A two-stage catalytic process for the production of galactonic acid and lactonic acid using whey as a substrate

Abstract

This invention discloses a two-stage catalytic method for the production of galactobionic acid and lactobionic acid using whey as a substrate. The method utilizes whey as the substrate and engineered *E. coli* expressing β-galactosidase and engineered *Pseudomonas putida* expressing a PQQ-dependent membrane-bound dehydrogenase of the gluconeo-shikimic acid family as catalysts. First, the contents of engineered *E. coli* cells, after ultrasonic disruption and heat treatment, hydrolyze whey into glucose and galactose. Then, engineered *P. putida* is inoculated into the whey hydrolysate. This strain uses glucose as a carbon source for cell proliferation and simultaneously oxidizes all galactose to galactobionic acid, achieving a 100% galactobionic acid yield. The proliferated *P. putida* cells are recovered and inoculated into a high-concentration whey solution for highly efficient catalytic production of lactobionic acid, also achieving a 100% lactobionic acid yield. In this invention, whey is used not only for cell proliferation but also for product production, offering advantages such as low production cost, simple process, high substrate utilization, and high product yield.

Description

Technical Field

[0001] This invention relates to the field of biocatalytic conversion, specifically to a two-stage catalytic method for the production of galactobionic acid and lactobionic acid using whey as a substrate. Background Technology

[0002] Whey is a byproduct of dairy production; nine kilograms of whey are produced for every kilogram of cheese produced. As demand for dairy products increases, whey production also increases. Because whey has high biological and chemical oxygen demand (BOD), it pollutes the environment whether discharged directly or after dilution. Whey powder, as an inexpensive industrial waste, can be used to produce high-value products.

[0003] Whey powder is 70% lactose, and while the market demand for lactose is relatively limited, it can be converted into more valuable derivatives through chemical, physical, or enzymatic methods. For example, the β-1,4-glycosidic bond of lactose can be hydrolyzed to yield glucose and galactose, both of which have wide applications. The selective oxidation of the anomeric hydroxyl group of galactose yields galactobionic acid, which can be used in the development of sweeteners, pharmaceutical intermediates, and dispersants. The selective oxidation of the anomeric hydroxyl group of lactose yields lactobionic acid, which can be used as a moisturizer in cosmetics, an additive in the food industry, and a drug carrier in the pharmaceutical industry.

[0004] Galactobionic acid and lactobionic acid both belong to the aldonic acid family and are mainly synthesized through chemical methods. There are few reports on the biological production of aldonic acids; most strains can only oxidize them to produce galactobionic acid or lactobionic acid. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to address the shortcomings of the prior art. The present invention provides a two-stage catalytic method for the production of galactobionic acid and lactobionic acid using whey as a substrate, and the yields of both galactobionic acid and lactobionic acid can reach 100%.

[0006] To address the aforementioned technical problems, this invention discloses a two-stage catalytic method for the production of galactobionic acid and lactobionic acid using whey as a substrate, comprising the following steps:

[0007] (1) Culture engineered Escherichia coli expressing β-galactosidase to obtain bacterial cells, wherein the nucleotide sequence of the β-galactosidase is shown in SEQ ID NO.1;

[0008] (2) The bacterial cells obtained in step (1) by ultrasonic disruption are centrifuged and the supernatant is taken to obtain a crude enzyme solution containing β-galactosidase. The crude enzyme solution is subjected to heat denaturation treatment, centrifuged to remove the heat denatured protein, and the supernatant is removed to obtain the processed cell contents.

[0009] (3) Using lactose or whey as a substrate, the substrate is completely hydrolyzed using the cell contents obtained in step (2) to prepare a hydrolysate containing glucose and galactose.

[0010] (4) The engineered *Pseudomonas putida* expressing the PQQ-dependent membrane-bound dehydrogenase of the gluconeo-shikimic acid family was inoculated into the hydrolysate containing glucose and galactose obtained in step (3). The engineered *Pseudomonas putida* used glucose in the hydrolysate as a carbon source for cell proliferation and oxidized all galactose to galacturonic acid. The nucleotide sequence of the PQQ-dependent membrane-bound dehydrogenase of the gluconeo-shikimic acid family is shown in SEQ ID NO.2.

