Mutant strain for efficient biotransformation synthesis of miglitol, screening method and application

Abstract

The invention relates to the technical field of biological pharmacy, and discloses a mutant strain for efficient biotransformation synthesis of miglitol, a screening method and application. A compound mutation strategy of ARTP mutagenesis and NTG mutagenesis is utilized, a rapid screening method based on carbonyl color development is combined, a mutant strain capable of efficiently converting and synthesizing a miglittol key intermediate is obtained, the mutant strain is named as Gluconobacter oxydans G1036, and the enzyme activity of the mutant strain is 61.7 U / mL and is improved by about 48% compared with that of an original strain. A process for producing 6NSL by catalyzing NEHG through resting cells of the mutant strain is researched, 73.53 g / L of 6NSL is produced by catalyzing 80 g / L of a substrate within 48 h, and compared with 80.37% of an original strain, the 6NSL is improved by 19.63%. According to the present invention, the research on the process for repeatedly and catalytically producing the 6NSL by using the resting cells of the mutant strain in multiple batches shows the good catalytic performance, and 3 batches can be efficiently utilized to accumulatively produce 220.59 g / L of the 6NSL within 144 h;

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to mutant strains, screening methods, and applications for the efficient biotransformation synthesis of miglitol. Background Technology

[0002] Miglitol (1-(2-hydroxyethyl)-2-(hydroxymethyl)-3,4,5-piperidintriol) is a glucose structural analogue and a novel α-glucosidase inhibitor developed by Bayer AG, Germany. It has a high affinity for pancreatic amylase and α-glucosidase, inhibiting the hydrolysis of disaccharides, polysaccharides, and complex sugars, and delaying the absorption of glucose and other monosaccharides. It has a significant hypoglycemic effect and significantly lower toxicity than sulfonylureas and bismuth subsalicylates. By delaying the breakdown and absorption of carbohydrates in the intestine, it effectively reduces postprandial blood glucose. Its clinical application is widespread and demand continues to grow, making it a key oral hypoglycemic agent for the treatment of type 2 diabetes.

[0003] Using hydroxyethylglucosamine as a substrate, the stereoselective dehydrogenation of bioenzymes to synthesize key intermediates of miglitol, combined with further chemical hydrogenation steps, has gradually become the mainstream route for miglitol synthesis. This route overcomes the limitations of traditional chemical synthesis methods, such as numerous steps, low product yield, and severe environmental pollution. The membrane-localized sorbitol dehydrogenase mSLDH in *Gluconobacter oxydans* can selectively and asymmetricly oxidatively dehydrogenate the 4-hydroxy group of the substrate N-hydroxyethylglucosamine to form a carbonyl group. This is a core step in the catalytic synthesis of the key intermediate 6-deoxy-6-amino(N-hydroxyethyl)-α-L-furanose (6NSL). Chinese patent application CN 110016455 A discloses a method and strain for rapidly screening mutant strains for the synthesis of key intermediates of miglitol. This method enhances the catalytic activity of recombinant strains by overexpressing mSLDH using free plasmids; however, the enzyme activity stability of the strain is poor, affecting the stability of production batches. Furthermore, the strain cultivation process provided by this patent requires the addition of antibiotics, which is detrimental to downstream wastewater purification treatment.

[0004] Classic mutagenesis breeding strategies have played a crucial role in improving the production of industrial-grade bacterial strains. The selective asymmetric dehydrogenation capabilities of *Gluconobacter oxidans* can catalyze the dehydrogenation of reducing sugars, and high-activity strains can be obtained through simple Fehling's reagent colorimetric method. Utilizing this design method, an efficient composite mutagenesis strategy can be constructed to screen for high-performance target strains. This is of great significance for promoting the industrial application of miglitol biotransformation, reducing production costs, and enhancing product competitiveness. Summary of the Invention

[0005] To address the technical problems of low transformation efficiency, poor substrate tolerance, and antibiotic-dependent screening of recombinant expression strains in existing miglitol biotransformation strains, this invention provides a mutant strain, screening method, and application for efficient biotransformation synthesis of miglitol. This invention combines ARTP and NTG combined mutagenesis with rapid colorimetric screening technology to enhance the mutagenesis and selection of *Gluconobacterium oxysporum*, obtaining a mutant strain for efficient biotransformation synthesis of miglitol. The application of this strain in miglitol biosynthesis enables the green and efficient industrial production of miglitol.

