to about 38% of the enzyme activity of the 1 st catalysis when the 4 th catalysis is carried out (1, 5-pentanediamine is prepared by using the fixed L-lysine decarboxylase cells, Jiangli and the like, fine chemical industry, 2007, 24(11), 1080-. In summary, the currently prepared immobilized cells containing lysine decarboxylase mostly adopt natural polymer gel as a carrier, and in actual operation, the immobilized cells have the problems of low strength, easy microbial decomposition, deformation, breakage or dissolution in the transformation process, enzyme or cell leakage, low reuse efficiency of the immobilized cells and the like. Currently, few reports are made on the preparation and stability studies of immobilized lysine decarboxylase, and no report is made on the study on the improvement of the use efficiency of immobilized lysine decarboxylase. Therefore, it is desired to develop a method suitable for immobilizing lysine decarboxylase and capable of improving the reuse efficiency of immobilized lysine decarboxylase.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an immobilized lysine decarboxylase, a preparation method thereof and a method for producing pentamethylene diamine.
In one aspect, the present invention provides the use of tannin in the preparation of an immobilized enzyme, said use being the treatment of the immobilized enzyme with tannin.
Preferably, the active state of the enzyme is multimeric.
More preferably lysine decarboxylase, ornithine decarboxylase, arginine decarboxylase or glutamic acid decarboxylase.
As a preferred embodiment of the invention, the enzyme is CadA lysine decarboxylase or LdcC lysine decarboxylase.
The tannin is a water-soluble phenolic compound, and can be any one or more of hydrolyzed tannin or condensed tannin.
The tannin preferably comprises any one or more of gallotannin, tara tannin or persimmon tannin.
Lysine decarboxylase can catalyze the decarboxylation of lysine to generate 1, 5-pentanediamine and carbon dioxide, and is a key enzyme for synthesizing pentanediamine by a biological method. In order to improve the use efficiency of lysine decarboxylase and realize repeated use of multiple batches with high catalytic activity, the inventors tried to prepare immobilized lysine decarboxylase by covalent bond method in previous research, however, the inventors found that the catalytic stability of immobilized lysine decarboxylase still needs to be further improved although the use efficiency of immobilized lysine decarboxylase is improved compared with that of free lysine decarboxylase. The active lysine decarboxylase is in a polymer state formed by polymerization of a plurality of monomers, and the inventor finds that not every monomer can be combined with a carrier to form a covalent bond in the preparation process of immobilized enzyme, and partial monomers can fall off in the repeated use process of the immobilized lysine decarboxylase to cause the reduction of the catalytic activity of the enzyme. Therefore, the binding efficiency of the multimeric protein structure to the immobilization carrier is a key factor affecting the immobilization efficiency and catalytic stability.
Tannins are a class of water-soluble phenolic compounds, and tannin molecules contain a large number of phenolic hydroxyl groups and can interact with proteins, but studies have shown that tannin treatment may inactivate enzyme molecules. In the course of research for improving the immobilization efficiency of lysine decarboxylase, the inventors discovered that the binding efficiency of lysine decarboxylase to an immobilized carrier can be significantly improved by treating the immobilized enzyme with a macromolecular compound tannin, and the catalytic stability of the immobilized enzyme can be further improved.
Thus, in another aspect, the invention provides an immobilized lysine decarboxylase obtained by tannin treatment.
The tannin treatment is to add tannin to treat immobilized lysine decarboxylase after the lysine decarboxylase is combined with the immobilized carrier.
Furthermore, the inventor finds that the effect of improving the immobilization effect of the lysine decarboxylase is more remarkable when the tannin and the lysine decarboxylase are mixed according to a specific dosage ratio.
Specifically, the tannin treatment is to add 5000U-60000U lysine decarboxylase into each gram of tannin. Preferably 8000U-48000U lysine decarboxylase is added per gram tannin. More preferably 9000U-15000U lysine decarboxylase is added per gram of tannin.
The catalytic stability of the immobilized lysine decarboxylase is further improved by optimizing the reaction conditions and the treatment time of tannin treatment.
The tannin treatment is carried out at the temperature of 10-40 ℃ for 1-20 hours. Preferably, the treatment is carried out for 1 to 8 hours at a temperature of between 20 and 30 ℃. More preferably, the treatment is carried out at 20 to 25 ℃ for 2 to 3 hours.
