Method for preparing pentamethylene diamine by biological method

Abstract

The invention discloses a method for preparing pentamethylene diamine by a biological method. According to the method, inorganic metal salt is added to optimize a fermentation culture medium, the pH value is periodically controlled and induced to optimize the fermentation process, fermentation liquor is directly used as invertase, and the conversion pH value is maintained by feeding ethanol and controlling the pressure of a reaction kettle in the conversion process so as to reduce the emission of carbon dioxide, so that the pH value does not need to be controlled by additionally adding acid. Finally, the substrate conversion rate is greater than or equal to 98%, the concentration of the pentamethylene diamine product is greater than 200g / L, and the conversion completion time is less than or equal to 6h. For industrial production, the method simplifies the production steps, shortens the production cycle, reduces the use of acid and alkali, and achieves the purposes of reducing the cost and improving the efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of biocatalysis, and relates to a method for preparing pentanediamine by a biological method. Background Art

[0002] Pentanediamine, also known as 1,5-pentanediamine, 1,5-diaminopentane, cadaverine, is a kind of biogenic amine and is a colorless viscous liquid. It has a wide range of applications in the fields of agriculture, medicine, industry, etc. As an important industrial chemical raw material, 1,5-pentanediamine is a bio-based polyamide derived from renewable raw materials and is widely used in various polyamide products, polyurethanes, chelating agents, additives, etc. Currently, the more widely used polyamide products are polyamide PA66 and polyamide PA6, accounting for about 90% of the total global consumption of polyamide products. Polyamide PA66 is a product of the polymerization of hexamethylenediamine and adipic acid. It was developed earliest, has the widest application, and the largest output, and is widely used in the fields of automobiles, electronic appliances, aerospace, etc. At present, the raw material adipic acid can be catalytically synthesized by biotechnology, but hexamethylenediamine depends on petrochemical synthesis. With the gradual depletion of petroleum resources and the increasing environmental pollution, there is an urgent need for a bio-synthesis technology that can achieve sustainable resource utilization and environmental protection to replace hexamethylenediamine in the production of high molecular polymer monomers. Pentanediamine can replace the traditional hexamethylenediamine produced by chemical methods. The polymerization reaction with dibasic acid can synthesize high-quality polymer materials - new nylon, which is an environmentally friendly, sustainable development, and high-temperature resistant bioplastic with broad application prospects.

[0003] As one of the bio-based raw materials, the current industrial production method of pentanediamine is to deacidify and convert L-lysine salt to pentanediamine salt under weakly acidic or neutral conditions. In the extraction stage, a large amount of strong alkaline chemical reagents need to be added to displace the pentanediamine in the conversion solution, and then an extractant is added or direct distillation is carried out to obtain the finished product pentanediamine. The use of a large amount of strong alkaline chemical reagents often brings a large cost burden, and at the same time, a large amount of industrial waste salt will be produced, increasing the raw material consumption and pollution treatment costs of enterprises. Specifically, there is no price competitiveness in the price competition with the same type of petrochemical products, which also limits the application and promotion of this product in the market. Therefore, reducing costs and increasing the competitiveness of products are urgent problems to be solved in the industry for this product. Summary of the Invention

[0004] To solve the problem of the excessively high production cost of pentanediamine in the prior art, the present invention provides a method for synthesizing pentanediamine by a biological method. The present invention directly uses the fermentation broth as a reaction catalyst without centrifuging to collect the bacterial cells; the present invention also involves controlling the reaction tank pressure by controlling the release rate of carbon dioxide during the conversion process without regulating the pH value during the conversion process. It has a low-cost, low-consumption, and highly feasible industrialization prospect.

[0005] The technical solution adopted by the present invention is as follows: A method for preparing pentamethylenediamine by a biological method, which is a method for obtaining L-lysine decarboxylase through biological fermentation using Escherichia coli as a fermentation cell and converting L-lysine hydrochloride into pentamethylenediamine under the action of L-lysine convertase.

[0006] Preferably, the method specifically includes the following steps: S1: Fermentation broth culture: The strain is fermented and cultured in a fermentation medium to obtain a fermentation broth; S2: Enzymatic catalytic conversion reaction: The fermentation broth is added to the conversion system for conversion to obtain pentamethylenediamine.

