Method for improving enzyme activity of nucleoside phosphorylase

Abstract

The invention discloses a method for improving enzyme activity of nucleoside phosphorylase, which comprises the following steps of: inoculating activated escherichia coli engineering bacteria into a seed culture medium for primary strain culture, and then inoculating the primary strain obtained by culture into a fermentation culture medium according to a proportion for fermentation culture to prepare nucleoside phosphorylase enzyme liquid; in the fermentation culture process, the culture temperature before fed-batch sugar is 37 DEG C, the fed-batch sugar is carried out after bottom sugar is used up, the total concentration of glucose and lactose in the fed-batch sugar is 90 g / L, the sugar adding rate of the fed-batch sugar is controlled to be 225-325 ml / h, the induction temperature after the fed-batch sugar is 25-32 DEG C, and the dissolved oxygen in the fermentation process is 20-40%. Specific escherichia coli engineering bacteria serve as production strains, the enzyme activity of the nucleoside phosphorylase is improved by controlling the concentration of base sugar, controlling dissolved oxygen, controlling the fed-batch sugar adding ratio, the sugar adding rate and the induction temperature, the enzyme activity can reach 500 U / g or above, and the quality requirement for the enzyme activity in the I + G conversion process can be met.

Description

Technical field:

[0001] The invention belongs to the field of flavor nucleotide production, and particularly relates to a method for improving the activity of nucleoside phosphorylase. Background technology:

[0002] I+G is a new generation of food flavor enhancer, which is a mixture of 5'-inosinic acid (IMP) and 5'-guanylic acid (GMP) in a ratio of 1:1. It is widely used in the field of food processing. Nucleoside phosphorylase is a key enzyme in the salvage pathway of purine biosynthesis that can catalyze the reversible phosphorylation of nucleosides or deoxynucleosides. The enzyme catalysis method for producing I+G does not use expensive chemical reagents, and has significant advantages such as strong specificity, simple operation process, and mild reaction conditions. It is gradually replacing the chemical synthesis method to produce I+G.

[0003] Excellent production strains are a prerequisite for enzyme production by fermentation, but if you want to further improve the enzyme activity, you need to explore suitable enzyme production fermentation processes, which should take into account both the growth needs of the bacteria and the improvement of enzyme activity. At present, in the process of synthesizing nucleoside phosphorylase using Escherichia coli engineered bacteria, due to the influence of metabolic pathways, the design of substrate sugar concentration, dissolved oxygen control, flow sugar addition ratio and sugar addition rate, and induction temperature are not reasonable, resulting in unsatisfactory enzyme activity of the prepared nucleoside phosphorylase, which cannot meet the quality requirements of enzyme activity in the I+G conversion process. Summary of the invention:

[0004] In order to further improve the enzyme activity of nucleoside phosphorylase, the present invention uses a specific strain of Escherichia coli engineered bacteria as a production strain, and improves the enzyme activity of nucleoside phosphorylase by controlling the substrate sugar concentration, dissolved oxygen control, flow sugar addition ratio and sugar addition rate, and induction temperature, thereby providing a method for improving the enzyme activity of nucleoside phosphorylase.

[0005] The present invention is implemented by the following technical scheme: a method for improving the activity of nucleoside phosphorylase, wherein activated Escherichia coli engineering bacteria are inoculated into a seed culture medium for primary bacterial strain culture, and then the primary bacterial strain obtained by culture is inoculated into a fermentation culture medium in proportion for fermentation culture to prepare a nucleoside phosphorylase enzyme solution; during the fermentation culture process, the culture temperature is 37°C before the flow sugar addition, and when the base sugar is exhausted, the flow sugar addition is performed, the total concentration of glucose and lactose in the flow sugar addition is 90g / L, the flow sugar addition rate is controlled at 225-325ml / h, the induction temperature after the flow sugar addition is 25-32°C, and the dissolved oxygen in the fermentation process is 20-40%.

[0006] The sugar addition rate is a key factor affecting the growth and enzyme production of E. coli engineered bacteria. If the sugar addition rate is too high, the glucose content in the culture medium is high, and glucose is preferentially used, resulting in growth without enzyme production or low enzyme activity; if the sugar addition rate is too low, the strain will use lactose in the absence of glucose, which will only induce enzyme production and inhibit growth. Therefore, it is necessary to control the sugar addition rate in accordance with the process conditions to balance the relationship between bacterial growth and enzyme production.