Further, the inventors have found that the lysine decarboxylase bound to the immobilized carrier by covalent bonding is stronger, and the tannin treatment is more effective in this case.
The immobilized lysine decarboxylase is characterized in that a plurality of groups of the lysine decarboxylase are combined with an immobilized carrier in a covalent bond mode.
Preferably, the immobilization carrier is an epoxy group-containing polymer carrier or a hydroxyl group-containing polymer carrier.
More preferably, the epoxy-containing polymer carrier is obtained by reacting one or more of a polymer containing an amino group, a polymer containing an amino group and a polymer containing a hydroxyl group with an epoxidizing agent.
The hydroxyl group-containing high molecular substance includes: cellulose, polyvinyl alcohol, cellulose acetate hydrolysate, cellulose butyrate hydrolysate, hydroxyl-containing polyester hydrolysate, absorbent cotton, bagasse, cotton, straw or a combination of one or more thereof.
The high molecular substance containing the aniline group comprises: the hydroxyl-containing high molecular substance and the aniline-containing etherifying agent are subjected to etherification reaction to obtain the substance.
The amine group-containing high molecular substance includes: the hydroxyl-containing high molecular substance and the amino-containing etherifying agent are subjected to etherification reaction to obtain a substance, and/or one or more of the following substances: hydrolysis products of polyacrylonitrile, hydrolysis products of polyesters containing amine groups, and hydrolysis products of polyamides.
The etherifying agent containing the aniline group comprises p-beta-sulfuric ester ethyl sulfuryl aniline;
the amine group-containing etherifying agent includes a silylating agent.
The preparation method of the epoxy group-containing polymer carrier can refer to the preparation method of the epoxy group-modified polymer carrier disclosed in chinese patent CN 107779445A.
Preferably, the lysine decarboxylase is derived from a wild-type or mutagenized or genetically engineered bacterium, fungus, plant or animal, including, but not limited to, Bacillus alkalophorans (Bacillus halodurans), Bacillus subtilis (Bacillus subtilis), Escherichia coli (Escherichia coli), Streptomyces coelicolor (Streptomyces coelicolor), Hafnia alvei (Hafniaalvei), Thermoplasma acidophilum (Thermoplasma acidophilum), Thermococcus halophilus (Pyrococcus abyssi), or Corynebacterium glutamicum (Corynebacterium glutamicum); more preferably, the lysine decarboxylase is derived from Hafnia alvei or Escherichia coli.
The lysine decarboxylase can be crude extraction enzyme or purified enzyme.
Furthermore, the invention provides a preparation method of the immobilized lysine decarboxylase, which is to adopt tannin to treat the immobilized lysine decarboxylase after the lysine decarboxylase and a carrier are immobilized.
Specifically, the preparation method of the immobilized lysine decarboxylase provided by the invention comprises the following steps:
(1) mixing the lysine decarboxylase solution with an immobilized carrier, and preparing immobilized lysine decarboxylase by covalent bond combination;
preferably, the immobilization carrier is a polymer carrier containing an epoxy group or a polymer carrier containing a hydroxyl group;
(2) treating the immobilized lysine decarboxylase obtained in the step (1) by using tannin, wherein the tannin treatment is to add 5000U-60000U lysine decarboxylase into each gram of tannin and treat the tannin at the temperature of 10-40 ℃ for 1-20 hours;
preferably, the tannin treatment is that 8000U-48000U lysine decarboxylase is added into each gram of tannin, and the treatment is carried out for 1-8 hours at the temperature of 20-30 ℃.
More preferably, the tannin treatment is to add 9000U-15000U lysine decarboxylase into each gram of tannin and treat the tannin at the temperature of 20-25 ℃ for 2-3 hours.
Specifically, the preparation of the immobilized lysine decarboxylase in the step (1) comprises the following steps: mixing the lysine decarboxylase solution with the immobilized carrier according to the proportion that the enzyme amount is 2000U-8000U added to each gram of carrier, and carrying out covalent bond combination reaction for 12-72 hours under the conditions that the reaction temperature is 4-30 ℃, the pH value is 5.0-11.0, and the ion concentration of a reaction system is 0.1-2.0 mol/L.
