Fermentation medium for producing marine low-temperature (+) gamma-lactamase strain

Abstract

The invention belongs to the technical field of bioengineering, and discloses a fermentation medium for producing marine low-temperature (+) gamma-lactamase strains. The culture medium is prepared from yeast extract, magnesium sulfate, soluble starch, KH2PO4, Na2HPO4, CaCl2, FeSO4 and NH4Cl. By adopting the fermentation culture medium, the enzyme activity of (+) gamma-lactamase can be remarkably improved, the resolution efficiency of (+ / -) gamma-lactam is improved, the optical purity of a product is improved, and the enzyme activity is improved by 2.28 times compared with that before the culture medium is adopted.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and relates to a fermentation medium for a strain producing marine low-temperature (+) γ-lactamase. Background Art

[0002] Since optically pure (+), (+) γ-lactam plays an important role in biomedicine, and there are drawbacks in directly using the racemate for drug production, and the biological method for resolving racemic γ-lactam has advantages such as high catalytic efficiency, mild conditions, and environmental friendliness, the research on γ-lactamase has become a hot issue.

[0003] Since the 1980s and 1990s when γ-lactamase first appeared in the academic community, after decades of development, enzymes with excellent reaction performance have already been applied industrially, but they cannot meet the industrial production requirements. In order to utilize natural enzymes with low catalytic performance, poor thermal stability, and low stereoselectivity, researchers have adopted various methods in an attempt to improve various aspects of the performance of natural enzyme molecules.

[0004] It has been found in research that the composition of the culture medium plays a crucial role in product yield, quality, and production cost. Changing the different combinations of carbon sources and nitrogen sources can maximize the growth rate of microorganisms and the yield of metabolites, and ultimately improve the quality and productivity of the product. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, the present invention provides a fermentation medium for a strain producing marine low-temperature (+) γ-lactamase. Using this fermentation medium can significantly enhance the enzyme activity of (+) γ-lactamase, improve the resolution efficiency of (±) γ-lactam, and enhance the optical purity of the product. The enzyme activity is increased by 2.28 times compared with that before the medium.

[0006] The above object of the present invention is achieved by the following technical solutions:

[0007] A fermentation medium for a strain producing marine low-temperature (+) γ-lactamase, the medium comprising yeast extract, magnesium sulfate, soluble starch, KH2PO4, Na2HPO4, CaCl2, FeSO4, NH4Cl.

[0008] Further, the specific medium formula is: by mass concentration: yeast extract 15%, magnesium sulfate 0.6%, soluble starch 30%, KH2PO4 4%, Na2HPO4 1.5%, CaCl2 0.02%, FeSO4 0.015%, NH4Cl 15%.

[0009] The specific preparation method of the above fermentation medium is:

[0010] In order to utilize natural enzymes with low catalytic performance, poor thermal stability, and low stereoselectivity and improve their enzyme activity, the strain producing marine low-temperature (+)-γ-lactamase is Bacillus thuringiensis LNH-5-3B producing (+)-γ-lactamase, which is derived from deep-sea mud.

[0011] The present invention also claims the application of the above fermentation medium in the fermentation of Bacillus thuringiensis LNH-5-3B.

[0012] The beneficial effects of the present invention compared with the prior art are as follows:

[0013] The present invention provides a fermentation medium for a strain producing marine low-temperature (+)-γ-lactamase. After fermentation with this fermentation medium, the enzyme activity is increased by 2.28 times compared with that before using the medium. Compared with the chemical method, the biological method has the advantages of high stereoselectivity, high resolution efficiency, easy purification of products, mild reaction conditions, and little environmental pollution. It is beneficial to fill the domestic technical gap in the production of optically pure γ-lactam, relieve the economic pressure, and save the production cost, and has great research value. Description of the Drawings

[0014] Figure 1 It is the high-performance liquid chromatography diagram of the substrate (±)-γ-lactam in the present invention. In the figure: 1—epi-benzamide; 2—epi-(+)-γ-lactam; 3—epi-(-)-γ-lactam.

[0015] Figure 2 It is the contour line and response surface diagram of yeast extract and magnesium sulfate in the present invention. Figure a is the response surface diagram, and Figure b is the contour line diagram.

[0016] Figure 3 It is the contour line and response surface diagram of yeast extract and ammonium chloride in the present invention. Figure a is the response surface diagram, and Figure b is the contour line diagram.