[0006] The specific technical solution of this invention is as follows:

[0007] As a first aspect of the present invention, a mutant strain for the efficient biotransformation synthesis of miglitol is provided. The mutant strain is *Gluconobacter oxydans*, named *Gluconobacter oxydans* G1036, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252432, deposited on November 3, 2025, at Wuhan University, Wuhan, China, 430072, China.

[0008] The mutant strain *Gluconobacter oxydans* G1036 exhibits efficient and stable catalytic synthesis of the key intermediate 6NSL, miglitol. This mutant strain was obtained through a combined ARTP and NTG mutagenesis screening method. HPLC analysis showed that the enzyme activity of the original strain ZJB16009 was 41.7 U / mL, while the enzyme activity of the *Gluconobacter oxydans* G1036 mutant strain was significantly higher at 61.7 U / mL, representing an increase of approximately 48% compared to the original strain.

[0009] As a second aspect of the present invention, an application of a mutant strain of *Glucosamine oxyphylla* in the catalytic synthesis of miglitol intermediate 6NSL is provided.

[0010] As a preferred embodiment of the above application, the method of the application includes the following steps: using N-hydroxyethylglucosamine as a substrate and the mutant strain wet cells as a catalyst to carry out a catalytic reaction to obtain miglitol intermediate 6NSL.

[0011] As a preferred embodiment of the above application, in the catalytic reaction system, the amount of the mutant strain wet cells fed is 40~100g / L.

[0012] As a preferred embodiment of the above application, the concentration of N-hydroxyethylglucosamine in the catalytic reaction system is 40~80 g / L.

[0013] As a third aspect of the present invention, a method for screening mutant strains for efficient biotransformation synthesis of miglitol is provided, comprising the following steps:

[0014] The wild-type glucosinolate bacillus was subjected to ARTP and NTG combined mutagenesis to obtain a mutant strain, which was then fermented. The wet cells of the cultured mutant strain were used to catalyze the synthesis of miglitol intermediate 6NSL. After the reaction, the 6NSL content of the reaction solution was detected. Based on the 6NSL content, a high-activity mutant strain for synthesizing miglitol intermediate 6NSL was obtained.

[0015] This invention utilizes a combined mutagenesis strategy of ARTP and NTG mutagenesis, combined with a rapid screening method based on carbonyl colorimetry, to obtain a mutagenic strain capable of efficiently synthesizing key intermediates of miglitol. This strain is tolerant of high substrate concentrations, exhibits good catalytic stability in multiple batches of repeated transformations, and achieves a high space-time yield, enabling the green and efficient industrial production of miglitol.

[0016] As a preferred method for the screening, the reaction system for the catalytic synthesis of miglitol intermediate 6NSL is as follows: 40-100 g / L of wet mutant strain cells, 40-80 g / L of N-hydroxyethylglucosamine, 2-6 g / L of magnesium sulfate heptahydrate, and water as the solvent.

[0017] As a preferred method for the screening, the pH of the reaction system for the catalytic synthesis of miglitol intermediate 6NSL is 4.8 to 5.5.

[0018] As a preferred method for the screening, the wild-type Gluconobacter oxydans used for ARTP mutagenesis is Gluconobacter oxydans ZJB16009.

[0019] As a preferred method for the screening, the wild-type *Gluconobacterium oxysporum* subjected to NTG mutagenesis is a mutant strain obtained by ARTP mutagenesis.

[0020] Compared with the prior art, the present invention has the following technical effects:

[0021] (1) This invention provides a mutant strain that can efficiently and stably catalyze the synthesis of the key intermediate 6NSL of miglitol. The mutant strain was obtained by a combination of ARTP mutagenesis and NTG mutagenesis screening. HPLC detection showed that the enzyme activity of the original strain ZJB16009 was 41.7 U / mL, while the enzyme activity of the mutant strain of Gluconobacter oxidase obtained in this invention was 61.7 U / mL, which is about 48% higher than that of the original strain.

[0022] (2) This invention studies the process of producing 6NSL from NEHG using resting mutant cells. Within 48 h, 73.53 g / L of 6NSL was produced from 80 g / L substrate, which is 19.63% higher than the original strain's 80.37%. The process of producing 6NSL by repeated batches using G1036 resting cells was studied. G1036 resting cells showed good catalytic performance and could efficiently utilize 3 batches, accumulating a production of 220.59 g / L of 6NSL within 144 h. Attached Figure Description

[0023] Figure 1 Characterization results of sorbitol dehydrogenase activity of positive mutant strains obtained through ARTP mutagenesis selection.