[0007] Further preferably, in the step S1, an inorganic metal salt is added to the fermentation medium, and the inorganic metal salt includes one or more of sodium salt, magnesium salt, calcium salt, manganese salt, copper salt, iron salt (including ferrous), and cobalt salt. To promote the growth and metabolism of Escherichia coli cells and increase the activity of L-lysine decarboxylase, metal ions can also increase the solubility of carbon dioxide during conversion due to the "salting-in principle". Further preferably, the concentrations of sodium ions, magnesium ions, and calcium ions in the inorganic metal salt in the medium are 0.1% - 1.8% (W / V), and the concentrations of manganese ions, copper ions, iron ions (including ferrous), and cobalt ions are 0.00001% - 0.05% (W / V); The medium includes the following components: The fermentation medium is 15 - 25 g / L of corn steep liquor dry powder, 0.8 - 1.2 g / L of dipotassium hydrogen phosphate, 1.5 - 2.5 g / L of potassium dihydrogen phosphate, 3 - 5 g / L of ammonium sulfate, 10 - 12 g / L of glucose monohydrate, and the feed supplement is 45 - 55% of glucose monohydrate; preferably, the fermentation medium is 20 g / L of corn steep liquor dry powder, 1 g / L of dipotassium hydrogen phosphate, 2 g / L of potassium dihydrogen phosphate, 4 g / L of ammonium sulfate, 11 g / L of glucose monohydrate, and the feed supplement is 50% of glucose monohydrate.

[0008] The strain is Escherichia coli, Bacillus alcalophilus (Bacillus halodurans) , Bacillus subtilis (Bacillus subtilis) , Escherichia coli (Escherichia coli) , Selenomonas ruminantium (Selenomonas ruminantium) , Vibrio cholerae (Vibrio cholerae) , Vibrio parahaemolyticus (Vibrio parahaemolyticus) , Streptomyces coelicolor (Streptomyces coelicolor) , Streptomyces hirsutus (Streptomyces pilosus) , Eikenella corrodens (Eikenella corrodens) , Eubacterium acidaminophilum (Eubacterium acidaminophilum) or Corynebacterium glutamicum (Corynebacterium glutamicum) .

[0009] Further preferably, in the step S1, during the fermentation culture process, when the OD600 grows to 10 - 40, IPTG (isopropyl-β-D-thiogalactoside) is added for induction. Glucose is supplemented during the induction period to maintain the normal growth of the bacterial liquid. At the end of fermentation, the OD value is 60 - 150.

[0010] Further preferably, in the step S1, before the induction with IPTG (isopropyl-β-D-thiogalactoside), the pH value is controlled at 6.5 - 7.5; the induction duration is 10 - 16 h. Among them, the pH is not controlled during 0 - 10 h of induction. When the pH is lower than 4.5, the pH is controlled at 7.0 - 7.2 until the end of fermentation. By stagewise controlling the pH value during the fermentation stage, the accumulation of bacteria is ensured during the strain growth stage, and it is beneficial to the expression of L-lysine decarboxylase during the induction stage. In the step S1, the fermentation culture temperature is 33°C - 37°C, the induction temperature is 22 - 30°C, and the fermentation cycle is 24 - 30 h to prepare the fermentation broth.

[0011] Even more preferably, in the step S2, during the conversion process, the pressure in the reaction tank is controlled at 0.1 - 0.3 Mpa, and the gas used to control the pressure in the reaction tank during the conversion process is the carbon dioxide gas released during the conversion process.

[0012] Further preferably, in the step S2, during the conversion process, in the conversion system, the OD600 value of the fermentation broth is 5 - 15, the concentration of the conversion substrate L-lysine hydrochloride is 450 - 550 g / L; the concentration of the coenzyme 5'-pyridoxal phosphate is 0.05 mM - 0.5 mM; the conversion temperature is 25 - 50°C. L-lysine hydrochloride is used as the effective component of the substrate, and the L-lysine decarboxylase is the fermentation broth and can be directly used for conversion without re-centrifugation for collection.

[0013] Even more preferably, in the step S2, during the conversion process, ethanol is added dropwise. Preferably, ethanol starts to be added dropwise into the conversion system from the conversion stage, and the rate is 0.1% - 0.5% / L*h (V / V), and the total amount of ethanol added dropwise is controlled at 0.5% - 5% (V / V). This can promote the solubility of carbon dioxide in water and also increase cell permeability, which is beneficial to the catalytic reaction of L-lysine decarboxylase.

