Glucostarchase mutants and their use in the process of reducing sugar in glutamic acid mother liquor

Abstract

This invention belongs to the field of biotechnology and discloses a glucosylamylase mutant. The amino acid sequence of the glucosylamylase mutant is shown in SEQ ID NO: 1, or an amino acid sequence with more than 90% homology to the above sequence. Compared with the wild type, the glucosylamylase of this invention exhibits significantly improved pH stability, a wider temperature tolerance range, and significantly enhanced heavy metal resistance, making it more suitable for complex industrial environments, especially for hypoglycemic treatments involving heavy metals and large pH fluctuations, such as glutamate mother liquor, and has high industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a glucose amylase mutant and its application in the hypoglycemic process of glutamate mother liquor. Background Technology

[0002] Glutamic acid mother liquor is a high-concentration organic waste liquid generated during glutamic acid production. It has a high sugar content and complex composition, and its sugar reduction treatment is a crucial step in achieving resource utilization and compliant discharge. Currently, industrial glutamic acid production mainly uses glucose as the fermentation carbon source. In glutamic acid fermentation processes using glucose as the carbon source, the residual polysaccharide components in the yeast broth are relatively simple, mainly originating from three pathways: incompletely utilized fermentation medium components, microbial metabolic byproducts, and cell autolysis products. The sugars in the yeast broth are mainly glucose, maltose, maltotriose, and other miscellaneous sugars (including dextrin, bacterial glucan, etc.).

[0003] The commonly used hypoglycemic processes in existing technologies mainly include microbial hypoglycemic processes, physicochemical hypoglycemic processes, and enzymatic hypoglycemic processes. Each process has its own advantages and disadvantages.

[0004] Microbial hypoglycemic process: This process utilizes the metabolic activity of specific microorganisms to convert sugars in the mother liquor into useful products such as microbial proteins and organic acids, while simultaneously reducing sugar content. Using osmotically tolerant strains, such as *Staphylococcus aureus*, eliminates the need for sterilization and seed liquor replenishment; the glutamic acid mother liquor is directly fed into the fermentation system, achieving hypoglycemia through continuous feeding. Alternatively, a mixed fermentation of yeast and lactic acid bacteria can be used. Yeast preferentially utilizes monosaccharides such as glucose, while lactic acid bacteria convert remaining sugars into lactic acid, improving hypoglycemic efficiency. Advantages include: environmental friendliness with no secondary pollution; recovery of high-value byproducts such as microbial proteins and organic acids; strong adaptability, capable of handling high-concentration, high-viscosity mother liquors; and a high degree of automation in the continuous fermentation process, suitable for large-scale production. Disadvantages include: a longer fermentation cycle; strict requirements for environmental conditions such as temperature and pH; difficulty in strain compatibility, potentially leading to incomplete hypoglycemia if strains are not properly acclimatized; and the need for additional solid-liquid separation equipment for microbial protein separation, increasing investment costs.

[0005] Physicochemical hypoglycemic processes: These processes directly remove or transform sugars in the mother liquor using physical or chemical methods, including evaporation concentration, membrane separation, and oxidative degradation. Advantages include thorough hypoglycemic control, near-zero wastewater discharge, high resource utilization (simultaneous recovery of multiple byproducts), and strong adaptability (ability to treat glutamic acid mother liquors of varying properties). Disadvantages include complex processes, high equipment investment, high operational difficulty requiring specialized technical personnel, relatively high operating costs, and the need for precise control over the integration of different processes.

[0006] Enzymatic hypoglycemic process: Glucoamylase can accurately recognize the α-1,4 glycosidic bonds in polysaccharide molecules, rapidly breaking them down into glucose. This method is 3-5 times more efficient than chemical hydrolysis. The enzymatic reaction conditions are mild, requiring no strong acids or alkalis, thus avoiding secondary pollution. The enzyme preparation can be recovered and reused using immobilization technology after the reaction. However, heavy metal ions (such as Cu) in the wastewater... 2+ Pb 2+ High temperatures (>60℃) can inactivate the enzyme, requiring pretreatment to remove inhibitors or the use of heat-resistant enzyme preparations. Therefore, discovering new, high-performance glucoamylases is a major research direction.

