Trehalase mutant and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a trehalase mutant and application thereof. The amino acid sequence of the trehalase mutant is as shown in SEQ ID No. 4. Corresponding engineering bacteria are constructed by means of genetic engineering, fermentation performance verifies that the enzyme activity of the obtained trehalase mutant can reach 4000 U / mL or above, the catalytic pH and temperature range is wide, the optimal pH is 4.0, and the optimal temperature is 60 DEG C. The enzyme activity, heat-resistant stability and pH-resistant stability of the mutated trehalase are greatly improved, so that the production process and product metabolism of a yeast extract are more controllable, the utilization rate of raw materials in industrial production is effectively improved, and the trehalase mutant has a very good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a trehalase mutant and application thereof. Background Art

[0002] Glycoside hydrolases (GHs) are important functional enzymes in the sugar metabolism process, mainly responsible for the hydrolysis of disaccharides, polysaccharides and other carbohydrate complexes in organisms. Trehalase belongs to the GH15, GH37 or GH65 family in GHs. Trehalase is an enzyme that can hydrolyze trehalose and is widely distributed in organisms such as microorganisms, insects and mammals. Trehalase has extremely important physiological functions for organisms. For example, trehalose can provide energy for insects. By inhibiting the activity of trehalase, insect growth is restricted and stress response is blocked, so as to achieve the purpose of insect control. In plants, trehalase participates in the regulation of sugar metabolism (sucrose, starch, etc.), giving plants stress resistance. For fungi and bacteria, trehalase plays a variety of roles. Trehalase is an important functional enzyme for yeast to resist external pressure and a neutral osmotic protectant compatible with bacteria.

[0003] In the process of industrial production of amylase products, yeast can use carbohydrate raw materials to synthesize trehalose. Trehalose is the main component of unusable residual sugar in the fermentation broth. When too much trehalose is produced in the fermentation system, the utilization rate of the raw materials will decrease. Therefore, in industrial production, it is often necessary to add trehalase to the enzyme preparation to degrade trehalose into glucose that can be used to reduce the consumption of carbohydrate raw materials and achieve the purpose of increasing production. In addition, yeast extract is widely used as a natural culture medium in microbial fermentation. Its ingredients usually contain a high content of trehalose. At present, studies have shown that when trehalose is used as part of the culture medium for microbial fermentation, it not only has no beneficial effect on cell growth but may also interfere with the metabolism of microorganisms. Based on this premise, trehalase can be added in the production process of yeast extract to convert trehalose into glucose, which can reduce the impact on microorganisms, make the culture process and product metabolism more controllable, and improve the utilization efficiency of yeast powder.

[0004] Currently, there are few studies on trehalase, which mainly focus on the expression of recombinant proteins and the determination of enzymatic properties. In addition, the expression and enzyme activity of recombinant proteins are both low. E. coli BL21 was used as the expression host to express Microvirga spThe trehalase from Microvirga sp. strain MC18 has an enzyme activity of only 24 U / mg, an optimum temperature of 40°C, and an optimum pH of 7.0 (Chaonan D, Qiwen F, Xu L, et al. Expression and characterization of a novel trehalase from Microvirga sp. strain MC18[J]. Protein Expression and Purification, 2021, 182105846-105846.). The most studied is neutral trehalase, while acid trehalase is less studied. Mitsuhiro et al. E. coli BL21 was used as the expression host to express trehalase from Sulfolobus acidocaldarius, with an enzyme activity of 48.4 U / mg, an optimum temperature of 60-70°C, and an optimum pH of 3.7-4.0 (Mitsuhiro Y, Takeshi O, Masahiro K, et al. Two trehalose-hydrolyzing enzymes from Crenarchaeon Sulfolobus acidocaldarius exhibit distinct activities and affinities toward trehalose[J]. Applied microbiology and biotechnology, 2018, 102(10): 4445-4455.).

