A method for preparing a recombinant sucrase derived from aspergillus oryzae and use thereof

Abstract

The application discloses a preparation method and application of recombinant sucrose invertase derived from Aspergillus oryzae, wherein the amino acid sequence of the recombinant sucrose invertase is shown as SEQ ID NO. 2, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 1 or SEQ ID NO. 3. The optimal reaction pH of the recombinant sucrose invertase is 5.0, the optimal reaction temperature is 50 DEG C, K + , Na + , Zn 2+ , Mn 2+ and Co 2+ have activating effects on the enzyme activity, Fe 2+ , Mg 2+ and Cu 2+ have inhibiting effects on the enzyme activity. The recombinant sucrose invertase can rapidly and effectively hydrolyze sucrose in 70% sucrose syrup, and the sucrose hydrolysis rate can reach more than 98%. The enzyme has good pH stability, good thermal stability and anti-interference ability, and can well meet and adapt to the application requirements of the product in food, medicine and chemical industries.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, specifically to a recombinant sucrose invertase derived from Aspergillus oryzae, its preparation method, and its uses. Background Technology

[0002] Sucrase is a β-D-fructofuranoside hydrolase (EC 3.2.1.26) that hydrolyzes sucrose into glucose and fructose. Because the fructose produced by sucrase hydrolysis of sucrose is strongly dextrorotatory, while sucrose and glucose are levorotatory, sucrase is also called invertase. Sucrase plays a vital role in carbohydrate metabolism and growth and development in organisms, directly participating in plant growth, organogenesis, and sugar transport, and indirectly participating in the regulation of cell differentiation and plant development. Sucrase is specific for glycosidic bonds, catalyzing not only the hydrolysis of sucrose but also the hydrolysis of raffinose and stachyose.

[0003] Sucrase is widely distributed in nature, found in fungi, bacteria, and plants. Microbial sucrase, compared to animal or plant-derived sucrase, has many advantages, including a broader substrate spectrum, more stable enzymatic properties, and ease of production. Natural microbial producers of sucrase mainly include yeasts, molds, and bacteria. Common yeasts include *Hansenula polymorpha*, *Kluyveromyces marxianus*, and *Candida guilliermondii*; common molds include *Aspergillus caespitosus*, *Aspergillus niger*, and *Aspergillus terreus*; and common bacteria include *Arthrobacter globiformis* and *Bifidobacterium breve*.

[0004] In the food industry: Sucrase hydrolyzes sucrose to produce invert sugar syrup, which is sweeter than sucrose, less prone to crystallization, and enhances the texture, flavor, and color of food. Its hygroscopic properties effectively prevent sugar crystallization in almond meringues, gingerbread dough, or nut fillings, keeping the candy smooth and soft. It can also improve product taste and nutritional value by breaking down sucrose, and is widely used in the manufacture of juices, jams, creams, artificial honey, and chocolates. Adding sucrase during the production of fructose syrup can shorten the process and save production costs. Adding sucrase as a preservative to baked goods can maintain freshness, improve product stability, and extend shelf life. Sucrase can also act as a moisture retainer, increasing the water control ability of dairy products and making them easier to shape. Sucrase can be used in combination with other enzymes; for example, sucrase and fructosyltransferase acting together in sucrose solution can produce fructooligosaccharides; sucrase, glucose isomerase, and polyfructase react synergistically to prepare special dietary foods with a three-stage sequential energy supply.

[0005] In the feed industry, sucrase can break down sucrose and other oligosaccharides in feed, releasing monosaccharides and oligosaccharides that are more easily digested and absorbed by animals. This helps prevent bacterial infections and intestinal microbial fermentation caused by oxidation, thus improving the nutritional value of the feed. Furthermore, sucrase also helps prevent digestive system diseases caused by bacterial growth in the digestive tract, thereby increasing animal growth rate and improving overall health.

[0006] In biomedicine, sucrase not only plays a crucial role in carbohydrate digestion but also has a vital function in preventing human diseases, delaying aging, and restoring physical strength. Sucrase is a natural antibacterial and antioxidant agent. It accelerates the hydrolysis of polysaccharides such as sucrose in the stomach to produce monosaccharides. Monosaccharides remain in the stomach for a shorter time, insufficient to undergo toxic fermentation, thus helping to reduce gastric toxicity. Sucrase also has hygroscopic (moisturizing) properties, which can reduce bacterial infections and intestinal fermentation caused by food oxidation.