[0007] Studies have found that *Candida cruzi* exhibits strong adaptability to mother liquor and can rapidly utilize glucose and eliminate ammonia nitrogen from the mother liquor. However, its ability to utilize maltose, maltotriose, dextrin, and glucan is poor, thus it cannot completely degrade residual sugars in the mother liquor. Glucoamylase, on the other hand, can degrade maltose, maltotriose, and dextrin, and can be used in combination with *Candida cruzi*. However, existing technologies disclose various microbial glucamylases, but most suffer from drawbacks such as narrow pH and temperature operating ranges and susceptibility to inhibition by heavy metal ions when used in mother liquors. Therefore, improvements or optimizations of glucamylases are needed.

[0008] Each of the above processes has its own advantages and disadvantages. How to optimize the glutamic acid mother liquor hypoglycemic process to make it more resource-efficient and simpler is a technical problem that needs further research and solutions. Summary of the Invention

[0009] To address the technical problems existing in the prior art, this invention provides a glucose amylase mutant and its application in the glucose-lowering treatment process of glutamic acid mother liquor.

[0010] To achieve the above objectives, the present invention is implemented through the following technical solution: A glucose amylase mutant having an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having more than 90% homology with the above sequence.

[0011] Furthermore, the nucleotide sequence of the glucosylase is as shown in SEQ ID NO: 2, or a nucleotide sequence with more than 90% homology to the above sequence.

[0012] On the other hand, the present invention also relates to genes encoding the above-mentioned glucosylamylase mutants, expression cassettes, recombinant expression vectors, and recombinant cells.

[0013] Preferably, the recombinant cells are recombinant yeast cells.

[0014] On the other hand, the present invention also relates to the application of glucosylamylase mutants in the hydrolysis of polysaccharides.

[0015] Preferably, the polysaccharide is selected from one or more of starch, dextrin, maltose, and maltotriose.

[0016] The beneficial effects achieved by this invention mainly include, but are not limited to, the following aspects: The glucoamylase mutant GA-M obtained in this invention exhibits significant advantages over the wild-type GA-WT: First, its pH stability is greatly improved, maintaining over 80% relative enzyme activity in the pH range of 3.5-5.0, far exceeding the stability range of the wild type; second, it has a wider temperature tolerance range, maintaining over 70% activity in the 30-75℃ range, while the wild type only meets the standard in the 60-75℃ range; third, its heavy metal resistance is significantly enhanced, maintaining over 70% enzyme activity at a copper ion concentration of 20 μmol / L, while the wild type drops to below 20%; fourth, its substrate hydrolysis efficiency is significantly improved, achieving a 92% hydrolysis rate for polysaccharides in glutamate mother liquor within 30 minutes and complete hydrolysis within 60 minutes, far exceeding the 47% and 80% of the wild type. These characteristics make GA-M more suitable for complex industrial environments, especially for processing raw materials containing heavy metals and with large pH fluctuations, such as glutamate mother liquor, and it has extremely high industrial application value. Attached Figure Description

[0017] Figure 1 : Optimal pH detection of wild-type and mutant glucosylamylase.

[0018] Figure 2 : Detection of the optimal temperature for wild-type and mutant glucosylamylase.

[0019] Figure 3 The effect of copper ions on relative enzyme activity.

[0020] Figure 4 : Hydrolysis of polysaccharides in glutamate mother liquor by glucoamylase. Detailed Implementation

[0021] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0022] Unless otherwise defined in this invention, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0023] Example 1

[0024] The original sequence of glucoamylase was obtained from GenBank: AYA22369.1. Bioinformatics analysis yielded the mature peptide and cDNA sequences of glucoamylase. Conserved domain information of glucoamylase was obtained using the UniProt database. The protein's three-dimensional structure was predicted using the platform, and compared with the resolved crystal structures of homologous proteases to screen for key amino acid sites that might affect enzyme activity. A glucoamylase mutant (GA-M) with potential optimization properties was designed. The tyrosine (Y) at position 58 of the original glucoamylase sequence was mutated to aspartic acid (D), corresponding to a change from tac to gac; the glutamic acid (E) at position 240 was mutated to serine (S), corresponding to a change from gaa to tcc in the nucleotide codon.