Preferably, the preparation of the immobilized lysine decarboxylase in step (1) comprises the steps of: mixing a lysine decarboxylase solution with an immobilized carrier according to the proportion that the enzyme amount is 2500U-5000U per gram of carrier, oscillating and stirring at 50-200 rpm under the conditions that the reaction temperature is 10-30 ℃, the pH value is 7.0-9.0 and the ion concentration of a reaction system is 0.2-1.8 mol/L to carry out covalent bond bonding, reacting for 24-60 hours, separating the obtained solid-liquid mixture to obtain an immobilized enzyme, and leaching with a buffer solution to obtain the immobilized lysine decarboxylase.
In addition, the invention also provides a production method of the 1, 5-pentanediamine, and the method adopts the immobilized lysine decarboxylase provided by the invention or the immobilized lysine decarboxylase prepared by the preparation method provided by the invention to catalyze lysine or lysine salt.
Specifically, the production method of the 1, 5-pentanediamine comprises the following steps:
(1) adding the immobilized lysine decarboxylase into a solution containing lysine or lysine salt for catalytic reaction;
(2) after the reaction is completed, the immobilized enzyme and the reaction solution are separated to obtain a solution containing 1, 5-pentanediamine.
Preferably, the amount of the immobilized lysine decarboxylase is 200U to 2000U per gram of lysine or lysine salt. More preferably 800 to 2000U per gram of lysine or lysine salt.
Preferably, the catalytic reaction is carried out at the temperature of 20-40 ℃ and the pH value of the lysine solution of 6.0-7.0.
Preferably, the method for producing 1, 5-pentanediamine according to the present invention further comprises the step of adding a coenzyme, which may be selected from one or more of pyridoxal, pyridoxal phosphate, pyridoxine, and pyridoxamine, and more preferably, pyridoxal 5' -phosphate, at a concentration of 0.1mM to 0.5 mM.
The lysine or lysine salt of the invention can be any solution containing lysine or lysine salt, including but not limited to lysine solution, lysine salt solution or lysine fermentation liquor; preferably, the lysine salt is one or more of long-chain dibasic acid lysine salts such as lysine hydrochloride, lysine sulfate, lysine carbonate, lysine phosphate, lysine adipate, lysine sebacate and the like.
The present invention does not require any particular means of solid-liquid separation, including but not limited to centrifugation or filtration, and the particular equipment and process parameters can be determined by one skilled in the art.
Further, the invention provides the 1, 5-pentanediamine prepared by the production method.
The invention has the beneficial effects that:
(1) according to the invention, the prepared immobilized lysine decarboxylase is treated by tannin, so that the catalytic stability of the immobilized lysine decarboxylase in repeated use is obviously improved, and compared with the lysine decarboxylase in a free state (the conversion rate of catalytic lysine is reduced to 20% after repeated use of 3 batches) and the immobilized enzyme which is not treated by tannin (the conversion rate of catalytic lysine is reduced to below 90% after repeated use of 13 batches), the conversion rate of catalytic lysine of the immobilized enzyme provided by the invention is still higher than 90% after repeated use of 19 batches, the use efficiency of the lysine decarboxylase is greatly improved, the catalytic stability of repeated use is improved, and the problem of low use efficiency of the lysine decarboxylase in the traditional process is solved;
(2) the immobilized lysine decarboxylase provided by the invention is used for biological preparation of the 1, 5-pentanediamine, and the cost of raw materials of an enzyme preparation used in the catalysis process is obviously reduced, so that the production cost of the 1, 5-pentanediamine is greatly reduced, the separation step of pentanediamine solution and enzyme is simplified, the automation degree of pentanediamine production is improved, and the industrialization process of biological production of the pentanediamine is promoted.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents, etc. (e.g., lysine salts or lysine fermentation broth) used are commercially available, unless otherwise specified, wherein tannin is available from Jiangsu Proleutop bioengineering, Inc. The buffer used was 1mol/L phosphate buffer, pH 7.0.
The results of the experiments in the following examples are all the average of at least three parallel replicates.
The lysine decarboxylase solution can be purchased on the market or prepared by adopting the conventional technical means in the field, and can be a fermentation liquid containing lysine decarboxylase, a crude enzyme liquid containing more impurities or a refined enzyme liquid with higher purity.
The lysine decarboxylase-producing solution of the present invention can be prepared by methods conventional in the art. For example, the glycerol-preserved strain liquid of the strain expressing lysine decarboxylase is inoculated into a liquid seed culture medium and cultured at 25-40 ℃, and if the strain is cultured in a shaking stage, the rotation speed of the shaking flask is controlled at 150-250rpm and the strain is cultured for 15-30 hours to obtain the seed liquid of the strain producing lysine decarboxylase. Then inoculating the seed liquid into a fermentation culture medium for fermentation culture, controlling the temperature to be 30-45 ℃ and the pH to be 6.5-8.0, and performing fermentation culture for 10-50 hours.