[0005] In summary, the application and development of trehalase in industrial production is still limited by many factors. The main reasons are: 1. The expression level of the original strain is low and the enzyme production rate is low. 2. Trehalase has poor stability and is easy to inactivate. Therefore, improving the trehalase production level of enzyme-producing strains and improving the enzymatic properties of trehalase are issues that need to be solved urgently. Summary of the invention

[0006] In view of the above technical problems, the present application uses a strain of Pseudomonas CAO12-1 producing trehalase as the experimental material, and obtains a mutant trehalase-producing strain after mutagenesis and screening, which can produce trehalase with excellent and stable enzymatic properties, and further constructs the corresponding Pichia pastoris engineered strain through genetic engineering means to efficiently express the mutant trehalase gene. The trehalase obtained in the present application can meet the needs of industrial production of amylase, effectively improve the utilization rate of yeast extract, and reduce production costs, which is of great significance.

[0007] The technical solution of the present invention is as follows: One of the technical solutions provided by the present invention is a trehalase mutant, wherein the trehalase mutant is obtained by mutating the phenylalanine at position 76 to isoleucine, the glycine at position 230 to arginine, the proline at position 265 to leucine, and the lysine at position 439 to asparagine on the basis of the wild-type trehalase shown in SEQ ID NO.2, and the amino acid sequence of the trehalase mutant is shown in SEQ ID NO.4; The enzymatic properties of the trehalase mutant are as follows: (1) Optimum temperature: The enzyme activity is stable at 50℃-65℃, and the optimal temperature is 60℃; (2) Optimum pH: The enzyme activity is stable at pH 3.0-5.0, and the optimal action pH is 4.0; (3) Stability: After being treated at pH 4.0 and 60°C for 60 min, the enzyme activity still retained more than 85%, indicating good heat stability. After being treated at pH 2.0-4.0 for 60 min, the remaining enzyme activity could be maintained at more than 90%. After being treated at pH 5.0-7.0 for 60 min, the remaining enzyme activity could be maintained at more than 70%, indicating excellent acid resistance.

[0008] The present invention also provides a gene encoding the above-mentioned trehalase mutant; Furthermore, the nucleotide sequence of the gene encoding the trehalase mutant is shown in SEQ ID NO.3.

[0009] The second technical solution provided by the present invention is a recombinant vector or recombinant strain comprising the above-mentioned trehalase mutant encoding gene; Furthermore, the expression vector used in the recombinant vector is a pPIC9K plasmid; Furthermore, the host cell used by the recombinant strain is Pichia pastoris GS115; Furthermore, the recombinant bacteria is obtained by connecting the gene encoding the trehalase mutant shown in SEQ ID NO.3 to the expression vector pPIC9K and expressing it in Pichia pastoris GS115.

[0010] The third technical solution provided by the present invention is the application of the recombinant vector or recombinant strain described in the second technical solution, especially in the production of the trehalase mutant described in the first technical solution.

[0011] A fourth technical solution provided by the present invention is the application of the trehalase mutant described in the first technical solution, especially in the hydrolysis of trehalose; More particularly, in the application in the industrial production of amylase, by adding trehalase, the unusable trehalose produced in the production process of amylase is removed, and the trehalose is degraded into usable glucose, thereby improving the utilization rate of raw materials and greatly improving the yield of the target product; More particularly, it is used in the production of yeast extracts. Trehalase is added during the production of yeast extracts to convert trehalose into glucose, which reduces the impact of trehalose on microorganisms, makes the culture process and product metabolism more controllable, and improves the utilization efficiency of yeast powder.

[0012] The beneficial effects of the present invention are: 1. The present invention discloses a new trehalase mutant, which has the characteristics of high enzyme activity and good stability. The enzyme activity of the mutant fermentation broth can reach more than 4000U / mL.

[0013] 2. The optimum reaction pH of the trehalase obtained by the present invention is 4.0, and compared with the wild-type trehalase, the acid resistance is greatly improved.

[0014] 3. The trehalase obtained by the present invention retains more than 85% of its enzyme activity after being treated at pH 4.0 and 60°C for 60 minutes. The residual enzyme activity can be maintained at more than 90% after being treated at pH 2.0-4.0 for 60 minutes, and has excellent heat and acid resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the optimum temperature curve of wild-type trehalase and trehalase mutants.

[0016] Figure 2 This is the optimum pH curve of wild-type trehalase and trehalase mutants.

[0017] Figure 3 The relative enzyme activity change curve of trehalase mutants treated at different temperatures.

[0018] Figure 4 Comparison of relative enzyme activities of wild-type trehalase and trehalase mutants treated at 60°C.