[0007] In other industries, sucrase is used in the production of biofuels, cosmetics, and paper, as well as in the production of glycerol and lactic acid. Developing bioethanol, a renewable and clean energy source, is a crucial issue in the conversion and utilization of biomass energy. In this context, the production of first-generation bioethanol primarily relies on traditional sugar feedstocks, such as glucose and sucrose, derived from crops like corn, sugarcane, and sugar beets. However, with increasing demand for bioethanol, relying solely on first-generation biomass feedstocks is insufficient to meet the growing market demand and directly competes with food security issues. In second-generation bioethanol production, invertase plays a vital role, hydrolyzing raffinose, fructooligosaccharides, and highly polymerized inulin, enabling the use of various inexpensive substrates, including molasses and Jerusalem artichoke, for metabolic fermentation to produce bioethanol, reducing production costs and improving economic efficiency. The expression level and regulatory mechanism of sucrase are key factors affecting production efficiency and cost control.

[0008] Recent research has focused on site-directed mutagenesis and directed evolution of enzyme molecules, development of novel immobilization vectors, substrate action mechanisms, and screening of natural product inhibitors. However, current research still faces challenges such as a narrow substrate spectrum and susceptibility to product feedback inhibition, limited enzyme activity in natural strains, difficulty in balancing stability and high activity through molecular modification, insufficient stability in extreme environments, difficulties in industrializing immobilization technology, high costs of high-purity formulations, lagging regulatory approvals, and limited application scenarios. These factors collectively restrict the large-scale and high-value application of enzymes. Therefore, it is crucial to explore new high-quality strains and gene resources, screen for high-performance enzyme proteins, construct recombinant engineered strains for efficient expression of sucrose invertase, and develop simplified, green, and low-carbon production processes to truly achieve efficient enzyme production and promote its widespread application in the food processing and renewable resource sectors. Summary of the Invention

[0009] The purpose of this invention is to provide a recombinant sucrose invertase derived from Aspergillus oryzae, its preparation method, and its application, so as to realize the industrial production and application promotion of sucrose invertase.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] The present invention first provides a recombinant sucrose invertase derived from Aspergillus oryzae, the amino acid sequence of which is shown in SEQ ID NO.2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.1 or SEQ ID NO.3.

[0012] Furthermore, the above-mentioned recombinant invertase has the following physicochemical properties:

[0013] ① The fermentation product level can reach 31600 U / mL;

[0014] ② The effective reaction pH is 4.0-6.0, with the highest point being 5.0;

[0015] ③ The effective reaction temperature is 40-50℃, with the highest point being 50℃;

[0016] ④ After treatment at 40-55℃ for 5 minutes, the residual enzyme activity can be maintained at over 90%;

[0017] ⑤ Treatment at pH 4.0-7.0 for 1 hour can maintain more than 80% of the residual enzyme activity;

[0018] ⑥ K + Na + Zn 2+ Mn 2+ and Co 2+ It has an activating effect on enzyme activity, Ca 2+ and Fe 3+ It had no significant effect on enzyme activity, Fe 2+ Mg 2+ and Cu 2+ It has an inhibitory effect on enzyme activity.

[0019] ⑦ Recombinant sucrose invertase can rapidly and effectively hydrolyze sucrose in 70% sucrose syrup, with a sucrose hydrolysis rate of over 98%.

[0020] The present invention also provides a recombinant expression vector carrying the above-mentioned coding gene.

[0021] The present invention also provides a recombinant genetically engineered strain comprising the above-mentioned recombinant expression vector, wherein the recombinant genetically engineered strain uses Pichia pastoris X33 as the host cell.

[0022] This invention also provides a method for preparing the above-mentioned recombinant sucrose invertase, comprising the following steps:

[0023] 1) RNA was extracted from Aspergillus oryzae and cDNA was obtained by reverse transcription;

[0024] 2) The obtained cDNA was used as a template for PCR amplification to obtain the encoding gene AOInv;

[0025] 3) The coding gene AOInv-OP was obtained through sequence optimization, and the optimized gene was synthesized;

[0026] 4) The PCR amplification products encoding the gene AOInv or AOInv-OP are digested stepwise with restriction endonucleases Avr II and Not I, and then ligated with the pPICZα expression vector digested with the same enzymes to form a recombinant expression plasmid.