[0025] The mutant described in this invention is represented as "Y58D+E240S", meaning that the 58th amino acid in the wild-type amino acid sequence is mutated from tyrosine (Y) to aspartic acid (D), and the 240th amino acid is mutated from glutamic acid (E) to serine (S). The specific amino acid sequence is shown in SEQ ID No. 1, and the nucleotide sequence is shown in SEQ ID No. 2. The gene fragments of glucosyl amylase wild-type (GA-WT) and mutant (GA-M) were routinely amplified using primers with XhoI + EcoRI double restriction sites and protective bases. The cDNA sequence of glucosyl amylase was cloned downstream of the pPIC9K signal peptide using XhoI + EcoRI double digestion. The recombinant plasmid was linearized with SalI and then electrotransformed into Pichia pastoris GS115 competent cells. Positive clones were screened using MD / -His auxotrophic plates, and multi-copy integrating strains were screened using G418. Positive strains were inoculated into BMGY medium and cultured at 30°C and 250 rpm until OD600 = 6. The culture was then transferred to BMMY medium at pH 6.0, and 1.0% methanol was added for induction. Methanol was added every 24 hours to maintain a stable induction concentration. Culture was stopped after 96 hours of induction. The supernatant was collected by centrifugation at 6000 rpm for 3 min and concentrated through ultrafiltration to obtain the crude enzyme solution. SDS-PAGE electrophoresis revealed a significant band at approximately 66 kDa, consistent with the expected molecular weight of glucosylase, indicating successful expression of glucosylase.

[0026] Take the crude enzyme solution, slowly add saturated ammonium sulfate to 40% saturation, let stand at 4℃ for 2 hours, then centrifuge and discard the precipitate; continue adding ammonium sulfate to the supernatant to 70% saturation, let stand overnight, centrifuge to collect the precipitate, dissolve it with a small amount of buffer and dialyze to remove salt. Load the dialysate onto a pretreated DEAE-cellulose column, elute with low-concentration buffer to remove impurities, then elute the target enzyme with a gradient buffer containing 0-0.5 mol / L NaCl, and collect the active peak fraction. Load the active fraction onto a Sephadex G-75 column, elute with the same buffer, collect in separate tubes, detect enzyme activity, and combine the active peak fractions to obtain purified glucosylamylase.

[0027] DNS colorimetric assay for enzyme activity: 1% Soluble Starch Solution: Take 1g of soluble starch, mix with a small amount of distilled water to form a paste, add boiling water to 100mL, boil for 2 minutes, and then cool. DNS Reagent: Weigh 3.5g DNS, 5g NaOH, and 100g potassium sodium tartrate, dissolve in distilled water, and bring the volume to 1000mL. Store in a brown bottle protected from light. Glucose Standard Solution: Prepare a 0.1mg / mL glucose standard solution. Enzyme Activity Assay: Take 1mL of appropriately diluted enzyme solution, add 1mL of 1% soluble starch solution, react in a water bath for 15 minutes, immediately add 2mL of DNS reagent to terminate the reaction, and follow the same steps as the standard curve. Calculate the reducing sugar content based on absorbance. Enzyme Activity Calculation: Under conditions of 75℃ and pH 4.5, the amount of enzyme required to catalyze the production of 1mg of glucose per minute is defined as 1 enzyme activity unit (U). The enzyme activities of wild-type (GA-WT) and mutant (GA-M) glucoamylase were measured to be 64,300 U / ml and 71,600 U / ml, respectively.

[0028] Example 2 Enzymatic properties of glucoamylase 1. Determination of the optimal pH: The enzyme activities of wild-type (GA-WT) and mutant (GA-M) glucoamylase were detected under different pH buffers (2.0–8.0) to explore the optimal pH value. Enzyme activity was tested at 75°C within the pH range of 2.0–8.0. Three parallel samples were set up for each pH value, and the average value was calculated. The relative enzyme activity was used as a reference for the optimal enzyme activity. The final results are shown below. Figure 1 As shown in the figure. Experimental results show that the optimal pH for both wild-type (GA-WT) and mutant (GA-M) glucoamylase is 4.5. The mutant can maintain more than 80% of the relative enzyme activity between pH 3.5 and 5.0, which is higher than that of the wild-type.