[0014] Even more preferably, in the step S2, after the enzymatic conversion, the substrate conversion rate ≥ 98%, and the concentration of the cadaverine product > 200 g / L. The conversion can be completed without adjusting the conversion pH value. When the conversion is carried out for 6 h, the substrate conversion rate ≥ 98%, and the concentration of the cadaverine product > 200 g / L. The beneficial effects of the present invention are as follows: 1) Reduction in acid usage: There is no need to add extra acid to control the pH value, reducing the dependence on acid, decreasing the use of chemicals, and lowering the raw material cost for conversion and extraction.

[0015] 2) Shortening the production cycle: Without affecting the conversion cycle, directly using the fermentation broth as the enzyme catalyst to shorten the entire cycle process of preparing pentanediamine by the biocatalytic method, saving labor and equipment costs. 3) Environmentally friendly: After removing the use of acid in the conversion, the use of alkali in extraction will be reduced. Reducing the use of acid and alkali is more environmentally friendly and meets the requirements of sustainable development. Description of the Drawings

[0016] Figure 1 It is a reaction diagram for the formation of pentanediamine from L-lysine hydrochloride. Detailed Embodiments

[0017] The following further elaborates on the technical solutions of the present invention in combination with embodiments, but the scope of protection of the present invention is not limited thereto. The specific embodiments described herein are only for the purpose of illustration and explanation of the present disclosure and are not used to limit the present disclosure. Equivalent substitutions or corresponding improvements made to the content of the present invention still fall within the scope of protection of the present invention.

[0018] The plate and shake flask seed culture methods for the strains in the following examples and comparative examples are as follows: Pick a single colony from the plate and inoculate it into 20 ml / 100 ml of LB medium. Culture it in a rotary shaker at 37 °C and 220 rpm for 16 h to prepare the first-stage shake flask seed. The OD600 of the bacterial solution is 3.4. Transfer 1 ml of the first-stage seed solution to 100 ml / 500 ml of LB medium and culture it in a rotary shaker at 37 °C and 220 rpm for 3.5 h. The OD600 of the bacterial solution is 1.02 to obtain the seed solution.

[0019] The fermentation medium in the fermenter includes the following components: 20 g / L of corn steep liquor dry powder, 1 g / L of dipotassium hydrogen phosphate, 2 g / L of potassium dihydrogen phosphate, 4 g / L of ammonium sulfate, 11 g / L of glucose monohydrate, and the feed supplement is 50% glucose monohydrate.

[0020] The strain is Escherichia coli.

[0021] Comparative Example 1: Fermentation broth culture process: Under flame protection, inoculate 2.0% of the seed solution into a 5 L fermenter with a liquid volume of 3 L of fermentation broth (basic medium). Culture at 35 °C, control the dissolved oxygen ≥ 30%, and add IPTG (isopropyl-β-D-thiogalactoside) for induction when OD600 grows to 21.8 during the fermentation culture process. Glucose is continuously added at a constant rate during the induction period to maintain the normal growth of the bacterial solution. The fermentation ends after 28 h of fermentation to obtain the fermentation broth, and the OD value is 70.5.

[0022] Enzymatic catalysis reaction: In the conversion system, the concentration of lysine hydrochloride is 450 g / L, the concentration of PLP (pyridoxal 5'-phosphate) is 0.1 mM, the conversion temperature is 30 °C, and the initial conversion volume is 2 L; fermentation broth is added to the conversion system. The OD600 value of the bacterial solution in the system is calculated to be 12, and the conversion starts. The pressure is not controlled during the process, and the pH value is not controlled during the conversion process; after 6 hours of conversion, the concentration of L-lysine hydrochloride in the system is detected by a biosensor to be 29.6 g / L, the substrate conversion rate is 92.11%, and the concentration of the product cadaverine detected by GC is 189.51 g / L.

[0023] Comparative Example 2 Fermentation broth culture: The culture method is the same as that in Comparative Example 1. The fermentation volume is 50 L. The fermentation ends after 28 hours, and the OD600 value is 68.6.