[0027] 5) Transform the recombinant expression plasmid into Escherichia coli DH5α, expand the culture, and then extract the recombinant expression plasmid from Escherichia coli DH5α;

[0028] 6) The extracted recombinant expression plasmid was linearized with restriction endonuclease Pme I, transformed into Pichia pastoris X33 competent cells by electroporation, and the recombinant strain was obtained after culturing and screening.

[0029] 7) The recombinant strain was fermented to express the sucrose invertase gene, and the expression product, recombinant sucrose invertase, was harvested.

[0030] The present invention also provides the application of the above-mentioned recombinant sucrose invertase in the hydrolysis of sucrose.

[0031] This invention also provides the application of the above-mentioned recombinant sucrose invertase in the food and feed industries.

[0032] The beneficial effects of this invention are as follows:

[0033] This invention obtained a novel sucrose invertase encoding gene, optimized it, and achieved its efficient recombinant expression in Pichia pastoris strain. Furthermore, this invention analyzed the optimal operating temperature, optimal operating pH, pH stability, thermal stability, and the influence of metal ions on enzyme activity of the recombinant sucrose invertase through enzymatic property testing. The results demonstrate that the recombinant sucrose invertase of this invention possesses good pH stability, thermal stability, and anti-interference ability. It can efficiently and thoroughly hydrolyze sucrose in high-concentration syrups, effectively meeting and adapting to the application requirements of the food, feed, and energy industries. Attached Figure Description

[0034] Figure 1 Electrophoresis diagram of the sucrose invertase gene from Aspergillus oryzae; lane M is the marker, and lanes 1 and 2 are the cloned sucrose invertase DNA.

[0035] Figure 2 Electrophoresis diagram of recombinant Pichia pastoris sucrose invertase fermentation broth; lane M is Marker, lanes 1, 2, 3, and 4 are fermentation samples of recombinant Pichia pastoris sucrose invertase.

[0036] Figure 3 Analysis of the optimal reaction temperature of recombinant sucrose invertase.

[0037] Figure 4Temperature tolerance analysis of recombinant sucrose invertase.

[0038] Figure 5 : Optimal reaction pH analysis of recombinant sucrose invertase.

[0039] Figure 6 pH tolerance analysis of recombinant sucrose invertase.

[0040] Figure 7 : Recombinant sucrose invertase hydrolysis reaction curve (45℃).

[0041] Figure 8 : Recombinant sucrose invertase hydrolysis reaction curve (50℃). Detailed Implementation

[0042] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0043] The experimental materials and methods used in the following examples are as follows:

[0044] 1. Strains and vectors

[0045] Escherichia coli DH5α was purchased from Anolun (Beijing) Biotechnology Co., Ltd.; Pichia pastoris X33 and expression vector pPICZα were purchased from Ingen Life Sciences, Inc., USA; Aspergillus oryzae GCMCC 3.800 was purchased from China General Microbiological Culture Collection Center.

[0046] 2. Enzymes and other biochemical reagents

[0047] Restriction endonucleases Avr II, Not I, Pme I, DNA Maker, Protein Maker, and T4 ligase were purchased from BioNTech Biotechnology (Dalian) Co., Ltd.; Pfu DNA synthase was purchased from Fuxinteis Biotechnology (Shenzhen) Co., Ltd.; the RNA extraction kit RNeasy Mini Kit was purchased from Qiagen Enterprise Management (Shanghai) Co., Ltd.; the Primescript doublestrand cDNA synthesis kit was purchased from BioNTech Biotechnology (Dalian) Co., Ltd.; the SanPrep Column PCR Product Purification Kit, SanPreP Column Plasmid Mini-Preps Kit, and SanPreP Column DNA Gel Extraction Kit were purchased from Bioengineering (Shanghai) Co., Ltd.; sucrose, raffinose, fructobiose, fructotriose, fructotetraose, isomaltulose, and inulin were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Zeocin was purchased from Shanghai Sobo Biotechnology Co., Ltd.; agarose was purchased from Ingenium Life Sciences, Inc.; other routine reagents were either domestically produced or imported.