[0007] The carbon source in the culture medium before the addition of sugar is glucose. By controlling 37°C, the sugar consumption rate and growth rate can be increased, and the fermentation enzyme production cycle can be shortened. Lactose is present in the culture medium after the addition of sugar. Selecting a suitable induction temperature can ensure the improvement of bacterial metabolism and enzyme production activity. After the addition of sugar is added to the culture medium, lactose will be present in the culture medium. At this time, lactose induction enzyme production is performed by temperature control. If the induction temperature is too high during the fermentation process, on the one hand, the enzyme activity in the cells will be enhanced, the growth metabolism will be accelerated, and the growth will be uncontrolled; on the other hand, the high induction temperature will cause the activity of the produced nucleoside phosphorylase to decrease or even be inactivated. Therefore, the present invention chooses to control the induction temperature between 20-35°C to ensure that both growth and enzyme production can achieve the expected results.

[0008] Furthermore, the concentration ratio of the glucose to the lactose in the flow-added sugar is 60g / L:30g / L-30g / L:60g / L.

[0009] Furthermore, the genetic composition of the engineered Escherichia coli contains a DNA molecule encoding a phosphotransferase mutant, and the amino acid sequence of the phosphotransferase mutant is shown in SEQ ID No.2, SEQ ID No.4 or SEQ ID No.6.

[0010] Furthermore, the nucleotide sequence of the DNA molecule is shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5.

[0011] Furthermore, the engineered Escherichia coli bacteria expresses a phosphotransferase mutant, and the amino acid sequence of the phosphotransferase mutant is shown in SEQ ID No.2, SEQ ID No.4 or SEQ ID No.6.

[0012] The Escherichia coli engineered strain of the present invention is a lactose operon. When there is glucose in the culture medium, the strain will preferentially utilize glucose, and the metabolites produced will inhibit adenylate cyclase, activate phosphodiesterase, reduce intracellular cAMP and make it unable to bind to CAP, and CAP cannot bind to the CAP binding site upstream of LacP, and cannot promote RNA polymerase to bind to the promoter, resulting in inability to transcribe. As the glucose in the culture medium is exhausted, the lactose in the culture medium will bind to the repressor protein, resulting in the repressor protein being unable to bind to the operon, and at this time, the RNA polymerase can bind to the promoter to normally transcribe and express nucleoside phosphorylase.

[0013] Advantages of the present invention:

[0014] The present invention uses a specific strain of Escherichia coli as a production strain, and improves the activity of nucleoside phosphorylase by controlling the substrate sugar concentration, dissolved oxygen control, flow sugar addition ratio and sugar addition rate, and induction temperature, and the enzyme activity can reach more than 500U / g, which can meet the quality requirements of the I+G conversion process for enzyme activity.

[0015] The method of the invention is simple and easy to implement, can steadily improve the activity of nucleoside phosphorylase, can significantly improve the conversion and extraction process indicators of I+G, and lays a foundation for large-scale industrial production of I+G. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the result graph of the enzymatic activity of different mutant phosphotransferases in the second step.

[0017] Figure 2 This is a diagram showing the results of fermentation production of 5'-IMP or 5'-GMP by the mutant strain in the third step. Specific implementation method:

[0018] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Example 1: A method for improving the activity of nucleoside phosphorylase, wherein activated Escherichia coli engineering bacteria are inoculated into a seed culture medium for primary bacterial culture, and then the primary bacterial culture obtained is inoculated into a fermentation culture medium in proportion for fermentation culture to prepare a nucleoside phosphorylase enzyme solution; the specific preparation method of this example is as follows:

[0020] Primary strain preparation process: Inoculate the activated Escherichia coli engineered bacteria on the plate into the shake flask seed culture medium. The seed culture medium consists of: 5-20g / L glucose, 3-10g / L yeast extract, 5-10g / L corn steep liquor, 2-5g / L peptone, 1-5g / L sodium glutamate, 1-2.5g / L magnesium sulfate, 0.5-1.0g / L potassium dihydrogen phosphate, 0.5-2mL / L defoamer, and the rest is water. Seed culture conditions: initial pH = 6.6-7.2, shaker speed 150-250rpm, temperature 30-37℃. Primary strain culture OD 600 After reaching 4.0-12.0, the seeds are inoculated into the fermentation medium at a 5-10% inoculation ratio.