[0024] Enzymatic catalysis reaction: In the conversion system, the concentration of lysine hydrochloride is 500 g / L, the concentration of PLP is 0.3 mM, the conversion temperature is 45 °C, and the initial conversion volume is 20 L; fermentation broth is added to the conversion system. The OD600 value of the bacterial solution in the system is calculated to be 7, and the conversion starts. The pressure is not controlled during the process, and the pH value is not controlled during the conversion process; after 6 hours of conversion, the concentration of L-lysine hydrochloride in the system is detected by a biosensor to be 46.6 g / L, the substrate conversion rate is 89.00%, and the concentration of the product cadaverine detected by GC is 205.4 g / L.

[0025] Example 1 Fermentation broth culture: The culture methods of the plate and shake flask seed solutions are the same as those in Comparative Example 1. In the fermentation medium, 0.9% sodium chloride, 1.2% anhydrous magnesium sulfate, 0.8% calcium chloride, 0.0005% copper sulfate, 0.003% manganese sulfate, 0.003% zinc sulfate, 0.002% ferric sulfate, 0.002% ferrous sulfate, and 0.004% cobalt chloride are added to the basic medium; the pH value is controlled at 7.0 during the fermentation culture stage. After inducing and feeding glucose, the pH is no longer regulated. When the OD600 reaches 21.8 during the fermentation culture process, IPTG (isopropyl-β-D-thiogalactoside) is added for induction. The on-line pH shows 4.5 after 7 hours of induction, and the self-controlled pH is 7.2. The fermentation ends after 28 hours, and the OD600 value at the end of fermentation is 78.6.

[0026] Enzymatic catalysis reaction: In the conversion system, the concentration of lysine hydrochloride is 500 g / L, the concentration of PLP is 0.15 mM, the conversion temperature is 37 °C, and the initial conversion volume is 2 L. Fermentation broth is added to the conversion system. When the OD600 value of the bacterial solution in the system is calculated to be 7, the conversion starts. During the process, 50% (V / V) ethanol solution is added dropwise at a flow rate of 8 ml / L·h. At the start of the conversion, as the conversion proceeds, the pressure in the reaction kettle gradually increases and is finally stably controlled at 0.15 Mpa. The pH value is not regulated during the conversion process. After 6 h of conversion, the concentration of L-lysine hydrochloride in the system is detected by a biosensor to be 7.8 g / L, the substrate conversion rate is 99.46%, and the concentration of the product cadaverine detected by GC is 246.88 g / L.

[0027] Example 2 Fermentation broth culture: The culture methods of the plate and shake flask seed solutions are the same as those in Comparative Example 1. In the fermentation medium, 1.1% sodium chloride, 1.5% anhydrous magnesium sulfate, 0.5% calcium chloride, 0.0004% copper sulfate, 0.004% manganese sulfate, 0.004% zinc sulfate, 0.0015% ferric sulfate, 0.0015% ferrous sulfate, and 0.005% cobalt chloride are added to the basic medium. During the fermentation culture stage, the pH value is controlled at 7.0. After inducing and adding glucose dropwise, the pH is no longer regulated. When the OD600 grows to 21.8 during the fermentation culture process, IPTG (isopropyl-β-D-thiogalactoside) is added for induction. After 8 h of induction, the online pH shows 4.5, and the automatic control pH is 7.0. The fermentation ends after 28 h of fermentation, and the OD value is 85.6.

[0028] Enzymatic catalysis reaction: In the conversion system, the concentration of lysine hydrochloride is 450 g / L, the concentration of PLP is 0.3 mM, the conversion temperature is 45 °C, and the initial conversion volume is 2 L. Fermentation broth is added to the conversion system. When the OD600 value of the bacterial solution in the system is calculated to be 10, the conversion starts. During the process, 50% (V / V) ethanol solution is added dropwise at a flow rate of 12 ml / L·h. At the start of the conversion, as the conversion proceeds, the pressure in the reaction kettle gradually increases and is finally stably controlled at 0.2 Mpa. The pH value is not regulated during the conversion process. After 6 h of conversion, the concentration of L-lysine hydrochloride in the system is detected by a biosensor to be 2.1 g / L, the substrate conversion rate is 99.46%, and the concentration of the product cadaverine detected by GC is 215.96 g / L. [[ID=Ten]]

[0029] Example 3 Fermentation broth culture: The culture methods of the plate and shake flask seed solutions are the same as those in Comparative Example 1. In the fermentation medium, 1.0% sodium chloride, 1.1% anhydrous magnesium sulfate, 0.9% calcium chloride, 0.0005% copper sulfate, 0.005% manganese sulfate, 0.004% zinc sulfate, 0.0015% ferric sulfate, 0.0015% ferrous sulfate, and 0.003% cobalt chloride are added to the basal medium; during the fermentation culture stage, the pH value is controlled at 6.8. After inducing the addition of glucose, the pH is no longer regulated. When the OD600 reaches 21.8 during the fermentation culture process, IPTG (isopropyl-β-D-thiogalactoside) is added for induction. After 6.5 hours of induction, the online pH shows 4.5 and the automatic control pH is 7.0. The fermentation ends after 30 hours of fermentation, and the OD value is 88.6.