[0048] 3. Culture medium

[0049] Czapek's agar medium: sucrose 30.0g, NaNO3 3.0g, MgSO4·7H2O 0.5g, KCl 0.5g, FeSO4·4H2O 0.01g, K2HPO4 1.0g, agar 15.0g, distilled water to a final volume of 1.0L, pH 6.0-6.5.

[0050] Aspergillus oryzae shake flask culture medium: 40.0g sucrose, 5.0g yeast powder, 1.5g KH2PO4, 0.5g MgSO4·7H2O, distilled water to a final volume of 1.0L, pH 5.5-6.0.

[0051] PTM1 Trace Element Solution: CuSO4·5H2O 7.5g, KI 0.08g, MnSO4·H2O 3.0g, Na2MoO4·2H2O 0.2g, H3BO3 0.02g, CoCl2·6H2O 0.6g, ZnCl2 20g, FeSO4·7H2O 65g, Biotin 0.25g, concentrated sulfuric acid 5mL, and distilled water to a final volume of 1.0L.

[0052] BSM fermentation basal medium: 40g glucose, 0.90g CaSO4, 18.7g K2SO4, 15.5g MgSO4·7H2O, 4.17g KOH, 26.5mL 85% phosphoric acid, and distilled water to a final volume of 1.0L; after sterilization, add 12mL / L of PTM1 trace element solution.

[0053] In addition to the culture media mentioned above, all other culture media used in the following examples were prepared in accordance with the Pichia pastoris operation manual of Ingenium Life Sciences, Inc.

[0054] 4. Experimental Methods

[0055] Unless otherwise specified in the following examples, the experimental methods were performed in accordance with the relevant chapters or sections of the following experimental manuals or literature, including: *Molecular Cloning: A Laboratory Manual* (3rd Edition) by J. Shambrook et al.; *Handbook of Biochemistry and Molecular Biology* (5th Edition) by Roger L. Lundbrard et al.; *Yeast Laboratory Manual* (4th Edition) by John M. Walker et al.; *Principles and Applications of Biochemical Techniques* by Zhao Yongfang et al.; *Biochemical Experiments* by Zhu Jian et al., or according to the kit instructions and product manuals.

[0056] In this invention, all related enzyme activities, enzyme viability, and enzyme activity refer to sucrose invertase activity, which is determined using the DNS spectrophotometric method. Unless otherwise specified, the reaction conditions for enzyme activity determination are: pH 4.5, temperature 20℃, substrate 5.4% (w / v) sucrose solution, and reaction time 30 min. Sucrose invertase hydrolyzes sucrose to produce glucose and fructose, both of which are reducing sugars. They can react with 3,5-dinitrosalicylic acid (DNS reagent) to produce a colorimetric reaction. The intensity of the color is directly proportional to the reducing sugar content. The absorbance is measured at a wavelength of 515 nm, and the reducing sugar equivalent is converted according to the standard curve (glucose) to calculate the sucrose invertase activity.

[0057] Example 1 Cloning of the sucrose invertase encoding gene

[0058] 1. RNA extraction

[0059] Take Aspergillus oryzae GCMCC 3.800 glycerol inoculum, inoculate it onto Czapek's agar plates, and incubate at 30°C for 3 days. Pick a single colony from the plate and inoculate it onto Aspergillus oryzae shake flask medium, and incubate at 30°C with shaking at 200 rpm for 24-28 hours (until the bacterial concentration OD) reaches zero. 600nmAfter reaching a concentration of 0.6 or higher, centrifuge at 12000 rpm for 10 min, discard the supernatant, and collect the bacterial pellet. Transfer the bacterial pellet to a mortar, add liquid nitrogen, and grind into powder. RNA extraction is performed using the RNeasy Mini Kit, and ds cDNA synthesis is performed using oligo dT primers and the Primescript double strand cDNA synthesis kit. Finally, the cDNA is purified using a gel extraction kit. The oligo dT primer sequences are as follows:

[0060] oligo dT:5'-d(TTTTTTTTTTTTTTTTTT)-3'