[0019] Figure 5 The relative enzyme activity changes of trehalase mutants treated at different pH values. DETAILED DESCRIPTION

[0020] The content of the present invention is further described below in conjunction with the examples, but the protection scope of the present invention is not limited thereto. The operating methods not described in detail in the examples are all conventional operating methods known to those skilled in the art.

[0021] The following definitions are used in the present invention: 1. Nomenclature of amino acid and DNA sequences The generally accepted IUPAC nomenclature for amino acid residues is used, either in the form of three-letter or one-letter codes. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0022] 2. Identification of Trehalase Mutants The mutated amino acid in the mutant is represented by "original amino acid + position + replaced amino acid". For example, Phe 76Ile means that the amino acid at position 76 is replaced by isoleucine from wild-type phenylalanine, and the position number corresponds to the amino acid sequence number of the wild-type trehalase in SEQ ID NO.2.

[0023] In the present invention, CAO-2 represents the original trehalase from Pseudomonas sp., and the lowercase italics are cao-2 Indicates the gene encoding the original trehalase CAO-2, CAO-4 indicates a trehalase mutant, in lowercase italics cao-4 Indicates the coding gene of trehalase mutant CAO-4, the specific information is as follows:

[0024] The amino acid sequence of the wild-type trehalase (CAO-2) derived from the Pseudomonas strain of the present invention is shown in SEQ ID NO.2: MGGRGRPSERGGAVLDSRVRVVTFSEARHPSRRRALTGFDEEARTSRKHSQDISEWEGLGLRLLREPRLGGSGLWFAGMAPSVLGLGGVWSGCLAQTLVIFLWLEGASGWWVMYPNHLHTQIVILSSCFWENRGGPPGVRSFYQCPRSTKFSIFIKQMLGLQHWVGREVGGVRRAGRAGAVGLWRGRRQPRSAGEGGSRSAHVRSIAAHPPLPLGESFYPPPAGPRRLIGLPLARVRASVHPPASGGGEEALPGSGSRGPENWPPPLGPAPQSYILLVFWAGLKADLVCGRCPAGELNRCLVKYMVGARAGGGDGQGAAAGRVQARFRLELPQEGRAEPDLHRALRGVEGRSEGLASEDRPGPGRIPSPLFPCYVTLGLFWRQEALALPKSLVVISKGAAVEAGRRPHPSPEGGGECCNTFLGVLCCLNSSLSRRWAGKSEKKASVSITSPVRALSTPMNASLLFACDFVLYIKFELEENYTHESNYRLLLLVIRLGVFSIEVIGGTRLPTDDRHAHPSFILSCLRSLLQYEITVSRVRLCKQNFNHFAQYFISISPAPSAALSKGILEHSLPHFHLLYWLIQPLDRALAFSLSSKSDDSQTDHTPQIEAVAGASTLQFFYNSLVHFLLILCLDPLHLTELLTINGFQVDVSSPNIT* The amino acid sequence of the trehalase mutant (CAO-4) involved in the present invention is shown in SEQ ID NO.4: * The present invention is described in detail below in conjunction with specific embodiments.

[0025] Example 1 Acquisition of Trehalase Mutants Culture medium: LB solid medium.

[0026] Seed medium: LB liquid medium.

[0027] Liquid fermentation medium: glucose 2%, peptone 0.6%, beef extract 0.3%, K2HPO4 0.12%, MgSO4·7H2O 0.03%, pH 7.2.

[0028] UV mutagenesis (1) Preparation of bacterial suspension: Transfer the colonies from the Pseudomonas CAO12-1 storage slant to the LB plate culture medium and culture at 37°C for 48 hours. Pick the fully grown activated Pseudomonas colonies and inoculate them into 5 mL of seed culture medium. Culture them at 37°C, 200 rpm for 6 hours. Take 50 μL of the above seed solution and culture it overnight in a 50 mL conical flask containing seed culture medium. Examine the cultured bacterial solution under a microscope. After the microscopic examination is correct, centrifuge the bacterial solution (3000 rpm, 15 min) and discard the supernatant. Resuspend the bacteria with sterile saline and wash 2 to 3 times. Finally, count the cells directly under a microscope using a hemocytometer and adjust the cell concentration to 10. 8 Pieces / mL.