[0024] Figure 2 The results of characterization of sorbitol dehydrogenase activity of strains obtained through NTG mutagenesis and secondary screening.

[0025] Figure 3 Results of the transformation process of 6NSL, a key intermediate in the synthesis of miglitol, catalyzed by different doses of resting cells.

[0026] Figure 4 Results of the transformation process for the synthesis of 6NSL, a key intermediate of miglitol, from resting cells in multiple batches. Detailed Implementation

[0027] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0028] Example 1: ARTP mutagenesis screening to enhance sorbitol dehydrogenase activity in G. oxydans

[0029] This embodiment uses ARTP (atmospheric pressure, room temperature plasma) mutagenesis to screen mutant strains and enhance sorbitol dehydrogenase activity. The procedure is as follows:

[0030] 1. Strains and culture media

[0031] The starting strain was a publicly available strain, specifically *Gluconobacter oxydans* ZJB16009 (accession number M201703), which could be purchased commercially. The seed culture medium consisted of: sorbitol 55 g / L, yeast extract 20 g / L, dipotassium hydrogen phosphate 5 g / L, and potassium dihydrogen phosphate 5 g / L, pH 6.5. The selection medium consisted of the same composition as the seed culture medium. The fermentation medium consisted of the same composition as the seed culture medium.

[0032] 2. ARTP mutagenesis treatment

[0033] ZJB16009 was inoculated into seed culture medium and cultured at 30℃ with shaking at 200 rpm for 14 h until the logarithmic growth phase. 1 mL of the bacterial culture was diluted to a 1:10⁻⁶ solution. 6 CFU / mL, take 10 μL and spread it evenly on a sterile slide. Place the slide in an ARTP mutagen and set the parameters: plasma power 120 W, treatment distance 2 mm, gas flow rate 10 L / min, and treat for 15 s, 30 s, 45 s, 60 s, and 75 s respectively. After treatment, elute the bacterial solution, serially dilute and spread it on selection medium, and incubate at 30 °C for 72 h.

[0034] 3. Fehling's reagent rapid colorimetric screening

[0035] Single colonies were picked and transferred to well 1 of a 96-well plate. 1.5 mL of fermentation medium was added, with ZJB16009 as a control. The plates were incubated at 30°C and 200 rpm for 48 h. 500 μL of the bacterial culture was transferred to well 2 of a 96-well plate and stored at 4°C. Well 1 of the 96-well plate was centrifuged (3000 rpm, 10 min), the supernatant was discarded, and the plates were washed with deionized water. 200 μL of substrate reaction solution (sorbitol 30 g / L, 2.4 g / L MgSO₄) was added. 4， The solvent was water. The transformation was performed at 30℃ for 30 min. After centrifugation, the transformation solution was subjected to Fehling's reagent staining; the darker the color, the higher the enzyme activity. Results showed that 2000 mutants were initially screened, with a positive mutation rate of 2.5%. The selected mutant strains exhibited increased enzyme activity compared to the original strain.

[0036] The substrate reaction solution consisted of 30 g / L sorbitol, 2.4 g / L MgSO4, pH 5.0, and water as the solvent.

[0037] The Fehling's reagent formulas are as follows: Solution A is prepared by dissolving 34.6 g of copper sulfate and 0.5 mL of 98% sulfuric acid in 500 mL of deionized water; Solution B is prepared by dissolving 125 g of sodium hydroxide and 137 g of potassium sodium tartrate tetrahydrate in 500 mL of deionized water.

[0038] The activity of sorbitol dehydrogenase is defined as the amount of enzyme that catalyzes the production of 1.0 μmol L-sorbose per minute in a phosphate buffer system of 20 g / L D-sorbitol at 30℃ and pH 6.0.

[0039] 4. High-viability strains were obtained through shake-flask re-screening.

[0040] After repeated screening in shake flasks, the corresponding positive mutant bacterial cultures obtained from the initial screening were aspirated from the 96-well plate (No. 2) and cultured in shake flasks at 30°C and 200 rpm for 24 h. After incubation, the cultures were centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the culture medium was removed by washing with deionized water to obtain the re-screened wet cells. A 10 mL reaction system (30 g / L sorbitol, 2.4 g / L MgSO4, pH 5.0, water as solvent) was established, and the re-screened wet cells were added to the system and reacted at 30°C for 30 min. After the reaction, samples were taken for carbonyl colorimetric analysis, photographed, and the amount of L-sorbose product was detected by HPLC (Aminex HPX-87H column, 5 mM sulfuric acid mobile phase, 60°C, differential detector). The positive mutant strains with high activity after re-screening were retained, and the results are as follows. Figure 1 The mutant strains shown are ZJB16009-1, ZJB16009-2, ZJB16009-3, ZJB16009-4, ZJB16009-5, and ZJB16009-6.