[0011] (5) The engineered Pseudomonas malodorans from step (4) is recovered and inoculated into the whey solution to oxidize all lactose into lactobionic acid.

[0012] In step (1), the engineered E. coli is constructed as follows: the nucleotide sequence of the β-galactosidase is ligated into the pETDuet-1 vector to obtain a recombinant plasmid, which is then transformed into E. coli BL21(DE3). The culture and expression method is as follows: the engineered E. coli is first cultured in LB liquid medium containing ampicillin, then IPTG is added to induce expression, followed by shaking culture. After induction, the cell pellet is collected by centrifugation, washed with physiological saline, and then resuspended in phosphate buffer for later use. In a preferred embodiment, the engineered E. coli can be constructed according to patent CN 201410390773.5.

[0013] In step (2), the conditions for ultrasonic disruption are: ultrasonic power of 300-500W, ultrasonic time of 15-30min, and after ultrasonic disruption, the disrupted liquid is centrifuged and the supernatant is collected.

[0014] In step (2), the heat denaturation treatment is to keep warm in a water bath at 50-65°C for 0.5-2 hours.

[0015] In step (3), the concentration of lactose or whey is 10 g / L to 100 g / L, and cell contents are added to the hydrolysis system to make the β-galactosidase activity 0.1 to 5 U / mL. The hydrolysis system also contains 50 to 100 mg / L of kanamycin sulfate.

[0016] Preferably, enzymatic hydrolysis is performed at 45–60°C and 50–300 rpm.

[0017] In step (4), the starting strain of the engineered Pseudomonas malodoris is Pseudomonas malodoris ATCC No. 47054.

[0018] In step (4), *Pseudomonas malodorosa* is introduced to make the initial OD of the bacteria 0.5-2.

[0019] In step (4), the reaction conditions are as follows: the reaction pH is controlled at 5.0 to 8.0, the air flow rate is 0.5 to 1.5 vvm, the stirring speed is 200 to 400 rpm, and the temperature is 27 to 35℃.

[0020] In step (5), the engineered Pseudomonas malodorans obtained in step (4) is added to make the bacterial OD 5-10, the reaction pH is controlled to be 5.0-8.0, the dissolved oxygen concentration of the reaction system is not less than 1.5 mg / L, and the lactose or whey concentration is 150 g / L-350 g / L.

[0021] Beneficial effects: Compared with the prior art, the advantages of the present invention are:

[0022] (1) The present invention does not require expensive culture medium for cell culture, nor does it require expensive catalytic substrates. Whey (including its hydrolysis products) is used as both a culture medium component and a catalytic substrate, which greatly reduces the reaction cost.

[0023] (2) Using the strain provided by the present invention, under the conditions given by the present invention, the yields of galactobionic acid and lactobionic acid can both reach 100%.

[0024] (3) In the biocatalytic method described in this invention, whey is used not only for cell proliferation but also for product production, and has the advantages of low production cost, simple process, high substrate utilization rate and high product yield. Attached Figure Description

[0025] Figure 1 This is a graph showing the results of galactobionic acid production in Example 3;

[0026] Figure 2 This is a diagram showing the results of lactobionic acid production in Example 4. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments. The embodiments will help to understand the present invention, but the scope of protection of the present invention is not limited to the following embodiments.

[0028] In the following embodiments, the galactobionic acid yield and lactobionic acid yield are calculated according to the following formulas:

[0029] Galacturonic acid yield (%) = Galacturonic acid concentration obtained from the reaction (mM) * 100 / Initial galactose concentration (mM)

[0030] Lactobionic acid yield (%) = Lactobionic acid concentration obtained from the reaction (mM) * 100 / Initial lactose concentration (mM)

[0031] β-galactosidase activity is defined as the amount of enzyme required per minute to hydrolyze and produce 1 micromole of glucose, which is 1 unit of activity (U).

[0032] Example 1: Preparation of whey hydrolysate.

[0033] The nucleotide sequence of β-galactosidase, synthesized by General Biosystems (Anhui) Co., Ltd., as shown in SEQ ID NO.1, was ligated into the pETDuet-1 vector to obtain a recombinant plasmid.