[0030] Enzymatic catalysis reaction: In the conversion system, the concentration of lysine hydrochloride is 500 g / L, the concentration of PLP is 0.1 mM, the conversion temperature is 35 °C, and the initial conversion volume is 20 L; fermentation broth is added to the conversion system, and the OD600 value of the bacterial liquid in the system is calculated to be 15, and then the conversion starts. During the process, 50% (V / V) ethanol solution is added dropwise at a flow rate of 20 ml / L·h; when the conversion starts, due to the progress of the conversion, the pressure in the reaction kettle gradually increases and is finally stably controlled at 0.2 Mpa. The pH is not regulated during the conversion process. After 6 hours of conversion, the concentration of L-lysine hydrochloride in the system is detected to be 5.2 g / L by a biosensor, the substrate conversion rate is 98.74%, and the concentration of the product pentamethylenediamine detected by GC is 229.78 g / L.

[0031] Example 4 is directed to Example 1, and the metal ion components added to the fermentation medium are changed. Based on Example 1, the metal ion components added to the fermentation medium are changed, and the others are the same as in Example 1.

[0032] Fermentation broth culture: The culture methods of the plate and shake flask seed solutions are the same as those in Example 1, and the ions added to the fermentation medium are changed: Example 4-1: 1.1% anhydrous magnesium sulfate, 0.9% calcium chloride, 0.0005% copper sulfate, 0.005% manganese sulfate, 0.004% zinc sulfate, 0.0015% ferric sulfate, 0.0015% ferrous sulfate, 0.003% cobalt chloride; Example 4-2: 1.0% sodium chloride, 0.9% calcium chloride, 0.0005% copper sulfate, 0.005% manganese sulfate, 0.004% zinc sulfate, 0.0015% ferric sulfate, 0.0015% ferrous sulfate, 0.003% cobalt chloride; Example 4-3: 1.0% sodium chloride, 1.1% anhydrous magnesium sulfate, 0.0005% copper sulfate, 0.005% manganese sulfate, 0.004% zinc sulfate, 0.0015% ferric sulfate, 0.0015% ferrous sulfate, 0.003% cobalt chloride; Example 4-4: Sodium chloride 1.0%, anhydrous magnesium sulfate 1.1%, calcium chloride 0.9%; Example 4-5: No metal ions are added during the fermentation process.

[0033] Enzymatic catalysis reaction: The same as in Example 1.

[0034] Table 1

[0035] It can be seen from Table 1 that metal ions have a certain promoting effect on the enzyme activity. By adding metal salts during the fermentation process, it has a certain promoting effect on the enzyme activity of L-lysine decarboxylase. Due to the "salting-in effect", the presence of salt ions can promote the solubility of carbon dioxide in the conversion solution; in Example 4-2, when magnesium ions are lacking in the fermentation medium, although the conversion rate is still higher than that in Example 4-5, the conversion effect of more than 98% cannot be completed within 6 hours. It is possible that magnesium ions, as the structural active center of the enzyme, have an impact on the stability and activity of the enzyme activity; the absence of sodium ions and calcium ions in other metal ions such as Example 4-1 and 4-3 also reduces the conversion rate at the same time, but the effect is not as obvious as that of magnesium ions. Similar phenomena are shown by other trace metal elements in Example 4-4. In the present invention, the addition of metal ions can effectively improve the substrate conversion rate under the same culture conditions.

[0036] Example 5 is directed to Example 1, and the pH is conditioned during the fermentation process. Based on Example 1, the difference is that the pH value is controlled at 7.0 during the fermentation culture stage, and the pH is not regulated after the induced addition of glucose. When the OD600 reaches 21.8 during the fermentation culture process, IPTG (isopropyl-β-D-thiogalactoside) is added for induction. After 7 hours of induction, the on-line pH shows 4.5, and the automatic control pH is 7.2. The fermentation ends after 28 hours. The others are the same as in Comparative Example 1.

