Method for high-yield fermentation of recombinant proline aminopeptidase and preparation of debittered rice peptide

Abstract

The present disclosure discloses methods for high-yield fermentation of recombinant proline aminopeptidase and preparation of debittered rice peptide, belonging to the fields of fermentation technology, enzyme preparation and food additives. The present disclosure utilizes fermentation kinetic analysis to determine the high-yield fermentation method of proline aminopeptidase by recombinant Bacillus subtilis, and improve the yield of proline aminopeptidase to reach 174.8 U/mL. Proline aminopeptidase cooperates with alkaline protease and leucine aminopeptidase to hydrolyze rice protein. The free amino acid content is 27.3 times the unhydrolyzed free amino acid content, and the small peptide content below 180 Da in hydrolysate reaches 44.70%. The exposed N-terminal proline residue is fully hydrolyzed, and the free proline content is 1,064.3 times that of the unhydrolyzed free proline content, which increases the degree of rice protein hydrolysis. The method of the present disclosure has a good application prospect in the fields of foods and beverages and processing and utilization of food protein resources.

Claims

1. A method for high-yield fermentation production of a proline aminopeptidase using a recombinant Bacillus subtilis, wherein the recombinant Bacillus subtilis comprises a gene encoding the proline aminopeptidase, comprising: (1) inoculating a seed medium with the recombinant Bacillus subtilis and culturing to prepare a seed solution; (2) inoculating a fermentation medium with the seed solution, introducing sterile air at an aeration rate of 1.2 to 1.5 vvm, wherein rotational speed is adjusted as: 200 rpm from 0 to 6 h, 400 rpm from 6 to 12 h, 500 rpm from 12 to 28 h, and 400 rpm from 28 h to the end of fermentation; wherein adjustment of pH is: applying no pH control from 0 to 12 h, setting pH as 7.0 from 12 to 16 h, applying no pH control from 16 to 28 h, and setting pH as 7.0 after 28 h; and wherein temperature is adjusted as: 40° C. from the beginning of fermentation to 8 h, 35° C. from 8 to 12 h, and 33° C. from 12 h to the end of fermentation.

2. The method of claim 1, wherein the step (2) comprises inoculating a 5 L fermenter with the seed solution at an inoculum size of 5%, wherein the 5 L fermenter is filled with a 3 L fermentation medium.

3. The method of claim 1, wherein the seed medium in the step (1) comprises: 10 g/L sodium chloride, 10 g/L tryptone, and 5 g/L yeast powder, and wherein after sterilization, and wherein final concentration reaches 50 μg/mL by adding filter-sterilized kanamycin.

4. The method of claim 1, wherein the fermentation medium in the step (2) comprises 20 g/L glucose, 60 g/L yeast extract, 18.75 g/L fish meal, 3.25 g/L ammonium chloride, 12.54 g/L K.sub.2HPO.sub.4, 2.31 g/L KH.sub.2PO.sub.4, and 0.1% (v/v) phytic acid, wherein the pH is 7.0.

5. The method of claim 1, wherein the step (2) comprises inoculating a 5 L fermenter filled with a 3 L fermentation medium with the seed solution at an inoculum size of 5% and introducing sterile air at an aeration rate of 1.5 vvm, adjusting the pH with 25% ammonia, and using silicone as a defoaming agent.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1: Effect of rotational speed on fermentation of recombinant bacteria.

(2) FIG. 2A-2F: Analysis of fermentation kinetics under different conditions (FIG. 2A: 1: 200 rpm, 2: 300 rpm, 3: 400 rpm, 4: 500 rpm, FIG. 2B: 1: 200 rpm, 2: 300 rpm, 3: 400 rpm, 4: 500 rpm; FIG. 2C: 1: no pH control, 2: pH 7.0, 3: pH 7.5, FIG. 2D: 1: no pH control, 2: pH 7.0, 3: pH 7.5; FIG. 2E: 1: 30° C., 2: 33° C., 3: 35° C., 4: 37° C., 5: 40° C., FIG. 2F: 1: 30° C., 2: 33° C., 3: 35° C., 4: 37° C., 5: 40° C.).

(3) FIG. 3: Determining a fermentation process curve under a fermentation process.

(4) FIG. 4: Hydrophobic amino acid in hydrolysate.