In a preferred embodiment of the present invention, the lysine decarboxylase solution is prepared by the following steps:
the preservation number of the expression lysine decarboxylase is CCTCC NO: m2018737 Hafnia alvei (Chinese patent CN 109536542A). The glycerol-preserved bacterial liquid was inoculated into a 500mL seed bottle containing 100mL of a liquid medium (LB medium, containing peptone 1%, yeast powder 0.5%, sodium chloride 1%, pH7.0), and shake-cultured at 37 ℃ and 200rpm for 15 hours to obtain a seed liquid. Adding 3L LB culture medium into 5L fermentation tank, sterilizing at 121 deg.C for 20min, inoculating the above seed liquid, fermenting at 30 deg.C and 300rpm, controlling ventilation amount to 0.3vvm, and tank pressure to 0.04 MPa; and controlling the pH value of the fermentation liquor to be 7.0 in the fermentation process, and stopping fermentation after fermenting for 25 hours. Centrifuging at 6000r/min for 10min after fermentation to collect thallus (to obtain wet thallus), breaking cell by ultrasonic disruption, centrifuging to collect enzyme solution to obtain lysine decarboxylase solution, and refrigerating for use.
The following examples are defined for lysine decarboxylase enzyme activity as follows:
definition of enzyme activity: lysine decarboxylase catalyzes the decarboxylation of lysine using lysine salt as a substrate, and the amount of enzyme required to produce 1. mu. mol of pentamethylenediamine per minute is defined as 1 activity unit U of the enzyme.
EXAMPLE 1 preparation of immobilized lysine decarboxylase by epoxy Carrier
The epoxy carrier is epoxy-modified cellulose acetate, and specifically, the epoxy-modified cellulose acetate prepared by the preparation method disclosed in step (1) of the embodiment of Chinese patent application CN107779445A is adopted.
The lysine decarboxylase solution is diluted by phosphate buffer solution until the concentration of the enzyme solution is 1000U/g. 20g of the epoxylated cellulose acetate was added to 100g of a 1000U/g lysine decarboxylase solution, pH was controlled at 7.0, and the immobilized enzyme was prepared at 30 ℃ for 36 hours while shaking and stirring at 100 rpm. And (3) filtering and separating the prepared immobilized lysine decarboxylase and residual enzyme liquid, washing the obtained immobilized enzyme by using the phosphate buffer solution to obtain the immobilized enzyme, and refrigerating for later use, wherein the residual enzyme liquid can be recycled for preparing the immobilized enzyme liquid next time. The enzyme activity measurement result shows that the enzyme activity of the immobilized lysine decarboxylase is 800U/g.
Weighing 20g of the prepared immobilized enzyme with the enzyme activity of 800U/g, adding the immobilized enzyme into 100g of lysine fermentation liquor with the concentration of 18% (the lysine content is 18%), controlling the pH to be 6.8, reacting for 3h at 37 ℃ and 100rpm, stopping the reaction, filtering and separating the immobilized enzyme and reaction liquid, extracting the reaction liquid for pentanediamine, adding the collected immobilized enzyme into the same amount of lysine fermentation liquor, catalyzing lysine to generate pentanediamine under the same condition, circularly catalyzing for 15 batches according to the conditions, and determining the conversion rate of the lysine to the pentanediamine.
In this example, a lysine decarboxylase solution diluted with a phosphate buffer solution and having a concentration of 1000U/g was used as a control group, and the control group is different from the experimental group only in that the lysine decarboxylase added to the lysine fermentation broth is 16g of the lysine decarboxylase-containing fermentation broth (the total amount of the lysine decarboxylase contained therein is 16000U, and the enzyme activity is 1000U/g), and the catalysis results are shown in table 1.
TABLE 1 catalytic stability of immobilized lysine decarboxylase
The results show that the catalytic stability and the use efficiency of the experimental group adopting immobilized lysine decarboxylase are obviously improved compared with the control group adopting the lysine decarboxylase which is not subjected to immobilized treatment, the lysine conversion rate is greater than 90% when the immobilized enzyme is repeatedly used for 13 batches, and the lysine conversion rate is reduced to 20% when the free cells are repeatedly used for 3 batches.