[0037] Example 1: The pH value is controlled at 7.0 during the fermentation culture stage, and the pH is not regulated after the induced addition of glucose. When the OD600 reaches 21.8 during the fermentation culture process, IPTG (isopropyl-β-D-thiogalactoside) is added for induction. After 7 hours of induction, the on-line pH shows 4.5, and the automatic control pH is 7.2. The fermentation ends after 28 hours; Example 5-1: The pH value is controlled at 7.0 during the fermentation culture stage. After the induced addition of glucose, when the OD600 reaches 21.8 during the fermentation culture process, IPTG (isopropyl-β-D-thiogalactoside) is added for induction, and the automatic control pH is 7.2. The fermentation ends after 28 hours; The conditions of Example 5-1 and 1 here are the same, both are 7.2. Here, 5-1 is not okay and needs to be adjusted again? Example 5-2: During the fermentation culture stage, the pH value was controlled at 7.0. After inducing the fed-batch addition of glucose, when the OD600 reached 21.8 during the fermentation culture process, IPTG (isopropyl-β-D-thiogalactoside) was added for induction, and the pH was automatically controlled at 6.0. The fermentation ended after 28 h. Example 5-3: During the fermentation culture stage, the pH value was controlled at 7.0. After inducing the fed-batch addition of glucose, when the OD600 reached 21.8 during the fermentation culture process, IPTG (isopropyl-β-D-thiogalactoside) was added for induction, and the pH was automatically controlled at 4.5. The fermentation ended after 28 h. Table 2

[0038] The optimal growth range of Escherichia coli is 6.8 - 7.2, and the isoelectric point of the vast majority of enzymes is neutral. Therefore, acidic conditions are more conducive to the soluble expression of enzymes and have better enzyme activity, but are not conducive to the growth of Escherichia coli. As shown in Table 2, in Example 5-1, the neutral condition can obtain the highest OD value of the cell mass, but may lead to the formation of enzyme inclusion bodies, which is not conducive to enzyme activity, so the overall conversion rate is relatively low. In Example 5-3, although a relatively low acidic condition can obtain high enzyme activity, due to the small amount of bacteria obtained, more fermentation broth needs to be added, which is still not conducive to cost reduction. In the present invention, without regulating the pH value during the early stage of induction, the pH value is automatically reduced to 4.5 to promote the secreted expression of the enzyme, and then increased to neutral in the later stage to facilitate the increase in the number of bacteria. Thus, higher enzyme activity can be obtained, and more strains can be obtained for use as catalysts.

[0039] Example 6 Based on Example 1, during the enzymatic reaction process, the conditions for the use of ethanol were studied, and the other conditions were the same as in Example 1.

[0040] Example 1: A 50% (V / V) ethanol solution was added dropwise during the process, and the flow rate was 8 ml / L·h. Example 6-1: At the beginning of the conversion, no ethanol solution was added dropwise during the process. Example 6-2: At the beginning of the conversion, a 50% (V / V) ethanol solution was added dropwise during the process, and the flow rate was 2 ml / L·h. Example 6-3: At the beginning of the conversion, a 50% (V / V) ethanol solution was added dropwise during the process, and the flow rate was 30 ml / L·h.

[0041] Table 3

[0042] In the process of L-lysine decarboxylase catalyzing the decarboxylation of L-lysine to form cadaverine, it is necessary to cross the cell membrane and enter the conversion solution to participate in the reaction. Ethanol can improve the cell membrane permeability, facilitating the contact between the enzyme and the substrate. At the same time, due to the presence of ethanol, it can also promote the solubility of carbon dioxide in the conversion solution. As shown in Table 3, in Example 6-2, when the ethanol concentration is too low, the stimulation effect on cells is not significant and the requirements cannot be met; in Example 6-3, when the ethanol concentration is too high, it will damage the structure of the enzyme, leading to the premature inactivation of the enzyme and the premature end of the conversion process. The present invention controls the addition rate and total amount of ethanol by a feeding method to improve cell permeability without causing enzyme inactivation, achieving the effect of promoting conversion.

[0043] The above embodiments are only the preferred technical solutions of the present invention and should not be regarded as limitations on the present invention. The embodiments in this application and the features in the embodiments can be arbitrarily combined with each other without conflict. The protection scope of the present invention should be the technical solutions recorded in the claims, including the equivalent replacement solutions of the technical features in the technical solutions recorded in the claims. That is, the equivalent replacement improvements within this scope are also within the protection scope of the present invention.