[0017] Figure 4 It is the contour line and response surface diagram of magnesium sulfate and ammonium chloride in the present invention. Figure a is the response surface diagram, and Figure b is the contour line diagram. Detailed Embodiments

[0018] The present invention will be described in detail below through specific examples, but the protection scope of the present invention is not limited. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0019] Method for Measuring Enzyme Activity

[0020] Definition of enzyme activity: Under the condition of 28°C, the amount of enzyme required to hydrolyze 1 μmol of (±)-γ-lactam per minute is defined as one enzyme activity unit.

[0021] Method for measuring enzyme activity: High performance liquid chromatography was used to measure the enzyme activity. Shimadzu LC-20A chromatograph from Japan, and the chromatographic column was the CHIRALPAK AS-H 250×4.6mm chiral chromatographic column from Daicel Chemical Industries, Ltd., Japan; the volume ratio of isopropanol to acetonitrile was 10:90; the flow rate was 0.6 mL / min; the wavelength was 230 nm; the internal standard was benzamide; the injection volume was 20 μL.

[0022] The enzyme activity was calculated by the internal standard method, and the internal standard substance was benzamide. Determination of the standard curve: Add 0.5 mL of 1 g / L benzamide standard solution to 0.5 mL of (±)γ-lactam solutions with different concentrations (0.1 g / L, 0.2 g / L, 0.5 g / L, 1 g / L, 2 g / L, 5 g / L, 10 g / L). Both the (±)γ-lactam solution and the benzamide standard solution were prepared with 0.05 mol / L sodium phosphate buffer solution at pH 7.0. HPLC was used to measure the peak areas of (±)γ-lactam and benzamide at different concentrations. The ratio of the peak area of γ-lactam to the peak area of benzamide was used as the abscissa, and the concentration of (±)γ-lactam was used as the ordinate to plot the standard curve.

[0023] Enzyme activity determination: Take 5 mL of the fermentation broth, centrifuge it at 8000 r / min for 10 min to obtain wet bacteria, wash the wet bacteria with 0.05 mol / L phosphate buffer solution at pH 7.0 until the fermentation broth is completely washed. Suspend the washed bacteria in 5 mL of (±)γ-lactam phosphate buffer solution with a mass concentration of 10 g / L, react at 30 °C and 160 r / min for 30 min, then centrifuge at 8000 r / min for 10 min. Take 0.5 mL of the supernatant and add 0.5 mL of 1 g / L benzamide solution, mix well. Take 0.75 mL of the above mixture and extract it with an equal volume of n-butanol. Then filter it through a 0.22 μm organic filter membrane to obtain the sample for injection. Do 3 groups of replicates. Analyze and detect the sample by chiral HPLC, and calculate the enzyme activity of 1 L of the fermentation broth.

[0024] The equation for calculating enzyme activity is:

[0025] U / L = (CA - CB) ÷ (30 × 5)

[0026] In the formula, CA——the molar concentration of (±)γ-lactam before the reaction, CB——the molar concentration of (±)γ-lactam after the reaction

[0027] Seed culture conditions

[0028] Under aseptic conditions, take 1 mL of the bacterial liquid in the glycerol tube and inoculate it into the seed medium. Incubate it in a shaker at 30 °C and 160 r / min for 40 h to obtain the seed. Then transfer 5% of the above seed into the seed medium and culture it at 30 °C and 160 r / min for 40 h to obtain the seed liquid.

[0029] The percentages in the examples are mass concentration percentages.

[0030] Example 1

[0031] Determine the components and contents of the culture medium

[0032] (1) Influence of carbon source on enzyme production

[0033] To develop the best carbon source for Bacillus thuringiensis LNH-5-3B, experiments were conducted using soluble starch, glucose, lactose, sucrose, xylose, and fructose at a concentration of 0.5% as the sole carbon source respectively. Cultivate for 48 h under the conditions of an inoculation amount of 5%, 30 °C, and 160 r / min. Determine the enzyme activity according to the enzyme activity determination method, and calculate the product e.e. value and conversion rate. Determine the best carbon source based on the experimental results.