[0061] 2. Gene cloning

[0062] Comparative analysis was performed on sucrose invertase gene sequences from the genus *Aspergillus* in the database, and upstream and downstream primers AOInv F and AOInv R were designed. The upstream and downstream primers contain Avr II and Not I restriction sites, respectively, and were synthesized by Shanghai Sangon Biotech. The primer sequences are as follows:

[0063] AOInv F: 5'-gggcctaggATGTATCGTAAACCATTCCG-3' (The lowercase part is the bases added to introduce the Avr II restriction site and to change the GC content and annealing temperature of the primers)

[0064] AOInv R: 5'-aaaaagcggccgcTCAGGCGCGGAACCTCTTAA-3' (The lowercase part is the bases added to introduce the Not I restriction site and to change the annealing temperature of the primer)

[0065] Using the above-mentioned cDNA as a template, PCR amplification was performed using pfu DNA synthase and primers AOInv F and AOInv R (template 0.5 μL, forward and reverse primers 1 μL each, total volume 50 μL). The PCR amplification conditions were: 95℃ for 3 min; 95℃ for 20 sec, 52℃ for 30 sec, 72℃ for 1 min 30 sec, 30 cycles; 72℃ for 6 min 30 sec. The PCR amplification products were subjected to 1% agarose gel electrophoresis (see...). Figure 1The target product band was recovered using a gel extraction kit. Then, the product was digested stepwise with restriction endonucleases Avr II and Not I. The digested products were recovered using a PCR extraction kit and ligated with the same digested plasmid pPICZα using T4 ligase. After overnight ligation at 16°C, the ligation product was transformed into *E. coli* DH5α competent cells. Positive colonies AOInv / pPICZα / DH5α were obtained by LB plate selection (using Zeocin as an resistant agent). Plasmids were extracted from the cultures of the positive colonies using a plasmid extraction kit and sent to Sangon Biotech Co., Ltd. for sequencing. The encoding gene AOInv for *Aspergillus oryzae* sucrose invertase was thus obtained. Its nucleotide sequence is shown in SEQ ID NO.1, and the corresponding amino acid sequence is shown in SEQ ID NO.2. It belongs to the GH32 family of sucrose invertases.

[0066] Example 2: Optimization of the sucrose invertase encoding gene

[0067] The nucleotide sequence of the cloned sucrose invertase gene AOInv was analyzed. Low-frequency codons used in Pichia pastoris were replaced with high-frequency codons through synonymous mutations. The nucleotide sequence was optimized by comprehensively considering and rationally weighing various optimization parameters (GC content, shared splice sites, hidden splice sites, SD sequence, unstable sequences, TATA box, termination signal, de novo mRNA synthesis and stability, transcription and translation efficiency, etc.). Simultaneously, the amino acid sequence of this sucrose invertase was analyzed. Synergistic optimization was achieved through sequence or structural optimization of the flexible loop region, hydrogen bond network optimization, consensus sequence design, and combined with molecular stability and molecular interaction analysis.

[0068] The sucrose invertase encoding gene AOInv is 1578 bp in length, encoding 525 amino acids and one terminator, with a GC content of 54.06% (G 26.49%, A 21.99%, T 23.95%, C 27.57%). The optimized sucrose invertase encoding gene is denoted as AOInv-OP, also 1578 bp in length, encoding 525 amino acids and one terminator, with a GC content of 52.15% (G 26.17%, A 23.38%, T 24.46%, C 25.98%). The nucleotide sequence of the AOInv-OP gene is shown in SEQ ID NO.3, and the amino acid composition remains unchanged before and after optimization.

[0069] Example 3 Construction of a recombinant engineered strain of Pichia pastoris containing sucrose invertase

[0070] Using the optimized sequence SEQ ID NO.3 from Example 2 as a template, PCR amplification was performed using pfu DNA synthase and primers AOInv-OP F and AOInv-OP R (template 0.5 μL, forward and reverse primers 1 μL each, total volume 50 μL). The PCR amplification conditions were: 95℃ for 3 min; 95℃ for 20 sec, 52℃ for 30 sec, 72℃ for 1 min 30 sec, 30 cycles; 72℃ for 6 min 30 sec. The PCR amplification products were subjected to 1% agarose gel electrophoresis, and the target product band was recovered using a gel recovery kit. Then, the restriction endonucleases Avr II and Not I were used for stepwise digestion. After the digestion products were recovered using a PCR recovery kit, they were ligated with the plasmid pPICZα, which had been digested with the same enzymes, using T4 ligase. After ligation overnight at 16℃, the ligation product was transformed into E. coli DH5α competent cells, and positive colonies DEX-OP / pPICZα / DH5α were obtained by LB plate selection (using Amp as an resistant agent).