[0041] The single colonies obtained from the secondary screening were inoculated into fermentation medium and fermented at 30℃ and 200 r / min for 36 hours with shaking to obtain positive mutant strains ZJB16009-1, ZJB16009-2, ZJB16009-3, ZJB16009-4, ZJB16009-5, and ZJB16009-6. Among them, strain ZJB16009-6 had the highest activity, which was 33.09% higher than that of the original strain.

[0042] Example 2: NTG mutagenesis screening for highly efficient strains

[0043] This embodiment uses NTG (nitrosoguanidine) mutagenesis to screen mutant strains for efficient conversion into miglitol. The procedure is as follows:

[0044] 1. Starting strain and culture

[0045] The ZJB16009-6 strain obtained through ARTP mutagenesis screening in Example 1 was used as the starting strain and named *Gluconobacter oxydans* G1030. *G. oxydans* G1030 was inoculated into seed culture medium and cultured at 30°C and 200 rpm to the logarithmic growth phase. The seed culture medium used in the following steps consisted of: 55 g / L sorbitol, 20 g / L yeast extract, 5 g / L dipotassium hydrogen phosphate, and 5 g / L potassium dihydrogen phosphate, pH 6.5. The screening medium consisted of the same composition as the seed culture medium. The fermentation medium consisted of the same composition as the seed culture medium.

[0046] 2. NTG mutagenesis treatment

[0047] G. oxydans G1030 was inoculated into seed culture medium and cultured to the logarithmic growth phase. NTG was dissolved in pH 6.0 phosphate buffer to prepare a 0.5 mg / mL NTG solution. The bacterial culture and NTG solution were mixed at a volume ratio of 4:1 and treated with shaking at 30°C and 200 rpm for 30 min. Then, sterile physiological saline was added to terminate the reaction, and the mixture was spread onto selection medium and incubated at 30°C for 72 h.

[0048] 3. Screening and Validation

[0049] Single colonies were selected and numbered NTG-ARTP-1~n, and then transferred to 96-well plates. Culture, transformation, and Fehling's reagent screening were performed using the same method as in Example 1. Strains showing an increase in enzyme activity of more than 10% were selected and subjected to shake-flask rescreening as described in Example 1.

[0050] 4. Comparison of enzyme activities between mutant strain G1036 and the original strain

[0051] Referring to the secondary screening method in Example 1, mutant strains with enhanced viability were further screened for secondary screening verification. The results showed that... Figure 2 The mutant strains G. oxydans G1031, G. oxydans G1032, G. oxydans G1033, G. oxydans G1034, G. oxydans G1035, and G. oxydans G1036 showed increased yields compared to the starting strain, with mutant strain G1036 showing a significant increase. Furthermore, the selected strain G1036 was passaged 10 times consecutively, and resting cell catalysis experiments were performed after each passage to detect 6NSL yield. The results showed that the 6NSL yield of this strain remained stable after 10 passages, indicating good genetic stability.

[0052] 5. Preservation and seed culture preparation of mutant strain G1036

[0053] The single colonies obtained from the secondary screening were inoculated into the fermentation medium and fermented at 30°C and 200 r / min for 36 hours to obtain the seed culture of the mutant strain G. oxydans G1036.

[0054] The obtained strain G. oxydans G1036 was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252432, on November 3, 2025. The address is: Wuhan University, Wuhan, China, 430072, China.

[0055] Example 3: Enzyme production and fine cell enzyme activity determination during 5L tank fermentation

[0056] In this embodiment, the mutant strain G. oxydans G1036 obtained in Example 2 was inoculated into a 5L tank for fermentation to produce enzymes, and its enzyme activity was characterized. The steps are as follows:

[0057] 1.5L tank fermentation for enzyme production

[0058] A 5L fermenter was used. The fermentation medium was initially loaded into the fermenter with 3L of medium, and the seed culture of the mutant strain G. oxydans G1036 obtained in Example 2 was inoculated into the fermenter at an inoculum rate of 4% (v / v). The temperature was maintained at 30℃ throughout the process, the aeration rate was 2 vvm, and the stirring speed was 400 rpm to ensure that DO ≥ 30%. The fermentation cycle was 16 hours, and fermentation was terminated when the sorbitol dehydrogenase activity reached its peak.