[0034] Following conventional molecular biology methods, the recombinant plasmid was transformed into E. coli BL21(DE3) and named E. coli BL21(pETDuet-bgl).

[0035] Escherichia coli BL21 (pETDuet-bgl) was inoculated into 10 mL of LB broth containing 100 μg / mL ampicillin and cultured at 37°C with shaking for 12 h. Then, it was transferred to 1 L of LB broth containing 100 μg / mL ampicillin and cultured at 37°C with shaking until the cells reached OD500. 600 When the β-galactosidase activity reached 0.6–0.8, IPTG was added to a final concentration of 0.5 mM to induce expression, and the cells were cultured at 37°C with shaking for 8 hours. After induction, the cells were centrifuged at 10,000 rpm for 5 minutes, and the cell pellet was collected. The pellet was washed twice with physiological saline, and the cell pellet was resuspended in 50 mM phosphate buffer at pH 6.5, and the cell OD was adjusted to 20. The cells were disrupted by sonication for 20 minutes, and the disruption buffer was centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected, incubated in a 50°C water bath for 2 hours, and then centrifuged at 8,000 rpm for 10 minutes to remove heat-denatured proteins, obtaining the treated cell contents. The β-galactosidase activity of the cells was then measured.

[0036] Whey powder was dissolved in phosphate buffer (pH 6.5) to a concentration of 70 g / L, at which point the lactose concentration was 47 g / L. Cell contents obtained from the previous treatment were added to achieve a β-galactosidase activity of 1 U / mL, and kanamycin sulfate was added to a concentration of 70 mg / L. The total reaction volume was 1 L, and enzymatic hydrolysis was performed at 50 °C and 150 rpm. After 7 hours, lactose was essentially completely hydrolyzed, and the glucose concentration was measured to be 24.2 g / L and the galactose concentration to be 24.0 g / L.

[0037] Example 2: Construction of *Pseudomonas engineerii*.

[0038] Genomic DNA was extracted from *Pseudomonas fragi* NL20W (this strain is deposited at the China Center for Type Culture Collection, March 17, 2021, accession number CCTCC NO: M 2021245) using a Promega genome extraction kit. The target gene was amplified from the genomic DNA using synthetic primers, and its nucleotide sequence is shown in SEQ ID NO. 2.

[0039] Following conventional molecular biology methods, using The Seamless Cloning and Assembly Kit (purchased from TransGen) was used to clone the target gene obtained by PCR amplification into the pBBR1MCS-2 plasmid (purchased from Addgene).

[0040] The specific steps are as follows:

[0041] (1) Design two pairs of primers, one for amplifying the target gene and the other for amplifying the plasmid;

[0042] (2) The primers for amplifying the target gene are:

[0043] Upstream primer: 5'-TTACTCAGCCAGTTTGAACG-3'

[0044] Downstream primer: 5'-ATGAGCACTGAAGGTGCTTT-3'

[0045] (3) The primers for amplifying the plasmid are:

[0046] Upstream primer: 5'-AAAGCACCTTCAGTGCTCATAGCTGTTTCCTGTGTGAAAT-3'

[0047] Downstream primer: 5'-CGTTCAAACTGGCTGAGTAAGCGTTAATATTTTGTTAAAA-3'

[0048] (4) The PCR product was purified using a purification kit, and then... The Seamless Cloning and Assembly Kit (Beijing TransGen Biotechnology Co., Ltd.) instruction manual connects the target fragment and plasmid.

[0049] (5) The obtained recombinant plasmid was electroporated into competent cells of *Pseudomonas putida* KT2440 (ATCC No. 47054). The methods for preparing competent cells and electroporation are described in patent CN113073072A. After electroporation, the bacterial culture in the electroporation vessel was transferred to a centrifuge tube and cultured on a shaker at 30°C for 1 hour to resuscitate the cells. After resuscitation, the cells were screened on LB agar plates containing kanamycin resistance. The growing colonies were picked and inoculated into LB liquid medium containing kanamycin resistance, cultured at 30°C until mid-logarithmic growth, and the cells were collected and stored in a low-temperature freezer for later use. This strain is the engineered *Pseudomonas putida* of this invention, named *P. putida* KT2440[pBB-GDH1].