[0021] 10L fermentation process: Fermentation medium composition: glucose 15-20g / L, yeast extract 5-10g / L, corn steep liquor 10-30g / L, ammonium sulfate 2-5g / L, potassium dihydrogen phosphate 2-5g / L, magnesium sulfate 0.2-0.6g / L, biotin 0.02-0.1g / L, VB10.01-0.05g / L, defoamer 0.5-2mL / L, and the rest is water. After the base sugar is exhausted, the flow sugar is added. The concentrations of glucose and lactose in the flow sugar are 45g / L and 45g / L respectively, and the flow sugar addition rate is 230-280ml / h. The culture temperature before the flow sugar addition is 37℃, and the induction temperature after the flow sugar addition is 25-30℃. The fermentation culture conditions are: tank pressure 0.05-0.07Mpa, ventilation volume 5-20L / min, stirring 200-800rpm, pH 6.6-7.2. The tank pressure, ventilation volume and stirring are adjusted in a cycle to maintain the dissolved oxygen at 25-35%, and the fermentation is terminated after culturing for 28 hours.

[0022] This example was repeated 3 times, and the OD of the nucleoside phosphorylase solution was measured at the end of the fermentation. 600 and enzyme activity (U / g), and conversion indices are shown in Table 1.

[0023] Example 2: A method for improving the activity of nucleoside phosphorylase, which is different from Example 1 in that:

[0024] 10L fermentation process: Fermentation medium composition: glucose 20-25g / L, yeast extract 5-10g / L, corn steep liquor 10-30g / L, ammonium sulfate 2-5g / L, potassium dihydrogen phosphate 2-5g / L, magnesium sulfate 0.2-0.6g / L, biotin 0.02-0.1g / L, VB10.01-0.05g / L, defoamer 0.5-2mL / L, and the rest is water. After the base sugar is exhausted, the flow sugar is added. The concentrations of glucose and lactose in the flow sugar are 50g / L and 40g / L respectively, and the flow sugar addition rate is 265-315ml / h. The culture temperature before the flow sugar addition is 37℃, and the induction temperature after the flow sugar addition is 27-32℃. The fermentation culture conditions are: tank pressure 0.05-0.07Mpa, ventilation volume 5-20L / min, stirring 200-800rpm, pH 6.6-7.2. The tank pressure, ventilation volume and stirring are adjusted in a cycle to maintain the dissolved oxygen at 25-35%, and the fermentation is terminated after culturing for 28 hours.

[0025] This example was repeated 3 times, and the OD of the nucleoside phosphorylase solution was measured at the end of the fermentation. 600 and enzyme activity (U / g), and conversion indices are shown in Table 1.

[0026] Example 3: A method for improving the activity of nucleoside phosphorylase, which is different from Example 1 in that:

[0027] Primary strain preparation process: The seed culture medium and culture conditions are the same as those in comparative experiment example 1.

[0028] 10L fermentation process: Fermentation medium composition: glucose 10-15g / L, yeast extract 5-10g / L, corn steep liquor 10-30g / L, ammonium sulfate 2-5g / L, potassium dihydrogen phosphate 2-5g / L, magnesium sulfate 0.2-0.6g / L, biotin 0.02-0.1g / L, VB10.01-0.05g / L, defoamer 0.5-2mL / L, and the rest is water. After the base sugar is exhausted, the flow sugar is added. The concentrations of glucose and lactose in the flow sugar are 52g / L and 38g / L respectively, and the flow sugar addition rate is 240-290ml / h. The culture temperature before the flow sugar addition is 37℃, and the induction temperature after the flow sugar addition is 25-30℃. The fermentation culture conditions are: tank pressure 0.05-0.07Mpa, ventilation volume 5-20L / min, stirring 200-800rpm, pH 6.6-7.2. The tank pressure, ventilation volume and stirring are adjusted in a cycle to maintain the dissolved oxygen at 20-30%. The fermentation is terminated after culturing for 28 hours.