EXAMPLE 2 treatment of immobilized lysine decarboxylase with solutions of tannin of different concentrations
Preparing 1%, 2%, 3%, 4%, 5% and 6% tannin aqueous solutions respectively, accurately measuring 50ml tannin solutions with different concentrations, adding the tannin solutions into 250ml triangular flasks, weighing 30g immobilized lysine decarboxylase (with the enzyme activity of 800U/g) prepared in example 1 respectively, adding the immobilized lysine decarboxylase into the tannin solutions with different concentrations, controlling the reaction temperature to be 20 ℃ and the rotation speed to be 100rpm, reacting for 2 hours, filtering and separating the tannin solutions and immobilized enzyme particles, washing the immobilized enzyme particles for 5 times by using a phosphate buffer solution, collecting the immobilized enzyme particles, and displaying the enzyme activity determination result, wherein the enzyme activity of the immobilized lysine decarboxylase treated by the tannin solutions with different concentrations is 800U/g.
Respectively weighing 20g of lysine decarboxylase immobilized enzyme obtained by treating tannin solutions with different concentrations, adding the immobilized enzyme into 100g of lysine fermentation liquor (pH 6.8) with the concentration of 18%, reacting for 3h at 37 ℃ and 100rpm, stopping the reaction, filtering and separating the immobilized enzyme and the reaction liquid, extracting pentanediamine from the reaction liquid, adding the collected immobilized enzyme into the same amount of lysine fermentation liquor, catalyzing lysine to generate pentanediamine under the same conditions, and circularly catalyzing 19 batches according to the conditions, and measuring the conversion rate of the lysine to the pentanediamine, wherein the results are shown in Table 2.
TABLE 2 catalytic stability of immobilized enzymes treated with different concentrations of tannin solutions
The results show that, under the same amount of immobilized enzyme, the immobilized enzyme treated by tannin solution has a lysine conversion rate in the initial 9 catalytic batches which is equivalent to or reduced from that of the immobilized enzyme not treated by tannin solution (experimental group in example 1), but the catalytic stability of the immobilized enzyme is obviously improved, and the lysine conversion rate is still as high as 90% when 19 batches are repeatedly used after tannin treatment, while the conversion rate of the immobilized enzyme not treated by tannin solution is lower than 90% after 13 batches are repeatedly used. In addition, when the immobilized enzyme is treated by tannin solutions with different concentrations, the lysine conversion rate of the initial batch of the low-concentration tannin treatment group is equivalent to that of the initial batch of the group without tannin treatment, and the catalytic stability is obviously improved compared with the group without tannin treatment but is lower than that of the group with high-concentration tannin treatment; when the concentration of tannin treatment is too high, the conversion rate is obviously reduced although the catalytic stability is higher. Therefore, the tannin treatment with proper concentration can ensure higher catalytic conversion rate and catalytic stability, and the catalytic effect is obviously improved compared with the immobilized enzyme circulating catalytic effect without the tannin treatment.
EXAMPLE 3 Effect of tannin solution treatment time on immobilized lysine decarboxylase
50ml of tannin solution with the concentration of 4 percent is accurately measured, 30g of immobilized enzyme prepared in the embodiment 1 is added into the tannin solution to react under the conditions of 20 ℃ and 100rpm, the reaction lasts for 1, 2, 3, 4, 5 and 8 hours respectively, the tannin solution and immobilized enzyme particles are separated by filtration, the immobilized enzyme particles are washed for 5 times by buffer solution, the immobilized enzyme particles are collected, and the enzyme activities of the immobilized lysine decarboxylase treated by the tannin solution for different times are all 800U/g.
Respectively weighing 20g of the lysine decarboxylase immobilized enzyme obtained by treating the tannin solution for different time, adding the immobilized enzyme into 100g of lysine fermentation liquor (pH 6.8) with the concentration of 18%, reacting for 3h at 37 ℃ and 100rpm, stopping the reaction, filtering and separating the immobilized enzyme and the reaction liquid, extracting the reaction liquid for pentanediamine, adding the collected immobilized enzyme into the same amount of lysine fermentation liquor, catalyzing lysine to generate pentanediamine under the same condition, circularly catalyzing 15 batches according to the conditions, and measuring the conversion rate of the lysine to the pentanediamine, wherein the results are shown in Table 3.