[0029] 2. Determining the optimal temperature: The enzyme activities of wild-type (GA-WT) and mutant (GA-M) glucoamylase were detected under conditions ranging from 30°C to 80°C and pH 4.5. The results are as follows: Figure 2 As shown, the optimal temperature for both wild-type (GA-WT) and mutant (GA-M) glucoamylase is 75℃. The mutant (GA-M) maintains more than 70% relative enzyme activity between 30-75℃, while the wild-type maintains 70% relative enzyme activity between 60-75℃, and the relative enzyme activity decreases significantly outside this temperature range.

[0030] 3. Heavy metal ion tolerance test: The glutamate mother liquor contains trace amounts of heavy metal ions such as copper, mainly originating from corrosion of fermentation equipment or impurities in raw materials. These heavy metal ions may inhibit enzyme activity. To verify the effect of copper ions on enzyme activity, a copper ion concentration gradient was set up: 0.1 mol / L CuSO4 standard solution was diluted with acetate-sodium acetate buffer (pH 4.5) to the following concentration gradients: 0 μmol / L, 1 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L, and 50 μmol / L. Under optimal conditions of 75℃ and pH 4.5, the relative enzyme activities of wild-type (GA-WT) and mutant (GA-M) glucosylamylase were measured in buffers with different copper ion concentrations. Figure 3 As shown, the wild-type (GA-WT) enzyme activity decreased by more than 50% at a copper ion concentration of 10 μmol / L and by 80% at a copper ion concentration of 20 μmol / L. The mutant (GA-M) enzyme activity remained above 70% within a copper ion concentration range of 20 μmol / L, indicating that the mutant (GA-M) has good tolerance to heavy metal ions.

[0031] Example 3

[0032] Using a batch of glutamic acid mother liquor from the company as the research object, the polysaccharides mainly include dextrin, maltose, and maltotriose. The content of dextrin is 0.21 g / L, the content of maltose is usually 0.53 g / L, and the content of maltotriose is usually 0.19 g / L, with a total content of 0.93 g / L. 100 μL of glucoamylase was added per liter of mother liquor, and the total sugar hydrolysis rate (%) was measured at two time points: 30 min and 60 min. Figure 4 As shown, at 30 min, the hydrolysis rates of wild-type glucoamylase (GA-WT) and mutant (GA-M) were 47% and 92%, respectively. At 60 min, the hydrolysis rate of wild-type glucoamylase (GA-WT) was close to 80%, while the mutant (GA-M) achieved complete hydrolysis.

[0033] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the disclosed technical content without departing from the scope of the technical solution of the present invention, resulting in equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the scope of the technical solution of the present invention.

Claims

1. A glucoamylase mutant, characterized in that, The amino acid sequence of the glucosylamylase mutant is shown in SEQ ID NO: 1, or an amino acid sequence that is more than 90% homologous to the above sequence and contains the same mutation site.

2. The glucoamylase mutant of claim 1, wherein, The nucleotide sequence of the glucosylamylase mutant is shown in SEQ ID NO: 2, or a nucleotide sequence that is more than 90% homologous to the above sequence.

3. The gene encoding the glucosylamylase mutant of claim 1 or 2.

4. An expression cassette containing a gene encoding the glucosamine mutant of claim 3.

5. A recombinant expression vector containing the gene of claim 3 or the expression cassette of claim 4.

6. Recombinant cells containing the gene of claim 3, the expression cassette of claim 4, or the recombinant expression vector of claim 5.

7. The recombinant cell of claim 7, wherein, The recombinant cells are recombinant yeast cells.

8. The use of the glucosylamylase mutant according to any one of claims 1-2 in the hydrolysis of polysaccharides.

9. The use of the glucosylamylase mutant according to any one of claims 1-2 in the polysaccharide of hydrolyzed glutamate mother liquor.

10. Use according to claim 8 or 9, characterized in that, The polysaccharide is selected from one or more of starch, dextrin, maltose, and maltotriose.

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