Claims

1. A method for preparing pentanediamine by a biological method, characterized in that, The method obtains L-lysine decarboxylase through biological fermentation, and converts L-lysine hydrochloride into cadaverine under the action of L-lysine convertase.

2. The method according to claim 1, characterized in that: The method specifically includes the following steps: S1: Fermentation broth culture: The strain is fermented and cultured in a fermentation medium to obtain a fermentation broth; S2: Enzymatic catalytic conversion reaction: The fermentation broth is added to the conversion system for conversion to obtain cadaverine.

3. The method according to claim 2, characterized in that: In the step S1, inorganic metal salts are added to the fermentation medium, and the inorganic metal salts include one or several of sodium salts, magnesium salts, calcium salts, manganese salts, copper salts, iron salts, ferrous salts, and cobalt salts.

4. The method according to claim 3, wherein: In the inorganic metal salts, the concentrations of sodium ions, magnesium ions, and calcium ions in the fermentation medium are 0.1% - 1.8% (W / V), and the concentrations of manganese ions, copper ions, iron ions, ferrous ions, and cobalt ions are 0.00001% - 0.05% (W / V); The fermentation medium includes the following components: 15 - 25 g / L corn steep liquor dry powder, 0.8 - 1.2 g / L dipotassium hydrogen phosphate, 1.5 - 2.5 g / L potassium dihydrogen phosphate, 3 - 5 g / L ammonium sulfate, 10 - 12 g / L glucose monohydrate, and the feeding material is 45 - 55% glucose monohydrate; preferably, the fermentation medium is 20 g / L corn steep liquor dry powder, 1 g / L dipotassium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, 4 g / L ammonium sulfate, 11 g / L glucose monohydrate, and the feeding material is 50% glucose monohydrate; The strain is Escherichia coli, Bacillus alcalophilus, Bacillus subtilis, Escherichia coli, Selenomonas ruminantium, Vibrio cholerae, Vibrio parahaemolyticus, Streptomyces coelicolor, Streptomyces pilosus, Eikenella corrodens, Eubacterium acidaminophilum, or Corynebacterium glutamicum.

5. The method according to claim 2, wherein: In the step S1, during the fermentation culture process, when OD600 grows to 10 - 40, IPTG (isopropyl-β-D-thiogalactoside) is added for induction, and glucose is supplemented during the induction period to maintain the normal growth of the bacterial liquid. At the end of fermentation, the OD value is 60 - 150.

6. The method according to claim 5, characterized in that: In the step S1, before the induction with IPTG (isopropyl-β-D-thiogalactoside), the pH value is controlled at 6.5 - 7.5; the induction duration is 10 - 16 h, during which the pH is not controlled from 0 - 10 h, and when the pH is lower than 4.5, the pH is controlled at 7.0 - 7.2 until the end of fermentation; In the step S1, the fermentation culture temperature is 33°C - 37°C, the induction temperature is 22 - 30°C, and the whole fermentation cycle is 16 - 40 h to prepare the fermentation broth.

7. The method according to claim 2, wherein: In the step S2, during the conversion process, the pressure of the reaction tank is controlled at 0.1 - 0.3 Mpa, and the gas used to control the pressure of the reaction tank during the conversion process is the carbon dioxide gas released during the conversion process.

8. The method according to claim 2, characterized in that: In the step S2, during the conversion process, in the conversion system, the OD600 value of the fermentation broth is 5 - 15, the concentration of the conversion substrate L-lysine hydrochloride is 450 - 550 g / L; the concentration of the coenzyme 5'-pyridoxal phosphate is 0.05 mM - 0.5 mM; the conversion temperature is 25 - 50°C.

9. The method according to claim 8, characterized in that: In the step S2, during the conversion process, ethanol is added in a transformed manner. Preferably, ethanol starts to be added into the conversion system from the conversion stage at a rate of 0.1% - 0.5% / L*h (V / V) of the addition amount, and the total amount of added ethanol is controlled at 0.5% - 5% (V / V).

10. The method according to any one of claims 2-9, characterized in that: In the step S2, after the enzymatic catalytic conversion, the substrate conversion rate ≥ 98%, and the concentration of the pentanediamine product > 200 g / L.

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