[0050] Example 3: Production of galactobionic acid.

[0051] P. putida KT2440 [pBB-GDH1] was activated with LB medium and then transferred to whey hydrolysate medium to achieve an initial OD of 1. The whey hydrolysate medium was the same as that used in Example 1, without any added carbon, nitrogen, or inorganic salts. The experiment was conducted in a 3L bioreactor containing 1L of whey hydrolysate. The pH was maintained at approximately 6.5 by adding NaOH solution. The aeration rate was 1 vvm, the stirring speed was 350 rpm, and the temperature was 30°C.

[0052] After approximately 30 hours of reaction, both glucose and galactose were completely consumed, at which point the reaction was stopped. The results are as follows: Figure 1 As shown. The reaction yielded bacterial cells and galacturonic acid (salt) product solution. The bacterial cell OD reached 6.8, the galacturonic acid concentration reached 26.1 g / L, the utilization rate of glucose and galactose reached 100%, and the yield of galacturonic acid was close to 100%.

[0053] Example 4: Production of lactobionic acid.

[0054] The bacterial cells from Example 3 were collected by centrifugation, washed with physiological saline, and collected again. The recovered bacterial cells were added to 1 L of a solution containing 300 g / L whey powder for further catalysis, at which point the lactose concentration was 201 g / L. This was carried out in a 3 L bioreactor. The pH was maintained at approximately 6.5 by adding NaOH solution, and air was introduced to ensure the dissolved oxygen concentration in the reaction system was not less than 1.5 mg / L. After 80 hours of reaction, the lactose reaction was complete, at which point the reaction was stopped, yielding lactobionic acid (salt) product. The lactobionic acid concentration reached 209 g / L, the lactose utilization rate reached 100%, and the yield of lactobionic acid was close to 100%.

[0055] Comparative Example 1

[0056] Whey powder was dissolved in phosphate buffer (pH 6.5) to a concentration of 70 g / L, at which point the lactose concentration was 47 g / L. Commercially available β-galactosidase (Sigma, catalog number G3665, manufactured by Novozymes) was added to achieve an activity of 1 U / mL, and kanamycin sulfate was added to a concentration of 70 mg / L. The total reaction volume was 1 L, and enzymatic hydrolysis was performed at 35°C and 150 rpm. After 7 hours, approximately 62% of the lactose was hydrolyzed.

[0057] Comparative Example 2

[0058] The technical solution is the same as that of Comparative Example 1, but the enzymatic hydrolysis temperature is set to 50°C, which is consistent with the enzymatic hydrolysis technology in Example 1. Since the commercially available β-galactosidase comes from mesophilic bacteria, it quickly loses its activity under the reaction conditions of 50°C. After 7 hours, only no more than 10% of the lactose is hydrolyzed.

[0059] Comparative Example 3

[0060] After activation, P. putida KT2440 was transferred to the whey hydrolysate culture medium obtained in Example 1, and the rest of the specific technical solutions were the same as in Example 3.

[0061] After 30 hours of reaction, glucose and galactose were not completely consumed; after 40 hours of reaction, glucose and galactose were almost completely consumed, at which point the reaction was stopped. The OD and galacturonic acid concentration of the bacterial cells obtained from the reaction were basically the same as in Example 3.

[0062] The bacterial cells were collected by centrifugation and used for whey powder catalysis. The remaining specific technical procedures were the same as in Example 4. After approximately 80 hours of reaction, the lactobionic acid concentration did not exceed 20 g / L.

[0063] Comparative Example 4

[0064] The technical solution is the same as in Example 4, but the reaction pH is maintained at 4.0. After 80 hours of reaction, the lactose utilization rate is less than 10%.

[0065] Comparative Example 5

[0066] The technical solution is the same as in Example 4, but air is not introduced. After 80 hours of reaction, the lactose utilization rate is less than 40%.