[0034] (2) Determination of the optimal concentration of the carbon source

[0035] Using soluble starch as the sole carbon source, change the concentration of soluble starch in the culture medium (0.1%, 0.5%, 1%, 1.5%, 2.0%, 2.5%, 3.0%), and cultivate for 48 h under the conditions of an inoculation amount of 5%, 30 °C, and 160 r / min, then measure the enzyme activity.

[0036] (3) Influence of nitrogen source on enzyme production

[0037] Use yeast extract, beef extract, peptone, yeast extract powder, corn steep liquor paste, urea, ammonium acetate, potassium nitrate, diammonium hydrogen phosphate, and ammonium chloride as nitrogen sources respectively, and control the nitrogen source concentration at 5% for single-factor experiments. Cultivate for 48 h under the conditions of an inoculation amount of 5%, 30 °C, and 160 r / min, then measure the enzyme activity, and preliminarily determine the best nitrogen source according to the experimental results.

[0038] (4) Determination of the optimal concentration of the organic nitrogen source

[0039] The above experiment determined that yeast extract is the best organic nitrogen source. To further determine the concentration of yeast extract, set the concentrations of yeast extract to 0.5%, 1%, 1.5%, 2%, 2.5%, and 3% respectively, and cultivate for 48 h under the conditions of an inoculation amount of 5%, 30 °C, and 160 r / min, then measure the enzyme activity.

[0040] (5) Determination of the optimal concentration of the inorganic nitrogen source

[0041] The above experiment determined that ammonium chloride is the best inorganic nitrogen source. To determine the best concentration of ammonium chloride, set the concentrations of ammonium chloride to 0.5%, 1%, 1.5%, 2%, 2.5%, and 3% respectively, and cultivate for 48 h under the conditions of an inoculation amount of 5%, 30 °C, and 160 r / min, then measure the enzyme activity.

[0042] (6) Determination of the optimal concentration of inorganic salts

[0043] Under the condition of determining the optimal nitrogen source, carbon source and their concentrations, the concentrations of KH2PO4 (0.10%, 0.2%, 0.3%, 0.40%, 0.5%), Na2HPO4 (0.05%, 0.1%, 0.15%, 0.2%, 0.25%), MgSO4 (0.01%, 0.02%, 0.03%, 0.04%, 0.05%), CaCl2 (0.001%, 0.002%, 0.003%, 0.004%, 0.005%), and ferrous sulfate (0.0005%, 0.001%, 0.0015%, 0.002%, 0.0025%) in the medium were changed. The culture was carried out at an inoculum size of 5%, 30 °C, and 160 r / min for 48 h, and the enzyme activity was measured.

[0044] Determination of the most significant factors in the medium components by Plackett - Burman experiment

[0045] Based on the results of the single - factor experiment, taking 8 components in the fermentation medium as the investigation factors and the enzyme activity of (+)-γ - lactamase produced by Bacillus thuringiensis LNH - 5 - 3B as the response value, a P - B experiment was carried out, and each group of experiments was repeated 3 times. The factors and levels of the P - B experiment design are shown in Table 1.

[0046] Table 1 Design of factors and levels for Plackett - Burman test

[0047]

[0048] Through the P - B experiment, it was found that yeast extract, ammonium chloride, and magnesium sulfate had the most significant effects on enzyme production.

[0049] Determination of the optimal response region by the steepest ascent experiment

[0050] According to the results of the P - B experiment, since yeast extract, ammonium chloride, and magnesium sulfate had the most significant effects on the enzyme activity of (+)-γ - lactamase, a steepest ascent experiment was carried out with these three factors to determine the optimal response region, and 3 groups of repeated experiments were set, and their average value was taken as the final value. The experimental design and results of the steepest ascent experiment are shown in Table 2.

[0051] Table 2 Experimental design and results of the steepest ascent experiment

[0052]

[0053] Optimal medium formula

[0054] According to the results of the steepest ascent experiment, the central group of the Box-Behnken experiment was determined as 2.5% yeast extract, 0.03% magnesium sulfate, and 3% soluble starch. The Box-Behnken experiment was designed using software (Design-Expert). Each group of experiments was repeated 3 times, and the average value was taken as the final value. The factors and levels of the response surface test are shown in Table 3, and the analysis of variance is shown in Table 4.