DETAILED DESCRIPTION

(5) Biomaterial sample: the purification and preparation of recombinant Bacillus subtilis (Bacillus subtilis WB600, a recombinant strain of histidine-tagged proline aminopeptidase, and the plasmid used is PMA5) and proline aminopeptidase are described in Chinese patent No. CN105925650A.

(6) Method for determination of proline aminopeptidase activity: using L-proline p-nitroaniline as a substrate (a substrate stock solution is prepared with Tris-HCl 7.5 at a concentration of 4.25 mM), reacting a reaction mixture including 1 mL of enzyme solution as diluted, 2 mL of Tris-HCl 7.5 buffer and 1 mL of substrate stock solution in a water bath at 50° C. for 10 min, adding 1 mL of 50% (v/v) glacial acetic acid solution to terminate the reaction, and measuring the absorbance at 405 nm.

(7) The enzyme activity unit (U) is defined as the amount of enzyme required to decompose L-proline p-nitroaniline to produce 1 μM of p-nitroaniline at 50° C. per minute.

(8) The enzyme activity unit of alkaline protease is defined as the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine per minute at 40° C.

(9) The enzyme activity unit of leucine aminopeptidase is defined as the amount of enzyme required to decompose L-leucine p-nitroaniline to produce 1 μM of p-nitroaniline at 50° C. per minute.

(10) Fermentation tank used: T&J Type A 5 L fermenter (T&J Bio-engineering (Shanghai) Co., LTD)

(11) Detection of polypeptide molecular weight distribution: Waters 600 high performance liquid chromatographic instrument (equipped with a 2487 UV detector and an Empower workstation).

(12) Determination of free amino acids in the hydrolysate: the determination is carried out by using an amino acid chromatographic instrument (Agilent 1100).

(13) Determination of α-glucosidase inhibition rate: preparing 1.5 U/mL α-glucosidase solution with potassium phosphate buffer at pH 6.8, mixing 70 μL of the enzyme solution with 70 μL of the sample, incubating in a water bath at 37° C. for 10 min, and then adding 70 μL of potassium phosphate buffer at pH 6.8 containing 10 mM pNPG, reacting in a water bath at 37° C. for 1 h. After that, 70 μL of 1 M Na.sub.2CO.sub.3 solution is added to terminate the reaction. Absorbance A is measured at 405 nm. The α-glucosidase inhibition rate is calculated as follows:

(14) $\mathrm{Inhibition}\mathrm{rate}=\left(1-\frac{A_i-A_j}{A_0}\right)\times 100\%,$

(15) wherein A.sub.0 is the control, A.sub.i is the absorbance of sample, and A.sub.j is the absorbance of sample control.

Example 1: Fermentation Culture of Recombinant Bacillus subtilis Under Different Rotational Speeds

(16) A seed medium was inoculated with the recombinant Bacillus subtilis preserved with a glycerol tube at −40° C., and cultured by using a rotary constant temperature shaker at a rotational speed of 220 r/min and a culture temperature of 37° C. for 20 h. A 5 L fermenter filled with a 3 L fermentation medium was inoculated with the seed solution at an inoculum size of 5%, and sterile air was introduced at an aeration rate of 1.5 vvm during the fermentation process. The fermentation temperature was 37° C., pH 7.0. The rotational speed was set at 200 rpm, 300 rpm, 400 rpm, 500 rpm respectively, and the fermentation time was 36 h. During the fermentation, samples were taken every 2 h to measure the dry weight of the cells, the activity of extracellular proline aminopeptidase and the activity of intracellular proline aminopeptidase, etc.

(17) As shown in FIG. 1, the rotational speed was increased from 200 rpm to 500 rpm, and the proline aminopeptidase activity was also increased from 38.0 U/mL to 97.5 U/mL.

Example 2: Fermentation Culture Under the Guidance of Fermentation Kinetics

(18) A 5 L fermenter filled with a 3 L fermentation medium was inoculated with the seed solution at an inoculum size of 5%, and sterile air was introduced at an aeration rate of 1.5 vvm during the fermentation process. The specific growth rate (μ.sub.cell) and specific PAP production rate (ρ.sub.PAP) were analyzed based on fermentation kinetics.

(19) As shown in FIGS. 2A and 2B, the μ.sub.cell reached a maximum under an agitation speed of 200 rpm during the first 6 h of fermentation culture; the ρ.sub.PAP reached the highest value when the agitation speed was set at 400 rpm, and the μ.sub.cell was maintained at higher level from 6 h to 12 h; the ρ.sub.PAP reached the maximum at 500 rpm from 12{tilde over ( )}28 h and at 400 rpm after 28 h.