[0029] This example was repeated 3 times, and the OD of the nucleoside phosphorylase solution was measured at the end of the fermentation. 600 and enzyme activity (U / g), and conversion indices are shown in Table 1.

[0030] Example 4: A method for improving the activity of nucleoside phosphorylase, which is different from Example 1 in that:

[0031] Primary strain preparation process: The seed culture medium and culture conditions are the same as those in comparative experiment example 1.

[0032] 10L fermentation process: Fermentation medium composition: glucose 15-20g / L, yeast extract 5-10g / L, corn steep liquor 10-30g / L, ammonium sulfate 2-5g / L, potassium dihydrogen phosphate 2-5g / L, magnesium sulfate 0.2-0.6g / L, biotin 0.02-0.1g / L, VB10.01-0.05g / L, defoamer 0.5-2mL / L, and the rest is water. After the base sugar is exhausted, the flow sugar is added. The concentrations of glucose and lactose in the flow sugar are 46g / L and 44g / L respectively, and the flow sugar addition rate is 225-275ml / h. The culture temperature before the flow sugar addition is 37℃, and the induction temperature after the flow sugar addition is 26-31℃. The fermentation culture conditions are: tank pressure 0.05-0.07Mpa, ventilation volume 5-20L / min, stirring 200-800rpm, pH 6.6-7.2. The tank pressure, ventilation volume and stirring are adjusted in a cycle to maintain the dissolved oxygen at 20-30%. The fermentation is terminated after culturing for 28 hours.

[0033] This example was repeated 3 times, and the OD of the nucleoside phosphorylase solution was measured at the end of the fermentation. 600 and enzyme activity (U / g), and conversion indices are shown in Table 1.

[0034] Example 5: A method for improving the activity of nucleoside phosphorylase, which is different from Example 1 in that:

[0035] Primary strain preparation process: The seed culture medium and culture conditions are the same as those in comparative experiment example 1.

[0036] 10L fermentation process: Fermentation medium composition: glucose 10-15g / L, yeast extract 5-10g / L, corn steep liquor 10-30g / L, ammonium sulfate 2-5g / L, potassium dihydrogen phosphate 2-5g / L, magnesium sulfate 0.2-0.6g / L, biotin 0.02-0.1g / L, VB10.01-0.05g / L, defoamer 0.5-2mL / L, and the rest is water. After the base sugar is exhausted, the flow sugar is added. The concentrations of glucose and lactose in the flow sugar are 48g / L and 42g / L respectively, and the flow sugar addition rate is 250-325ml / h. The culture temperature before the flow sugar addition is 37℃, and the induction temperature after the flow sugar addition is 27-32℃. The fermentation culture conditions are: tank pressure 0.05-0.07Mpa, ventilation volume 5-20L / min, stirring 200-800rpm, pH 6.6-7.2. The tank pressure, ventilation volume and stirring are adjusted in a cycle to maintain the dissolved oxygen at 30-40%. The fermentation is terminated after culturing for 28 hours.

[0037] This example was repeated 3 times, and the OD of the nucleoside phosphorylase solution was measured at the end of the fermentation. 600 and enzyme activity (U / g), and conversion indices are shown in Table 1.

[0038] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that:

[0039] 10L fermentation process: Fermentation medium composition: glucose 15-20g / L, yeast extract 5-10g / L, corn steep liquor 10-30g / L, ammonium sulfate 2-5g / L, potassium dihydrogen phosphate 2-5g / L, magnesium sulfate 0.2-0.6g / L, biotin 0.02-0.1g / L, VB10.01-0.05g / L, defoamer 0.5-2mL / L, and the rest is water. After the base sugar is exhausted, the flow sugar is added. The concentrations of glucose and lactose in the flow sugar are 45g / L and 45g / L respectively, and the flow sugar addition rate is 250-300ml / h. The culture temperature before the flow sugar addition is 37℃, and the induction temperature after the flow sugar addition is 20-25℃. The fermentation culture conditions are: tank pressure 0.05-0.07Mpa, ventilation volume 5-20L / min, stirring 200-800rpm, pH 6.6-7.2, and the dissolved oxygen is maintained at 5-15% by adjusting the tank pressure, ventilation volume, and stirring in a cycle in sequence, and the fermentation is terminated after culturing for 28 hours. Among them, the induction temperature after the addition of sugar is 20-25°C and the dissolved oxygen is maintained at 5-15%, which is beyond the protection scope of the claims of the present invention.