[0055] Table 3 Factors and levels of the response surface test

[0056]

[0057] Table 4 Analysis of variance of the Box-Behnken experiment

[0058]

[0059]

[0060] "*" indicates a significant effect on the result (P < 0.05), and "**" indicates a highly significant effect on the result (P < 0.01)

[0061] Using Design-Expert software to analyze the results of the Box-Behnken experiment (Table 4), the regression equation of the (+)γ-lactamase produced by Bacillus thuringiensis LNH-5-3B was obtained: Enzyme activity = 290.1 - 9.11A - 1.02B - 27.51C - 17.08AB + 1.36AC + 2.92BC - 156.49A 2 - 41.12B 2 - 124.16C 2 As can be seen from Table 4, the fitting model was highly significant (P value < 0.01), the lack-of-fit term was not significant (P value = 0.48), and R2 = 0.9946, greater than 0.9, indicating that the experimental data was reliable and conformed to statistical laws, and the model could be used. Among them, the P values of A, C, A 2 , C 2 were all less than 0.01, indicating that these terms had a highly significant effect on the (+)γ-lactamase produced by Bacillus thuringiensis LNH-5-3B; the P values of B, B 2 terms were less than 0.05, indicating that these terms had a significant effect on enzyme production.

[0062] According to the regression equation model, contour plots and response surface plots were drawn using Design-Exper software ( Figure 2 , Figure 3 , Figure 4 ) to observe the interaction relationship between the three factors. In the contour plot, the horseshoe-shaped and elliptical contour lines both indicate a significant interaction between the two factors, and the circular contour line indicates no significant interaction. For exampleFigure 2 As shown, the interaction between yeast extract and magnesium sulfate is significant. When the content of magnesium sulfate is fixed, the enzyme activity of (+)γ-lactamase increases with the increase of the yeast extract content. When the addition amount of yeast extract is 1.487%, the enzyme activity reaches the maximum. According to Figure 3 As shown, the two-way interaction between yeast extract and soluble starch is not significant. When the content of yeast extract is fixed, the enzyme activity increases with the increase of the addition amount of soluble starch. When the content of ammonium chloride is 1.789%, the enzyme activity reaches the maximum. As Figure 4 As shown, the interaction between magnesium sulfate and soluble starch is significant. When the content of soluble starch is fixed, the enzyme activity first increases and then decreases with the increase of the magnesium sulfate content. When the addition amount of magnesium sulfate is 0.06%, the enzyme activity reaches the maximum value. Using Design-Expert software to calculate, the optimal fermentation medium formula for Bacillus thuringiensis LNH-5-3B is: yeast extract 1.487%, magnesium sulfate 0.06%, ammonium chloride 1.789%, and the remaining components are the same as those in the single-factor experiment. The optimal medium formula is determined as yeast extract 1.5%, magnesium sulfate 0.06%, soluble starch 3%, KH2PO4 0.4%, Na2HPO4 0.15%, CaCl2 0.002%, FeSO4 0.0015%, NH4Cl 1.5%.

[0063] The above-described embodiments are only the preferred embodiments of the present invention, and not all the feasible embodiments of the present invention. For those of ordinary skill in the art, any obvious changes made without departing from the principle and spirit of the present invention should be considered to be included within the protection scope of the claims of the present invention.

Claims

1. A fermentation medium for producing a marine low-temperature (+)-γ-lactamase strain, characterized in that the medium It includes yeast extract, magnesium sulfate, soluble starch, KH2PO4, Na2HPO4, CaCl2, FeSO4, and NH4Cl.

2. The fermentation medium for producing a marine low-temperature (+)-γ-lactamase strain according to claim 1, characterized in that, The specific medium formulation is as follows: by mass concentration: yeast extract 1.5%, magnesium sulfate 0.06%, soluble starch 3%, KH2PO4 0.4%, Na2HPO4 0.15%, CaCl2 0.002%, FeSO4 0.0015%, and NH4Cl 1.5%.

3. The preparation method of a fermentation medium for producing a marine low-temperature (+)-γ-lactamase strain as described in claim 2, characterized in that, Take yeast extract, magnesium sulfate, soluble starch, KH2PO4, Na2HPO4, CaCl2, FeSO4, and NH4Cl in proportion, make up the volume with distilled water, and adjust the pH to 7.

0.

4. Application of a fermentation medium for a marine low-temperature (+)-γ-lactamase-producing strain according to any one of claims 1-2 in the fermentation of Bacillus thuringiensis LNH-5-3B.

Images