[0029] (2) Screening for optimal mutagenesis conditions: Prepare LB plates in advance and turn on the UV light 20 minutes in advance to stabilize the light. 8 The bacterial suspension was diluted to 10 2 / mL, draw 100µL of bacterial suspension and drop it in the center of the plate, place the plate 20cm away from the 30W UV lamp, and cover it with a culture dish lid. Open the lid and start the timing immediately. Irradiate under the UV lamp for 0s, 30s, 60s, 90s, 120s, and 150s respectively. The timing ends when the lid is closed. Three culture media are set as repeats at each time. The UV treatment process needs to be protected from light. After the treatment, wrap the culture dish with a light-shielding cloth immediately, and culture it upside down in a 28℃ light-proof constant temperature incubator for 48 h. Count the number of colonies in each culture dish, and use the plate directly cultured without UV treatment after coating as a control. Calculate the lethality according to formula 1, and select the mutagenesis conditions corresponding to the lethality of 75%~80% as the best mutagenesis conditions.

[0030] Lethality rate (%) = 1-(number of surviving strains in the treatment group / number of surviving strains in the control group) × 100% Formula 1 (3) UV mutagenesis treatment: dilute the bacterial suspension to 10 2 / mL, aspirate 100µL of bacterial suspension and add it to LB solid medium plate, and perform UV mutagenesis treatment on the strain according to the optimal conditions determined by the above experiment. Compared with the control plate, select the colonies with good growth for preliminary screening.

[0031] Initial screening of mutant strains: Pick the colonies that grew well in the previous step and inoculate them into 100 mL LB liquid fermentation medium. Cultivate them in a shaker at 37°C and 200 rpm for 72 h, and collect the bacteria by centrifugation at 5000 rpm for 20 min. Wash twice with 0.03 mol / L sodium phosphate buffer at pH 7.2, resuspend the bacteria with 9.8 mL sodium phosphate buffer, add 0.2 mL toluene, and react at 37°C and 200 rpm for 2 h. Add 10 mL of 10% trehalose solution, react at 37°C and 200 rpm for 10 h, and then inactivate the enzyme in a boiling water bath for 10 min. Collect the supernatant after centrifugation at 5000 rpm for 20 min. Qualitative analysis of sugars in the supernatant was performed by thin layer chromatography.

[0032] Rescreening: The strains that were qualitatively analyzed to contain trehalase in the initial screening were rescreened and inoculated into 100 mL of liquid fermentation medium. They were cultured at 37°C and 200 rpm for 72 h, and centrifuged at 5000 rpm for 20 min to collect the bacteria, weigh the wet weight, and measure the moisture content. The sugar content in the supernatant was quantitatively analyzed by HPLC, and the sample volume was 10 μL. Six strains with the lowest trehalose content were screened out.

[0033] Shake flask rescreening: The 6 mutant strains screened in the previous step were inoculated into fermentation shake flasks for fermentation at 30°C, 200 rpm, and the fermentation enzyme activity of each mutant strain was determined by spectrophotometry after 62 hours of culture. After the shake flask fermentation test, a strain CAO12-2 with a high trehalase production was screened.

[0034] The trehalase protein molecules produced by the original strain CAO12-1 and the mutant strain CAO12-2 were subjected to protein amino acid sequence analysis (Beijing Biotech Biotechnology Co., Ltd.), and the amino acid sequence of the trehalase from the wild-type Pseudomonas was obtained as shown in SEQ ID NO.2, named CAO-2; the amino acid sequence of the trehalase mutant from the mutagenic strain Pseudomonas CAO12-2 was as shown in SEQ ID NO.4, named CAO-4. The nucleotide sequence of the coding gene of the trehalase mutant CAO-4 was obtained by gene comparison and software analysis as shown in SEQ ID NO.3.

[0035] Shenzhen BGI Co., Ltd. was commissioned to synthesize the nucleotide sequence of the wild-type trehalase shown in SEQ ID NO.1 and the nucleotide sequence of the mutant trehalase shown in SEQ ID NO.3, which were named cao-2 and cao-4 , and conduct subsequent experiments.