[0040] This comparative example was repeated 3 times, and the OD of the nucleoside phosphorylase solution in the tank was measured at the end of the fermentation. 600 and enzyme activity (U / g), and conversion indices are shown in Table 1.

[0041] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that:

[0042] 10L fermentation process: Fermentation medium composition: glucose 15-20g / L, yeast extract 5-10g / L, corn steep liquor 10-30g / L, ammonium sulfate 2-5g / L, potassium dihydrogen phosphate 2-5g / L, magnesium sulfate 0.2-0.6g / L, biotin 0.02-0.1g / L, VB10.01-0.05g / L, defoamer 0.5-2mL / L, and the rest is water. After the base sugar is exhausted, the flow sugar is added. The concentrations of glucose and lactose in the flow sugar are 60g / L and 30g / L respectively, and the flow sugar addition rate is 200-250ml / h. The culture temperature before the flow sugar addition is 37℃, and the induction temperature after the flow sugar addition is 30-35℃. The fermentation culture conditions are: tank pressure 0.05-0.07Mpa, ventilation volume 5-20L / min, stirring 200-800rpm, pH 6.6-7.2, and the dissolved oxygen is maintained at 15-25% by adjusting the tank pressure, ventilation volume, and stirring in a cycle in sequence, and the fermentation is terminated after culturing for 28 hours. Among them, the sugar addition rate of the flow addition is 200-250ml / h, the induction temperature after the flow addition is 30-35°C, and the dissolved oxygen is maintained at 15-25%, all of which are beyond the protection scope of the claims of the present invention.

[0043] This comparative example was repeated 3 times, and the OD of the nucleoside phosphorylase solution in the tank was measured at the end of the fermentation. 600 and enzyme activity (U / g), and conversion indices are shown in Table 1.

[0044] Comparative Example 3: The difference between Comparative Example 3 and Example 1 is that:

[0045] 10L fermentation process: Fermentation medium composition: glucose 20-25g / L, yeast extract 5-10g / L, corn syrup 10-30g / L, ammonium sulfate 2-5g / L, potassium dihydrogen phosphate 2-5g / L, magnesium sulfate 0.2-0.6g / L, biotin 0.02-0.1g / L, VB10.01-0.05g / L, defoamer 0.5-2mL / L, and the rest is water. After the base sugar is exhausted, the flow sugar is added. The concentrations of glucose and lactose in the flow sugar are 40g / L and 50g / L respectively, and the flow sugar addition rate is 250-300ml / h. The culture temperature before the flow sugar addition is 37℃, and the induction temperature after the flow sugar addition is 25-30℃. The fermentation culture conditions are: tank pressure 0.05-0.07Mpa, ventilation volume 5-20L / min, stirring 200-800rpm, pH 6.6-7.2, and the dissolved oxygen is maintained at 40-50% by adjusting the tank pressure, ventilation volume, and stirring in a cycle in sequence, and the fermentation is terminated after culturing for 28 hours. Among them, maintaining the dissolved oxygen at 40-50% is beyond the protection scope of the claims of the present invention.

[0046] This comparative example was repeated 3 times, and the OD of the nucleoside phosphorylase solution in the tank was measured at the end of the fermentation. 600 and enzyme activity (U / g), and conversion indices are shown in Table 1.