[0059] The fermentation medium consisted of: 80 g / L D-sorbitol, 20 g / L yeast extract, 5 g / L dipotassium hydrogen phosphate, 5 g / L potassium dihydrogen phosphate, and 50 mg / mL cefoxitin sodium.

[0060] 2. Cell collection and enzyme activity characterization

[0061] The fermentation broth was centrifuged at 8000 rpm for 10 min, and the cells were collected and washed. Sorbitol dehydrogenase activity was determined according to the method in Example 1. After 16 h of fermentation and enzyme production, the cells were collected by centrifugation, yielding a total of 21.9 g of resting wet cells. Enzyme activity assays showed that the sorbitol dehydrogenase activity of G1036 in the fermentation broth reached a peak of 61.7 U / mL, with a total enzyme activity of 1851 U.

[0062] Example 4: Effect of different wet weight cell dosages on the catalytic reaction process

[0063] In this embodiment, resting wet cells of the mutant strain G. oxydans G1036 obtained in Example 3 were used to conduct catalytic reactions with different addition amounts to investigate the effect of different wet weight cell dosages on the catalytic reaction process. The steps are as follows:

[0064] 1. Transformation System

[0065] Add 80 g / L of N-hydroxyethylglucosamine, 2.4 g / L of MgSO4, and resting wet cells of the mutant strain G1036 obtained from fermentation culture in Example 3 to a shake flask equipped with a baffle. Prepare a 30 mL reaction system using water as the reaction medium. The amount of wet cells added is 20, 40, and 60 g / L, respectively. The reaction system is prepared as follows: dissolve N-hydroxyethylglucosamine and MgSO4 in water, first adjust to a slightly acidic condition of pH 3.0 with HCl, then adjust to pH 5.0 with 2M NaOH, and then add the target concentration of resting wet cells of G1036. An N-hydroxyethylglucosamine solution with a concentration of 80 g / L was prepared.

[0066] 2. Experimental conditions

[0067] Temperature: 15℃; Rotation speed: 220 rpm; Adjust the alkali with 2M NaOH every 6 hours to maintain the pH of the conversion solution at 5.0. Samples were taken every 12 hours, the supernatant was collected by centrifugation, and the sample yield was determined by liquid chromatography (chromatographic conditions: C18 reversed-phase column (250 mm × 4.6 mm, 5 μm); mobile phase was methanol-water (2:98), the aqueous phase contained 4 mM sodium heptanesulfonate and 10 mM dipotassium hydrogen phosphate (pH 3.5); flow rate 0.5 mL / min; column temperature 30℃; injection volume 20 μL; differential detector).

[0068] 3. Results Analysis

[0069] The substrate conversion rate and product yield were investigated under different wet weight cell loading conditions of 20, 40, and 60 g / L. The results are as follows: Figure 3 As shown, the substrate conversion rate reached over 97% under different wet-weight cell dosages. In terms of product yield analysis, yields of 20 g / L, 40 g / L, and 60 g / L wet-weight cell dosages reached over 90%, with the best yields of over 99.45% achieved at 40 g / L and 60 g / L. Considering overall cost, the optimal cell dosage was selected as 40 g / L, yielding 98.02%.

[0070] Example 5: Single-batch catalytic synthesis of 6NSL from resting cells at different substrate concentrations

[0071] In this embodiment, resting wet cells of the mutant strain G. oxydans G1036 obtained in Example 3 were used to conduct catalytic reactions with different substrate concentrations to investigate the effect of different substrate concentrations on the catalytic reaction process. The steps are as follows:

[0072] 1. Construction of the transformation system

[0073] Using the G1036 resting cells prepared in Example 3, a 30 mL transformation system was constructed: the substrate N-hydroxyethylglucosamine (NHEG) concentrations were 80 g / L and 100 g / L, the cell input was 40 g / L, MgSO4 was added to a final concentration of 2.4 g / L, the pH was 5.0, the reaction volume was 30 mL, and the reaction was catalyzed at 15 °C for 48 h.

[0074] 2. Reaction conditions

[0075] The Erlenmeyer flask was placed in a shaker at 30°C and 200 rpm for 48 hours, during which the pH was adjusted to 5.0 with 6M sodium hydroxide every 6 hours.

[0076] 3. Product testing

[0077] After the reaction was completed, a sample was taken, centrifuged, and the supernatant was collected for HPLC analysis.