[0071] AOInv-OP F: 5'-gggcctaggATGTATCGTAAACCTTTTCG-3' (The lowercase part is the bases added to introduce the Avr II restriction site and to change the GC content and annealing temperature of the primers)

[0072] AOInv-OP R: 5'-aaaaagcggccgcTCAGGCTCGGAAGCGCTTAA-3' (The lowercase part is the bases added to introduce the Not I restriction site and to change the annealing temperature of the primer)

[0073] The prepared AOInv-OP / pPICZα / DH5α and the prepared AOInv / pPICZα / DH5α from Example 2 were inoculated into LB liquid medium and cultured overnight at 37°C. The plasmids AOInv-OP / pPICZα and AOInv / pPICZα were then extracted using a plasmid extraction kit. Subsequently, the plasmids were digested with the restriction endonuclease Pme I, and the large fragments were purified by gel extraction to obtain the linear DNA containing the mutant gene required for yeast transformation. Pichia pastoris strain X33 was transformed using electroporation, and after screening and identification, recombinant Pichia pastoris strains AOInv-OP / pPICZα / X33 and AOInv / pPICZα / X33 were obtained.

[0074] Example 4: Preparation of recombinant invertase by Pichia pastoris fermentation

[0075] The recombinant Pichia pastoris strain AOInv-OP / pPICZα / X33 or AOInv / pPICZα / X33 positive clones constructed in Example 3 were inoculated into 150 ml of YPD medium (2% glucose, 1% yeast extract, 2% peptone, the remainder being distilled water, pH 6.0-6.5) and cultured at 30°C with shaking at 250 rpm until OD500. 600nm =0.3~0.5 (about 20 hours), then transfer (8% inoculum) to a 5L fermenter containing 3L of BSM fermentation basal medium for fermentation.

[0076] Fermentation is divided into three stages, and the pH of the system is controlled throughout the process using concentrated ammonia (25%).

[0077] Cell growth stage (0~24h): Culture temperature 30℃; initial pH of BSM fermentation basal medium 6.0, adjusted to maintain pH 6.2~6.3 during the process, and PTM1 trace element solution continuously added at a rate of 4.0ml / hr. Stir and aerate for 20-24hr. As the cells grow, dissolved oxygen gradually decreases until the carbon source is exhausted, at which point dissolved oxygen rebounds sharply, and the wet weight of the cells can reach 92~95g / L.

[0078] Carbon source feeding stage (24-32h): The culture temperature is 30℃, and the pH of the system is maintained at 6.2-6.3 with concentrated ammonia. A solution containing 25% (w / v) glucose and 12 ml / L PTM1 trace element solution, prepared with distilled water, is continuously added at a rate of 25-30 ml / hr for 5-6 hours, while adjusting the aeration rate and stirring speed to ensure dissolved oxygen (DO) > 25%. After feeding, the cells are starved for 2 hours to consume the remaining carbon source. By the end of this stage, the wet weight of the cells can reach 190-195 g / L.

[0079] Methanol induction phase (32~190h): The culture temperature was lowered to 26~28℃, the pH of the system was maintained at 6.2~6.3 with concentrated ammonia, and 100% methanol (containing 12mL / L PTM1 trace element solution) was used as the sole carbon source. The initial methanol feeding rate was 9mL / h, which was increased to 12~16mL / h after 3h of induction. The final concentration of methanol in the culture medium was controlled to not exceed 0.3% (v / v), and the aeration rate and stirring speed were adjusted to maintain dissolved oxygen at 20%. When fermentation lasted 190 hours, the wet weight of the cells reached 315-320 g / L. The expression levels of recombinant sucrose invertases AOInv-OP / pPICZα / X33 and AOInv / pPICZα / X33 (expressed as enzyme activity in the supernatant of the fermentation broth collected after centrifugation at 12000 r / min for 5 min at room temperature) reached 31600 U / mL and 12350 U / mL, respectively, indicating that the Aspergillus oryzae sucrose invertase gene was efficiently recombinantly expressed in Pichia pastoris.