(20) FIGS. 2C and 2D show that the change of μ.sub.cell and ρ.sub.PAP at different pH values. Recombinant B. subtilis had a longer log phase at pH 7.5 that was adverse to cell growth; the μ.sub.cell reached the maximum without pH control during the first 12 h of fermentation; however, ρ.sub.PAP reached the maximum at pH 7.0 from 12{tilde over ( )}14 h; from 14{tilde over ( )}28 h, ρ.sub.PAP reached the maximum without pH control, and pH 7.0 was beneficial for PAP production after 28 h.

(21) FIGS. 2E and 2F show the effect of temperature on μ.sub.cell and ρ.sub.PAP during the fermentation process. The recombinant B. subtilis quickly reached μ.sub.max at 40° C. in the first 8 h of fermentation; the μ.sub.cell reached the maximum and ρ.sub.PAP was maintained at a higher level at 35° C. from 8{tilde over ( )}12 h, and ρ.sub.PAP continuously maintained a higher level at 33° C. after 12 h.

(22) The fermentation conditions are listed as follows: the adjustment of rotational speed was 200 rpm from 0 to 6 h, 400 rpm from 6 to 12 h, 500 rpm from 12 to 28 h, and 400 rpm from 28 h to the end of fermentation; the adjustment of pH was no pH control from 0 to 12 h, pH 7.0 from 12 to 16 h, no pH control from 16 to 28 h, and pH 7.0 after 28 h; the adjustment of temperature was 40° C. from the beginning of fermentation to 8 h, 35° C. from 8 to 12 h, and 33° C. from 12 h to the end of fermentation. During the fermentation, samples were taken every 4 h to measure the dry weight of the cells, the activity of extracellular proline aminopeptidase and the activity of intracellular proline aminopeptidase, etc.

(23) As shown in FIG. 3, the fermentation strategy under kinetic analysis could significantly increase the yield of proline aminopeptidase, and the proline aminopeptidase activity reached 174.8 U/mL.

Example 3 : Role of Proline Aminopeptidase from Recombinant Bacillus subtilis in Rice Protein Hydrolysis

(24) 5% rice protein solution was prepared with pure water buffer and pH was adjusted to 9.0 with a 2M NaOH solution. The mixture was incubated in a water bath at 90° C. for 30 min. After cooling, alkaline protease was added in an amount of 15,000 U/g rice protein, and the reaction was performed at 50° C. for 4 h and in a boiling water bath for 15 min. After cooling, leucine aminopeptidase powder was continually added in an amount of 3,000 U/g rice protein, and the reaction was performed at 50° C. for 2 h and in a boiling water bath for 15 min. After cooling, the enzyme solution of recombinant proline aminopeptidase was then added in an amount of 200 U/g rice protein, the reaction was performed at 50° C. for 2 h and in a boiling water bath for 15 min, and cooled. The hydrolysate was mainly in the form of small peptides and free amino acids. The content of small peptides below 180 Da reached 44.70%, and the exposed N-terminal proline residues were fully hydrolyzed. The free proline content was 0.149 mg/mL, which was 1064.3 times the unhydrolyzed free proline content.

(25) In addition, the inventors also compared the effects of different enzymatic hydrolysis methods on the hydrolysis effect of rice protein, and the results were shown in Table 1 and FIG. 4. The results showed that the content of small peptides below 180 Da was 13.68% when the hydrolysis was carried out by alkaline protease alone, and the content of small peptides below 180 Da was 42.21% when the hydrolysis was carried out by both alkaline protease and leucine aminopeptidase.

(26) TABLE-US-00001 TABLE 1 Effect of different enzymatic hydrolysis methods on the hydrolysis effect of rice protein Molecular Mass ratio of small peptide having weight respective molecular weight (%) (Da) 1 2 3 4 >5000 — 0.38 2.95 2.33 5000-3000 1.4 1.00 1.34 1.41 3000-2000 2.96 1.86 1.65 1.77 2000-1000 11.61 7.32 5.08 5.25 1000-500  26.16 18.26 11.06 10.92 500-180 48.85 57.50 35.70 33.63  <180 9.03 13.68 42.21 44.70 Note: 1: rice protein solution, 2: hydrolysate obtained by alkaline protease alone, 3: hydrolysate obtained by both alkaline protease and leucine aminopeptidase, 4: hydrolysate obtained cooperatively by synergy of proline aminopeptidase, alkaline protease and leucine aminopeptidase.