[0047] Comparative Example 4: The difference between Comparative Example 4 and Example 1 is that:

[0048] 10L fermentation process: Fermentation medium composition: glucose 10-15g / L, yeast extract 5-10g / L, corn steep liquor 10-30g / L, ammonium sulfate 2-5g / L, potassium dihydrogen phosphate 2-5g / L, magnesium sulfate 0.2-0.6g / L, biotin 0.02-0.1g / L, VB10.01-0.05g / L, defoamer 0.5-2mL / L, and the rest is water. After the base sugar is exhausted, the flow sugar is added. The concentrations of glucose and lactose in the flow sugar are 30g / L and 60g / L respectively, and the flow sugar addition rate is 100-150ml / h. The culture temperature before the flow sugar addition is 37℃, and the induction temperature after the flow sugar addition is 25-30℃. The fermentation culture conditions are: tank pressure 0.05-0.07Mpa, ventilation volume 5-20L / min, stirring 200-800rpm, pH 6.6-7.2, and the dissolved oxygen is maintained at 25-35% by adjusting the tank pressure, ventilation volume, and stirring in a cycle in sequence, and the fermentation is terminated after culturing for 28 hours. Among them, the flow sugar addition rate is 100-150ml / h, which exceeds the protection scope of the claims of the present invention.

[0049] This comparative example was repeated 3 times, and the OD of the nucleoside phosphorylase solution in the tank was measured at the end of the fermentation. 600 and enzyme activity (U / g), and conversion indices are shown in Table 1.

[0050] Comparative Example 5: The difference between Comparative Example 5 and Example 1 is that:

[0051] 10L fermentation process: Fermentation medium composition: glucose 5-10g / L, yeast extract 5-10g / L, corn steep liquor 10-30g / L, ammonium sulfate 2-5g / L, potassium dihydrogen phosphate 2-5g / L, magnesium sulfate 0.2-0.6g / L, biotin 0.02-0.1g / L, VB10.01-0.05g / L, defoamer 0.5-2mL / L, and the rest is water. After the base sugar is exhausted, the flow sugar is added. The concentrations of glucose and lactose in the flow sugar are 55g / L and 35g / L respectively, and the flow sugar addition rate is 450-500ml / h. The culture temperature before the flow sugar addition is 37℃, and the induction temperature after the flow sugar addition is 20-25℃. The fermentation culture conditions are: tank pressure 0.05-0.07Mpa, ventilation volume 5-20L / min, stirring 200-800rpm, pH 6.6-7.2, and the dissolved oxygen is maintained at 40-50% by adjusting the tank pressure, ventilation volume, and stirring in a cycle in sequence, and the fermentation is terminated after culturing for 28 hours. Among them, glucose is 5-10g / L, the flow sugar addition rate is 450-500ml / h, the induction temperature after the flow sugar addition is 20-25°C, and the dissolved oxygen is maintained at 40-50%, which are all beyond the protection scope of the claims of the present invention.

[0052] This comparative example was repeated 3 times, and the OD of the nucleoside phosphorylase solution in the tank was measured at the end of the fermentation. 600 and enzyme activity (U / g), and conversion indices are shown in Table 1.

[0053] Comparative Example 6: The difference between Comparative Example 6 and Example 5 is that the glucose concentration in the stream-added sugar is 90 g / L, and no lactose is added.

[0054] This comparative example was repeated 3 times, and the OD of the nucleoside phosphorylase solution in the tank was measured at the end of the fermentation. 600 and enzyme activity (U / g), and conversion indices are shown in Table 1.

[0055] Comparative Example 7: The difference between Comparative Example 7 and Example 5 is that the lactose concentration in the stream-added sugar is 90 g / L, and no glucose is added.

[0056] This comparative example was repeated 3 times, and the OD of the nucleoside phosphorylase solution in the tank was measured at the end of the fermentation. 600 and enzyme activity (U / g), and conversion indices are shown in Table 1.

[0057] Table 1: OD of the nucleoside phosphorylase solution in Comparative Examples 1-7 and Examples 1-5 600 Summary table of enzyme activity indicators

[0058]

[0059] Comparative Examples 1-5 set critical control points respectively beyond the scope of protection of the claims of the present invention, and investigated enzyme activity and growth OD, and found that enzyme activity index and OD and other fermentation indexes were low. It can be seen that key control points such as basal sugar concentration, flow sugar ratio, sugar addition rate, induction temperature and process dissolved oxygen control have a very important impact on fermentation enzyme activity and fermentation OD and other indicators.