[0080] The supernatant of recombinant invertase was collected and analyzed by SDS-PAGE. The results are as follows: Figure 2 As shown.

[0081] Example 5 Enzymatic characterization of recombinant sucrose invertase

[0082] The enzyme activity of the recombinant sucrose invertase (AOInv-OP / pPICZα / X33 fermentation broth supernatant) prepared in Example 4 was determined using different substrates (sucrose, raffinose, fructose-dissociate, fructotriose, fructotetraose, isomaltulose, and inulin). The specificity of the enzyme for each substrate was compared using relative enzyme activity (Table 1). The results showed that this recombinant sucrose enzyme preferentially recognized β-D-fructofuranosyl bonds. The longer the fructose oligosaccharide chain, the lower the relative enzyme activity, with the activity order being sucrose > raffinose > fructotriose > fructotetraose > inulin. The steric hindrance effect caused by the elongation of the fructose oligosaccharide chain hindered the binding of the substrate to the enzyme's active site. Fructose-dissociate contains only Fruβ2-1Fru glycosidic bonds and no glucose residues, making it difficult to fit the binding pocket of the enzyme's active site; only extremely low hydrolytic activity was detected. Isomaltulose lacks β-D-fructofuranosyl bonds and can hardly be hydrolyzed by this enzyme.

[0083] Table 1. Substrate Specificity of Recombinant Sucrose Invertase

[0084]

[0085] The recombinant sucrose invertase (AOInv-OP / pPICZα / X33 fermentation broth supernatant) prepared in Example 4 was subjected to enzymatic reactions in a 50 mM Na2HPO4-C6H8O7 (pH 5.0) buffer system at different temperatures (30℃-70℃) to determine the optimal reaction temperature. The results showed that the effective reaction temperature of the recombinant sucrose invertase was 40-50℃. Figure 3 The highest point was 50℃. Residual enzyme activity was measured after treatment at different temperatures (40℃-80℃) for 5 minutes to conduct a thermostability study. The results showed that after treatment at 40-55℃ for 5 minutes, the residual enzyme activity of the recombinant sucrose invertase remained above 90%. Figure 4 The above results indicate that the recombinant sucrose invertase has a good temperature range and heat resistance.

[0086] Enzymatic reactions were performed at 50℃ and different pH values ​​(50mM Na2HPO4-C6H8O7 buffer) to determine the optimal pH for recombinant invertase (AOInv-OP / pPICZα / X33 fermentation broth supernatant). The results showed that the effective reaction pH for invertase was 4.0-6.0. Figure 5The highest value was pH 5.0. The recombinant sucrose invertase prepared in Example 4 was diluted 5-fold with 50mM Na2HPO4-C6H8O7 buffer (pH 3.0-8.0) at different pH values, and then treated at room temperature for 1 hour before measuring residual enzyme activity to study the pH stability of the sucrose invertase. The results showed that after treatment at pH 4.0-7.0 for 1 hour, the recombinant sucrose invertase could retain more than 80% of its residual enzyme activity. Figure 6 The above results indicate that this sucrose invertase has a wide applicable pH range and good pH stability.

[0087] Different types of metal ions (K+, K+, K+, K+) were added to the substrate solution. + Na + Fe 2+ Zn 2+ Ca 2+ Mg 2+ Mn 2+ Cu 2+ Fe 3+ and Co 2+ The final concentrations of metal ions were set at 1 mM and 5 mM, respectively, with a blank control group without added metal ions. Enzyme activity was measured at pH 5.0 and 50℃ to analyze the effect of metal ions on the activity of recombinant sucrose invertase (AOInv-OP / pPICZα / X33 fermentation broth supernatant) (Table 2). The results showed that K... + Na + Zn 2+ Co 2+ The enzyme can be activated at both 1 mM and 5 mM concentrations; Zn 2+ The activation effect is concentration-dependent; at 1 mM, it has no significant effect on enzyme activity, while at 5 mM, it shows an activating effect; Ca 2+ and Fe 3+ It had no significant effect on enzyme activity within the tested concentration range; Fe 2+ Mg 2+ and Cu 2+ It has an inhibitory effect on recombinant sucrose invertase, and the effect of Fe increases with increasing ion concentration. 2+ with Fe 2+ The inhibitory effect is enhanced.