(27) In addition, the free amino acid content obtained by the method of the present disclosure was 3.484 mg/mL, which was 27.4 times the unhydrolyzed free amino acid content, wherein the proline content was 1,064.3 times the unhydrolyzed proline content, and the hydrophobic amino acid content was significantly improved. When the hydrolysis was carried out by alkaline protease alone, the free amino acid content was 1.121 mg/mL. When the hydrolysis was carried out by both the alkaline protease and leucine aminopeptidase, the free amino acid content was 3.567 mg/mL, which was basically balanced with the free amino acid content when the hydrolysis was cooperatively carried out by three enzymes; however, the hydrolysate content was 0.083 mg/mL, which was significantly lower the free proline content when the hydrolysis was cooperatively carried out by three enzymes.

(28) According to the present disclosure, the rice protein was cooperatively hydrolyzed by proline aminopeptidase, alkaline protease and leucine aminopeptidase. In the obtained hydrolysate, the content of free amino acid and the content of small peptide were greatly increased, the content of hydrophobic amino acid was also significantly improved, and not only was the degree of hydrolysis of rice protein greatly improved, but also the bitterness of the hydrolysate was removed or reduced.

Example 4: Method for Hydrolyzing Rice Protein

(29) The method for hydrolyzing rice protein of the present disclosure comprised preparing a rice protein solution having a mass concentration of 8%,

(30) (1) adding an appropriate amount of alkaline protease, reacting at 40° C. for 3.5 h and in a boiling water bath for 10 min;

(31) (2) after cooling, continually adding an appropriate amount of leucine aminopeptidase, reacting at 45° C. for 3 h and in a boiling water bath for 10 min;

(32) (3) after cooling, adding an appropriate amount of proline aminopeptidase, reacting at 45° C. for 3 h and in a boiling water bath for 10 min,

(33) wherein the additive amount of alkaline protease was 20,000 U/g rice protein; the additive amount of leucine aminopeptidase was 4,000 U/g rice protein; and the additive amount of proline aminopeptidase was 100 U/g rice protein.

(34) The polypeptide more than 2,000 Da in the obtained rice protein hydrolysate was basically absent, while the small peptide content below 180 Da reached 48.26%, the free amino acid content was 5.852 mg/mL, and the free proline content is 1,136.4 times the unhydrolyzed free proline content.

Example 5: Method for Separating and Purifying Active α-Glucosidase Inhibitory Peptide

(35) 50 mL of rice protein hydrolysate cooperatively hydrolyzed by alkaline protease, leucine aminopeptidase and proline aminopeptidase was centrifuged at 10,000 rpm for 15 min, and the supernatant was taken and freeze-dried. The freeze-dried powder was dissolved in 20 mM pH 7.5 PB buffer, and preliminary separation was performed by Hi Trip DEAE FF anion exchange chromatography. The loading volume was 10 mL, the flow rate was 1 mL/min, and the stepwise elution was performed with 150 mM, 350 mM, and 550 mM NaCl solution. 10 column volumes were eluted at each stage, and the eluate was collected, fully dialyzed, and concentrated by freeze-drying. The freeze-dried powder was rehydrated, and the α-glucosidase inhibition rate and the polypeptide concentration of each elution peak were determined. The elution peak F.sub.4 fraction was found to have the highest inhibition rate. The fraction was subjected to Sephadex G-15 gel chromatography, the loading volume was 1 mL, and the flow rate was 0.5 mL/min. Collection is conducted by each peak and the eluted peaks were freeze-dried. The freeze-dried powder was dissolved in 1 mL of pure water, and the α-glucosidase inhibition rate and the polypeptide concentration were determined. The F.sub.4 b fraction having the most potent α-glucosidase inhibitory effect was obtained, which had an IC.sub.50 of 132.6 μg/mL.

(36) Although the present disclosure has been disclosed in the above preferred embodiments, the present disclosure is not limited thereto, and various modifications and changes can be made without departing from the spirit and scope of the disclosure by anyone familiar with this technology. Therefore, the protection scope of the present disclosure should be determined by the claims.