[0078] The results showed that when the substrate concentration was 80 g / L, the NHEG conversion rate reached over 99.43%, and the product yield was 98.62%; when the substrate concentration was 100 g / L, the product yield was 95.85%, as shown in Table 1. Table 1 demonstrates the potential application value in the catalytic synthesis of the key intermediate 6NSL of miglitol. The symbol "*" in Table 1 indicates that the molecular weight of the conversion product 6NSL was determined by LC / MS because a standard sample was unavailable.

[0079] Table 1. Synthesis of 6NSL catalyzed by resting cells in shake-flask systems at different substrate concentrations.

[0080] Substrate concentration (g / L) 6NSL concentration (g / L) Conversion rate (%) Yield (%) 80 73.8 99.43 95.62* 100 89.4 97.11 90.85*

[0081] Example 6: Multiple batches of resting cells from mutant strain G1036 were repeatedly used to synthesize miglitol intermediate 6NSL.

[0082] In this embodiment, resting wet cells of the mutant strain G. oxydans G1036 obtained in Example 3 were repeatedly used to synthesize miglitol intermediate 6NSL in multiple batches to investigate the catalytic stability of G. oxydans G1036. The steps are as follows:

[0083] 1. Operational procedures for multiple batch transformations of resting cells

[0084] The "fermentation-conversion-cell recycling" cycle was adopted, and a total of 3 batches were run.

[0085] First catalysis: The reaction was carried out according to the method in Example 5 (substrate concentration of 80 g / L). After the reaction was completed, the reaction solution was centrifuged at 4°C and 8000 r / min for 10 min, and the bacterial pellet (i.e. resting cells) was collected. The supernatant was used for subsequent product separation. The bacterial pellet was washed twice with phosphate buffer.

[0086] For the second and third batches of catalysis: the recovered cells were inoculated into freshly prepared transformation buffer containing 80 g / L NHEG, and the same transformation parameters were maintained.

[0087] 2. Results

[0088] These resting cells can be reused for up to four batches. The NHEG conversion rate of the first three batches remained stable at over 99%. Even after adding 10% fresh cells to the fourth batch, the conversion rate remained at a high level. Figure 4 As shown, this multi-batch conversion strategy significantly reduces production costs and provides strong support for industrial applications.

[0089] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0090] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A mutant strain for efficient biotransformation synthesis of miglitol, characterized in that: It is deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20252432, deposited on November 3, 2025, at Wuhan University, Wuhan, China, 430072, China.

2. The application of the mutant strain as described in claim 1 in the catalytic synthesis of miglitol intermediate 6NSL.

3. The application as described in claim 2, characterized in that: The method of application includes the following steps: using N-hydroxyethylglucosamine as a substrate and the wetted mutant bacterial cells as a catalyst to carry out a catalytic reaction to obtain miglitol intermediate 6NSL.

4. The application as described in claim 2, characterized in that: In the catalytic reaction system, the amount of wet mutant bacterial cells fed is 40~100g / L.

5. The application as described in claim 2, characterized in that: In the catalytic reaction system, the concentration of N-hydroxyethylglucosamine is 40~80 g / L.

6. A method for screening mutant strains for efficient biotransformation synthesis of miglitol, characterized in that: Includes the following steps: The wild-type glucosinolate bacillus was subjected to ARTP and NTG combined mutagenesis to obtain a mutant strain, which was then fermented. The wet cells of the cultured mutant strain were used to catalyze the synthesis of miglitol intermediate 6NSL. After the reaction, the 6NSL content of the reaction solution was detected. Based on the 6NSL content, a high-activity mutant strain for synthesizing miglitol intermediate 6NSL was obtained.

7. The screening method as described in claim 6, characterized in that: The reaction system for the catalytic synthesis of miglitol intermediate 6NSL is as follows: 40~100 g / L of wet mutant strain cells, 40~80 g / L of N-hydroxyethylglucosamine, 2~6 g / L of magnesium sulfate heptahydrate, and water as the solvent.

8. The screening method as described in claim 6, characterized in that: The pH of the reaction system for the catalytic synthesis of miglitol intermediate 6NSL is 4.8–5.

5.

9. The screening method as described in claim 6, characterized in that: The wild-type Gluconobacterium oxidase that underwent ARTP mutagenesis was Gluconobacterium oxidase ( Gluconobacter oxydans ZJB16009.

10. The screening method as described in claim 6, characterized in that: The wild-type *Glucosobacterium oxysporum* strain subjected to NTG mutagenesis was a mutant strain obtained through ARTP mutagenesis.

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