[0088] Table 2 Effects of ions on recombinant sucrose invertase

[0089]

[0090] Example 6 Hydrolysis of sucrose by recombinant invertase

[0091] Hydrolysis conditions: sucrose solution mass concentration: 70% (m / m), reaction pH: 5.0, and reaction temperatures set at 45℃ and 50℃, respectively.

[0092] After the sugar solution was prepared, the pH was adjusted to 5.0. Once the system temperature rose and stabilized at 45℃ / 50℃, the recombinant sucrose invertase (AOInv-OP / pPICZα / X33 fermentation broth supernatant) prepared in Example 4 was added to the sugar solution. The enzyme dosage was 30 U / g sugar solution. The hydrolysis reaction was carried out under the above conditions for 24 hours. Samples were taken midway to determine the reducing sugar content and calculate the sucrose conversion rate. The results are as follows: Figure 7 and Figure 8 As shown in the figure. The results indicate that this recombinant sucrose invertase can rapidly and effectively hydrolyze sucrose in 70% sucrose solution, with a sucrose hydrolysis rate of over 98%, making it well-suited for the production of fructose syrup from sucrose.

[0093] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A recombinant invertase derived from Aspergillus oryzae, characterized in that: The amino acid sequence of the recombinant sucrose invertase is shown in SEQ ID NO.

2.

2. A gene encoding the recombinant sucrose invertase as described in claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.1 or SEQ ID NO.

3.

3. A recombinant expression vector, characterized in that: The recombinant expression vector carries the gene as described in claim 2.

4. A recombinant genetically engineered strain, characterized in that: The recombinant genetically engineered strain comprises the recombinant expression vector as described in claim 3.

5. The recombinant genetically engineered strain according to claim 4, characterized in that: The recombinant genetically engineered strain uses Pichia pastoris as the host cell.

6. A method for preparing recombinant invertase, characterized in that: Fermentation culture of the recombinant genetically engineered strain according to any one of claims 4 to 5 induces the expression of recombinant sucrose invertase in the recombinant genetically engineered strain.

7. The preparation method according to claim 6, characterized in that: The fermentation culture includes seed culture, cell growth stage, carbon source feeding stage, and methanol induction stage: The seed culture was carried out by inoculating the recombinant genetically engineered strain into YPD culture medium and incubating it at 30°C with shaking at 250 rpm until the OD reached its limit. 600nm It is 0.3~0.5; The cell growth stage is as follows: the seed culture is transferred to BSM fermentation basal medium at an inoculation rate of 8%, and cultured at 30°C. The pH of the system is maintained at 6.2-6.3 by adjusting the concentration of concentrated ammonia water. PTM1 trace element solution is continuously added at a rate of 4.0 mL / h until the carbon source is exhausted. The carbon source feeding stage is as follows: the culture temperature is 30℃, the pH of the system is maintained at 6.2~6.3 with concentrated ammonia water, a solution containing 25% glucose and 12mL / L PTM1 trace element solution is added at a rate of 25~30mL / h, and the addition is continued for 5~6h. The aeration rate and stirring speed are adjusted to make dissolved oxygen (DO) > 25%. After the feeding is completed, the system is starved for 2h to consume the remaining carbon source. The methanol induction phase is as follows: the culture temperature is lowered to 26-28℃, the pH of the system is maintained at 6.2-6.3 with concentrated ammonia, methanol containing 12 mL / L PTM1 trace element solution is used as the sole carbon source, the initial methanol feeding rate is 9 mL / h, and it is increased to 12-16 mL / h after 3 h of induction. The final methanol concentration in the culture medium is controlled to not exceed 0.3%, and the aeration rate and stirring speed are adjusted to maintain dissolved oxygen at 20% to induce the expression of recombinant sucrose invertase.

8. The application of the recombinant sucrose invertase as described in claim 1 in the hydrolysis of sucrose.

9. The application of the recombinant sucrose invertase as described in claim 1 in the food and feed industries.

Images