[0036] Example 2 Construction and transformation of recombinant vector Construction of the recombinant vector Primers were designed based on the full-length genes of SEQ ID NO.1 and SEQ ID NO.3, and then the pPIC9K plasmid was double-digested, and the wild-type Pseudomonas genome and the mutant Pseudomonas genome were used as templates, respectively, and connected using a seamless cloning kit to obtain the recombinant plasmid pPIC9K- cao-2 and pPIC9K- cao-4 .

[0037]

[0038] The PCR amplification system was as follows: total system 25 μL: 100 μM upstream primer 1.0 μL, 100 μM downstream primer 1.0 μL, template 1.0 μL, 5 U / μL phanta enzyme 12.5 μL, ddH2O 9.5 μL.

[0039] The PCR amplification conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 55 s, 30 cycles; and extension at 72°C for 5 min.

[0040] 2. Heat shock transformation The E. coli DH5α competent cells prepared in advance were taken out from -80℃, quickly placed on ice to thaw, and 10 μL of the recombinant plasmid pPIC9K- cao-2 or pPIC9K- cao-4 , gently mix, ice bath for 30min. Heat shock at 42℃ for 90s, then quickly cool in an ice bath for 2min. Add 900μL LB medium, 30℃, 200rpm / min shaking culture for 60min, so that the bacteria can return to normal growth state and express the antibiotic resistance gene encoded by the plasmid. Centrifuge at 2500g for 3min, discard 900μL supernatant, resuspend the bacteria with the remaining culture medium and evenly apply it on LB solid culture medium with resistance (ampicillin 100μg / mL). After the bacterial solution is sucked dry, invert the plate and culture at 37℃ for 12~16h. Select positive colonies and send them for sequencing. The sequence that meets the sequence of SEQ ID NO.1 and SEQ ID NO.3 is the correct recombinant strain. After the sequencing is correct, pick 10 single colonies, shake and culture at 30℃ for 6~8h, and add glycerol to preserve the strain.

[0041] Example 3 Expression and purification of recombinant vector in Pichia pastoris MD plate medium formula: 1.34% YNB; 4×10 -5 % biotin; 2% glucose; 2% agarose.

[0042] BMGY medium formula: 1% yeast extract, 2% tryptone, 1% glycerol, 10% potassium phosphate buffer, 1.34% YNB, 4×10-5 % Biotin.

[0043] BMMY medium formula: 1% yeast extract, 2% tryptone, 10% potassium phosphate buffer, 1.34% YNB, 4×10 -5 % Biotin.

[0044] Use a plasmid extraction kit to extract the correctly sequenced pPIC9K- cao-2 and pPIC9K- cao-4 Plasmid, restriction endonuclease BglⅡ linearized it, recovered the product, and transformed into Pichia pastoris GS115 competent cells using the method of Example 2. The obtained transformants were spread on MD plates, cultured in a 30°C incubator for 48 hours, and several clones were picked into a centrifuge tube containing 3mL BMGY medium. They were cultured at 30°C and 220rpm for 48 hours. The obtained bacterial solution was centrifuged at 4500rpm for 5 minutes, and the bacteria were resuspended in 1 mL of BMMY medium containing 0.5% methanol and continued to be induced for 48 hours. The obtained bacterial solution was spread on MD medium plates, positive colonies were selected, and samples were sent for sequencing. The sequence that met the column described in SEQ ID NO.3 was the correct recombinant strain GS115- cao-4 ; The sequence is consistent with the sequence of SEQ ID NO.1, which is the correct recombinant strain GS115- cao-2 .

[0045] Activation of strains: The trehalase mutant obtained in the previous step was recombined into strain GS115- cao-4 and wild-type trehalase recombinant strain GS115- cao-2 Each was inoculated into 50 mL of liquid YPD medium containing ampicillin at a 1% inoculum and cultured at 30 °C and 200 rpm with shaking for 72 h; Transfer of strains: 3 mL of the above activated strain was inoculated into 300 mL of BMGY medium containing ampicillin. After shaking culture at 30°C and 200 rpm for 48 h, the culture was centrifuged at 4500 rpm for 5 min. The cells were collected and transferred into 200 mL of BMMY medium. The culture was induced for 48 h, and 0.5% methanol was added every 24 h.