[0067] This invention provides a two-stage catalytic method for the production of galactobionic acid and lactobionic acid using whey as a substrate. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A two-stage catalytic method for the production of galactobionic acid and lactobionic acid using whey as a substrate, characterized in that, Includes the following steps: (1) Culture engineered Escherichia coli expressing β-galactosidase to obtain bacterial cells, wherein the nucleotide sequence of the β-galactosidase is shown in SEQ ID NO. 1; (2) The bacterial cells obtained in step (1) by ultrasonic disruption are centrifuged and the supernatant is taken to obtain a crude enzyme solution containing β-galactosidase. The crude enzyme solution is subjected to heat denaturation treatment, centrifuged to remove the heat denatured protein, and the supernatant is removed to obtain the processed cell contents. (3) Using lactose or whey as a substrate, the substrate is completely hydrolyzed using the cell contents obtained in step (2) to prepare a hydrolysate containing glucose and galactose; the concentration of lactose or whey is 10 g / L to 100 g / L, and cell contents are added to the hydrolysis system so that the β-galactosidase activity is 0.1 to 5 U / mL. The hydrolysis system also contains 50 to 100 mg / L of kanamycin sulfate, and the hydrolysis is carried out at 45 to 60 °C and 50 to 300 rpm. (4) The engineered *Pseudomonas putida* expressing the PQQ-dependent membrane-bound dehydrogenase of the gluconeo-shikimic acid family was inoculated into the hydrolysate containing glucose and galactose obtained in step (3). The engineered *Pseudomonas putida* used glucose in the hydrolysate as a carbon source for cell proliferation and oxidized all galactose to galacturonic acid. The nucleotide sequence of the PQQ-dependent membrane-bound dehydrogenase of the gluconeo-shikimic acid family is shown in SEQ ID NO.

2. The starting strain of the engineered *Pseudomonas putida* is *Pseudomonas putida* ATCC No. 47054. (5) The engineered Pseudomonas malodorans from step (4) is recovered and inoculated into the whey solution to oxidize all lactose into lactobionic acid; In step (5), the engineered Pseudomonas malodorans obtained in step (4) is added to make the bacterial OD 5~10, the reaction pH is controlled at 5.0~8.0, the dissolved oxygen concentration of the reaction system is not less than 1.5 mg / L, and the whey concentration is 150g / L~350g / L.

2. The method for two-stage catalytic production of galactobionic acid and lactobionic acid using whey as a substrate according to claim 1, characterized in that, In step (1), the engineered Escherichia coli was constructed by the following method: the nucleotide sequence of the β-galactosidase was linked to the pETDuet-1 vector to obtain a recombinant plasmid, and then the recombinant plasmid was transformed into Escherichia coli BL21(DE3). The culture and expression method was as follows: the engineered Escherichia coli was first cultured in LB liquid medium containing ampicillin, and then IPTG was added to induce expression. After shaking culture, the cell pellet was collected by centrifugation after induction, the cell pellet was washed with physiological saline, and then resuspended in phosphate buffer for later use.

3. The method for two-stage catalytic production of galactobionic acid and lactobionic acid using whey as a substrate according to claim 1, characterized in that, In step (2), the conditions for ultrasonic disruption are: ultrasonic power of 300~500W and ultrasonic time of 15~30 min. After ultrasonic disruption, the disrupted liquid is centrifuged and the supernatant is collected.

4. The method for two-stage catalytic production of galactobionic acid and lactobionic acid using whey as a substrate according to claim 1, characterized in that, In step (2), the heat denaturation treatment is to keep warm in a water bath at 50~65℃ for 0.5~2 hours.

5. The method for two-stage catalytic production of galactobionic acid and lactobionic acid using whey as a substrate according to claim 1, characterized in that, In step (4), *Pseudomonas malodorosa* is introduced to make the initial OD of the bacteria 0.5-2.

6. The method for two-stage catalytic production of galactobionic acid and lactobionic acid using whey as a substrate according to claim 1, characterized in that, In step (4), the reaction conditions are as follows: the reaction pH is controlled at 5.0~8.0, the air flow rate is 0.5~1.5vvm, the stirring speed is 200~400rpm, and the temperature is 27~35℃.

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