TABLE 34 catalytic stability of immobilized enzymes from different time periods of tannin solution treatment
The results show that when the immobilized enzyme is treated by 4 percent tannin solution, the catalytic stability is poor when the treatment time is short, and the conversion rate is obviously reduced when the treatment time is overlong.
Although the present invention has been described in the examples only for the method of producing an immobilized lysine decarboxylase using an epoxy-based carrier, and only for the catalytic efficiency of a lysine fermentation broth as a substrate, it will be understood by those skilled in the art that the present invention is not limited thereto, and can be applied to other carriers for producing immobilized enzymes, and the substrate form may be not only a lysine fermentation broth, but also other solutions containing lysine.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make various improvements and modifications without departing from the technical principle of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention.
Claims (8)
1. The immobilized lysine decarboxylase is characterized in that the immobilized lysine decarboxylase is obtained by combining lysine decarboxylase with an immobilized carrier in a covalent bond mode and then treating tannin;
the immobilized carrier is a macromolecular carrier containing epoxy groups;
the epoxy-containing polymer carrier is obtained by reacting one or more of a high polymer substance containing a phenylamino group, a high polymer substance containing an amino group and a high polymer substance containing a hydroxyl group with an epoxidizing reagent.
2. The immobilized lysine decarboxylase according to claim 1, wherein the tannin treatment is adding 5000U-60000U lysine decarboxylase per gram tannin.
3. The immobilized lysine decarboxylase according to claim 2, wherein the tannin treatment is adding 8000U-48000U lysine decarboxylase per gram tannin.
4. The immobilized lysine decarboxylase according to any one of claims 1 to 3, wherein the tannin treatment is at 10 ℃ to 40 ℃ for 1 to 20 hours.
5. The immobilized lysine decarboxylase according to claim 4, wherein the tannin treatment is at 20 ℃ to 30 ℃ for 1 to 8 hours.
6. A preparation method of immobilized lysine decarboxylase is characterized by comprising the following steps:
(1) mixing the lysine decarboxylase solution with an immobilized carrier, and preparing immobilized lysine decarboxylase by covalent bond combination;
the immobilized carrier is a macromolecular carrier containing epoxy groups; the epoxy-containing polymer carrier is obtained by reacting one or more of a high molecular substance containing an amino group, a high molecular substance containing an amino group and a high molecular substance containing a hydroxyl group with an epoxidizing reagent;
(2) treating the immobilized lysine decarboxylase obtained in the step (1) by using tannin, wherein the tannin treatment is to add 5000U-60000U lysine decarboxylase into each gram of tannin and treat the tannin at the temperature of 10-40 ℃ for 1-20 hours.
7. The method of claim 6, wherein the tannin treatment is performed by adding 8000U-48000U lysine decarboxylase into each gram of tannin and treating at 20-30 ℃ for 1-8 hours.
8. A method for producing 1, 5-pentanediamine, which is characterized in that the method adopts the immobilized lysine decarboxylase of any one of claims 1 to 5 or the immobilized lysine decarboxylase prepared by the preparation method of claim 6 or 7 to catalyze lysine or lysine salt.
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| CN101914514A (en) * | 2010-04-06 | 2010-12-15 | 福建师范大学 | Method for preparing immobilized alpha-amylase by using spherical tannin as carrier |
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| CN107779445A (en) * | 2016-08-25 | 2018-03-09 | 上海凯赛生物技术研发中心有限公司 | Immobilised lysine decarboxylase, its preparation, 1,5 pentanediamine preparation methods and product |
| CN110540986A (en) * | 2019-09-09 | 2019-12-06 | 华东理工大学 | Polyamine-assisted natural polyphenol rapid and stable modification magnetic nano immobilized enzyme carrier and application thereof |
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| CN101914514A (en) * | 2010-04-06 | 2010-12-15 | 福建师范大学 | Method for preparing immobilized alpha-amylase by using spherical tannin as carrier |
| CN105734036A (en) * | 2016-03-30 | 2016-07-06 | 江苏大学 | Preparation method of mesoporous silicon immobilized trypsin using tannic acid as template |
| CN107779445A (en) * | 2016-08-25 | 2018-03-09 | 上海凯赛生物技术研发中心有限公司 | Immobilised lysine decarboxylase, its preparation, 1,5 pentanediamine preparation methods and product |
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