[0046] Trehalase purification: After the induction culture, the fermentation broth was collected by centrifugation at 12000 rpm for 5 min. 200 mL of the fermentation broth was concentrated to 15 mL by rotary evaporation under reduced pressure and then concentrated to 10 mmol·L at pH 4.0. -1The enzyme was dialyzed overnight in a citric acid-sodium hydrogen phosphate buffer. Finally, the dialyzed enzyme solution was purified by an ion purification column, and the eluted protein was collected and the trehalase activity was determined (the definition of enzyme activity and the method for determining enzyme activity are described in detail in Example 5). The results are shown in the following table.

[0047] GS115- cao-2 and GS115- cao-4 Trehalase activity

[0048] Example 4 Verification of recombinant bacterial fermentation performance Seed medium formula: 1.0% yeast extract, 2.5% peptone, 1.0% diammonium phosphate, 1.34% YNB, 4×10 -5 % biotin, 1.0% glycerol, pH 6.0.

[0049] Fermentation medium formula: 1.5% yeast extract, 2.5% peptone, 1.0% diammonium phosphate, 0.5% dipotassium phosphate, 1.34%, YNB, 4×10 -5 % biotin, 1.5% methanol, pH 6.0.

[0050] The recombinant strain GS115- cao-2 and GS115- cao-4 The specific method is as follows: 3 mL of the recombinant strain culture liquid was inoculated into 300 mL of seed culture medium, and cultured at 30°C and 200 rpm until OD 600 When the fermentation temperature is about 6-8, 100 mL of seed liquid is added to a 30 L fermentation medium fermentation tank, and the culture is continued for 120 h at 30 ° C, pH 5.0, 500 r / min. After the fermentation liquid is centrifuged, the trehalase activity in the supernatant is determined.

[0051] Two recombinant Pichia yeasts and culture media were used for fermentation. The following table shows the fermentation cycle and enzyme activity results of the fermentation broth for 5 batches of fermentation. cao-4 The fermentation enzyme activity reached more than 4000U / mL, which is much higher than that of wild genotype recombinant bacteria.

[0052] 30L small tank fermentation experiment results

[0053] After the fermentation, the trehalase in the fermentation broth of the two groups of recombinant bacteria was extracted, 40% water and 0.3% calcium chloride were added according to the volume of the fermentation broth, and 3% perlite filter aid was added for filter pressing. The filtrate was concentrated by ultrafiltration to obtain the liquid trehalase preparations of the two recombinant bacteria, which were stored at room temperature (25°C).

[0054] Example 5 Determination of enzymatic properties of the produced trehalase The enzyme activity was determined by the DNS method. The specific method is as follows: 900 μL of the reaction substrate trehalose (1%) was added with 100 μL of appropriately diluted enzyme solution and 1 mL of pH 6.0 acetic acid buffer solution, placed in a 35°C water bath for 10 min, and then 1.5 mL of 3,5-dinitrosalicylic acid (DNS) was added to terminate the reaction. The reaction was boiled for 5 min and quickly cooled. The absorbance was measured at 540 nm. The control group was first added with the stop solution and then the enzyme solution. Each group had three parallels and one control. The amount of reducing sugar produced (mg) was calculated based on the standard curve prepared by the series of gradient concentrations of glucose colorimetric reaction.

[0055] Definition of unit trehalase activity (U): Under certain conditions (if not otherwise specified, the conditions are temperature 35°C, pH 6.0), the amount of enzyme required for 1 mL of enzyme solution to produce 1 μmol of glucose per minute is 1 activity unit (U).

[0056] The optimum temperature is based on the recombinant strain GS115- cao-2 and GS115- cao-4 The liquid trehalase preparation produced by was used as a sample, and the enzyme activity at 30-70°C (interval 5°C) was measured respectively. The relative enzyme activity was calculated with the highest trehalase activity of each of the two enzymes as the benchmark 100%. The experimental method was the same as above, and 3 parallel experiments were set for each gradient.

[0057] The results are as follows Figure 1 As shown, the trehalase mutant of the present invention has stable enzyme activity at 50-65°C, the optimum action temperature is 60°C, and more than 50% of the enzyme activity can remain between 35 and 70°C, belonging to a medium- and high-temperature enzyme. Compared with the wild-type trehalase, the optimum reaction temperature does not change, but the optimum temperature range is greatly improved.