[0060] In Comparative Examples 6-7, by adding glucose or lactose alone, it was found that the enzyme activity index and fermentation indexes such as OD were significantly lower than those in Example 5, indicating that only by adding glucose and lactose simultaneously can a more ideal fermentation enzyme activity and fermentation OD value be obtained.

[0061] The present invention adjusts and optimizes the key control points of the process, and finally determines the optimal key control points as (see Example 5): basal sugar concentration 10-15g / L, glucose 48g / L in the flow sugar addition, lactose concentration 42g / L, sugar addition rate 250-325ml / h, induction temperature 27-32°C, dissolved oxygen control 30-40%, at which time the fermentation enzyme activity is up to 526U / g. The repeatability of the key point optimization process of Examples 1-5 is good, and the fermentation enzyme activity reaches more than 500U / g.

[0062] The E. coli engineering bacteria used in Examples 1-5 and Comparative Examples 1-7 were constructed as follows:

[0063] Step 1: Acquisition of mutant phoC gene

[0064] Error-prone PCR is currently the simplest and most effective in vitro random gene mutagenesis technology. PCR itself has a certain error rate, generally 10 -6 -10 -5 . Use low-fidelity Taq enzyme and add Mg 2+ , Mn 2+ etc. to increase the mutation rate during mutation PCR amplification.

[0065] This step provides a method for obtaining a mutated phoC gene, and the specific steps are as follows:

[0066] The phoC gene of Enterobacter aerogenes (see SEQ ID No.7 for gene sequence and SEQ ID No.8 for corresponding amino acid sequence) was obtained by full gene synthesis and constructed into pUC19 plasmid. Primers phoC-21f / phoC-21r ((SEQ ID No.9 / SEQ ID No.10) were designed with this plasmid as template for error-prone PCR amplification.

[0067] Table 1 Primer sequences involved in the present invention

[0068] Primer name Sequence 5'→3' phoC-21f cagcaaatgggtcgcggatccATGAAAAAGCGCGTTCTCGC SEQ ID No.9 phoC-21r ctcgagtgcggccgcaagcttTTACTTCTGCGTTTTGGCGAA SEQ ID No.10

[0069] The amplification system was as follows: 5 μL of 10× error-prone PCR buffer (100 mmol / L Tris-HCl pH 8.3, 500 mmol / L KCl, 1% Triton, 20 mmol / L MgCl2, 5 μL of 10× dNTP mixture (1 mmol / L dGTP, 1 mmol / L dATP, 5 mmol / L dCTP, 5 mmol / L dTTP) was added to each 50 μL system; 50 pmol of primers phoC-21f and phoC-21r, 10 ng of plasmid DNA template, 5 μL of 5 mmol / L Mn 2+ , 2.5 μL of 15 U / μL Taq DNA polymerase, 5 μL of 25 mmol / L Mg 2+ , add sterilized ultrapure water to a total volume of 50 μL. The error-prone PCR program is: 95℃5min; 94℃1mi, 55℃1min, 72℃1min, 30 cycles; 72℃10min. The first PCR gel recovery product was used as a template for the next round of error-prone PCR, and this was repeated for a total of 5 rounds of error-prone PCR, and finally the phoC gene containing multiple different mutations was obtained.

[0070] Step 2 Construction of phoC mutant library and screening of highly active phosphotransferase mutants

[0071] This step provides a method for screening highly active phosphotransferase mutants, the steps are as follows:

[0072] The pET21a plasmid was extracted and linearized using BamHI / HindIII restriction endonucleases. The linearized vector and the PCR fragments containing various mutations obtained in the first step were assembled according to the instructions of the assembly kit and transformed into the BL21 strain by chemical transformation.

[0073] A large number of mutant PCR fragments were obtained from the first step. After the mutant PCR fragments were successfully assembled with the linearized vector and transformed into the BL21 strain, a total of 20 mutant strains were obtained through resistance and PCR verification screening, numbered 21a-1 to 20, and the 20 strains were induced to express, and 5 strains were screened out with higher enzyme activity than the control, among which the strain with the highest enzyme activity was 3.5 times higher than the control. Figure 1 shown.

[0074] The enzyme activity determination method is as follows: the activity of the phosphotransferase is determined in 1 ml of a standard reaction mixture (containing 100 μM sodium acetate buffer (pH 5.0), 40 μM inosine or guanosine, 100 μM tetrasodium pyrophosphate and enzyme solution). The reaction is incubated at 30°C for 10 minutes, and then 0.2 ml of 2N HCl is added to terminate the reaction. The quantitative determination of inosine, guanosine, 5'-IMP and 5'-GMP is performed by HPLC. One unit of enzyme activity refers to the amount of enzyme that catalyzes the reaction of inosine with sodium tripolyphosphate to produce 1 μmol of inosinic acid within 1 minute at a temperature of 30°C and a pH of 4.0, and is defined as 1U.

[0075] Step 3: Production of 5'-nucleotides using shake flasks

[0076] This step uses the strain with higher enzyme activity obtained in the second step to produce 5'-nucleotides, and the steps are as follows:

[0077] From the five strains with high enzyme activity obtained in the second step, three strains 21a-4, 21a-19, and 21a-20 were selected. The three strains were cultured overnight in LB shake flasks (LB medium formula (g / L): yeast powder 5, peptone 10, NaCl 10, pH 7.2-7.4), and inoculated into shake flask medium containing 30g / L inosine or guanosine and 150g / L disodium pyrophosphate (shake flask medium formula (g / L): glucose 30, yeast extract 12, peptone 18, NaCl 20, NaH2PO4·2H2O8, MgSO4·7H2O 5, (NH4)2SO4 8) at a 10% inoculation amount, and 1mM IPTG was added for induction, and fermentation was induced at 30°C for 18h. After the fermentation was completed, the content of inosinic acid or guanylic acid in the fermentation medium was determined by liquid phase. Results The inosinic acid or guanylic acid content of 21a-4, 21a-19, and 21a-20 was 8-13 times higher than that of the control BL21 (pET21a). Figure 2 .

[0078] The E. coli engineered bacteria used in Examples 1-5 and Comparative Examples 1-7 are strains 21a-20 with the highest enzyme activity, and the amino acid sequence of the phosphotransferase mutant expressed by them is shown in SEQ ID No. 6. The nucleotide sequence of the DNA molecule encoding the phosphotransferase mutant with the amino acid sequence shown in SEQ ID No. 6 is shown in SEQ ID No. 5.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for improving the enzymatic activity of nucleoside phosphorylase, characterized in that: The activated Escherichia coli engineering bacteria are inoculated into a seed culture medium for primary strain culture, and then the primary strain obtained by culture is inoculated into a fermentation culture medium according to a proportion for fermentation culture to prepare a nucleoside phosphorylase enzyme solution; during the fermentation culture process, the culture temperature is 37°C before the flow sugar addition, and when the base sugar is exhausted, the flow sugar addition is performed, the total concentration of glucose and lactose in the flow sugar addition is 90g / L, the flow sugar addition rate is controlled at 225-325ml / h, the induction temperature after the flow sugar addition is 25-32°C, and the dissolved oxygen in the fermentation process is 20-40%.

2. A method for improving the activity of nucleoside phosphorylase according to claim 1, characterized in that: The concentration ratio of the glucose to the lactose in the flow-added sugar is 60g / L:30g / L-30g / L:60g / L.

3. A method for improving the activity of nucleoside phosphorylase according to claim 1, characterized in that: The genetic composition of the engineered Escherichia coli bacteria contains a DNA molecule encoding a phosphotransferase mutant, and the amino acid sequence of the phosphotransferase mutant is shown in SEQ ID No. 2, SEQ ID No. 4 or SEQ ID No.

6.

4. A method for improving the activity of nucleoside phosphorylase according to claim 3, characterized in that: The nucleotide sequence of the DNA molecule is shown as SEQ ID No.1, SEQ ID No.3 or SEQ ID No.

5.

5. A method for improving the activity of nucleoside phosphorylase according to claim 1, characterized in that: The Escherichia coli engineered bacteria expresses a phosphotransferase mutant, and the amino acid sequence of the phosphotransferase mutant is shown in SEQ ID No.2, SEQ ID No.4 or SEQ ID No.6.

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