[0058] The optimal pH is the recombinant strain GS115- cao-2 and GS115- cao-4 The liquid trehalase preparation was used as the sample, and the enzyme activity in the buffers with pH 1.0-12.0 (interval 1) was measured at 60°C (wherein, pH 1-3 used 50 mmol / L glycine-HCl buffer; pH 3-9 used 50 mmol / L citric acid-K2HPO4; pH 9-12 used 50 mmol / L glycine-NaOH buffer), with the highest trehalase activity of the two enzymes measured as the benchmark 100%. The experimental method was the same as above, and 3 parallel experiments were set for each gradient.

[0059] The results are as follows Figure 2As shown, the optimum pH of the mutated trehalase of the present invention is 4.0. In the pH range of 3.0-5.0, the enzyme can maintain more than 80% of the enzyme activity. Compared with before mutation, the acid resistance is greatly enhanced and the pH range of action is wider.

[0060] Trehalase stability (1) The recombinant bacteria GS115 obtained in Example 4 cao-4 The liquid trehalase preparation was used as a sample to determine the heat stability of trehalase. The sample was placed at an optimum pH of 4.0 and kept in a constant temperature water bath at 60°C, 65°C, and 70°C for 0 min, 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, and 60 min. The residual enzyme activity was then determined, and the enzyme activity of the sample that had not been heat treated was set to 100%. The experimental method was the same as above, and 3 parallel experiments were set at each time point. The results are shown in the figure. Figure 3 As shown, from Figure 3 It can be seen that after treatment at 60°C for 60 min, the residual activity of the trehalase mutant of the present invention is still above 85%, and after treatment at 65°C for 60 min, the residual enzyme activity of the enzyme solution after treatment is 70%; when the treatment temperature is 70°C, the residual enzyme activity decreases faster, and when heated to 30 min, the residual enzyme activity can still be maintained at more than 40%.

[0061] (2) The recombinant bacteria GS115- cao-2 The liquid trehalase preparation was used as the sample, and the sample was placed at the optimum pH of 6.0 in a constant temperature water bath at 60°C. After 0 min, 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, and 60 min, the residual enzyme activity was measured, and the enzyme activity of the sample that had not been heat treated was set to 100%, and the relative enzyme activity of the wild-type trehalase and the trehalase mutant treated at 60°C was compared. The results are shown in Figure 4 As shown by Figure 4 It can be seen that the enzyme activity of the wild-type trehalase decreases rapidly after being treated at 60°C for 30 minutes. By comparison, the trehalase mutant has higher enzyme activity and thermal stability.

[0062] In summary, the heat resistance of the trehalase mutant is greatly improved, indicating that the enzyme mutant of the present invention has good heat stability.

[0063] (3) The recombinant bacteria GS115- cao-4The liquid trehalase preparation was used as the sample to determine the pH stability of trehalase. The samples were diluted with different pH 1.0-12.0 buffers and placed in a 37℃ constant temperature water bath for 1 hour. Then, the relative residual enzyme activity was determined at pH 4.0 and 60℃. The enzyme activity of the untreated sample was set as 100%.

[0064] The results are as follows Figure 5 As shown, the trehalase mutant of the present invention is an acidic trehalase, and the residual enzyme activity can be maintained at more than 90% in an extremely acidic environment (pH 1.0-4.0); in the pH range of 5.0-7.0, the residual enzyme activity can be maintained at more than 70%. As the pH moves toward the alkaline direction, its stability gradually weakens. The trehalase mutant of the present invention has good pH stability.

[0065] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A trehalase mutant, characterized in that: The amino acid sequence of the trehalase mutant is shown in SEQ ID No.

4.

2. A gene encoding the trehalase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence is shown in SEQ ID No.

3.

4. A recombinant vector or recombinant strain comprising the coding gene according to claim 2.

5. The recombinant vector according to claim 4, characterized in that The expression plasmid used was pPIC9K.

6. The recombinant strain according to claim 4, characterized in that The host bacteria used was Pichia pastoris GS115.

7. Use of the recombinant vector or recombinant strain according to claim 4 in producing the trehalase mutant according to claim 1.

8. Use of the trehalase mutant according to claim 1 in hydrolyzing trehalose.

9. The use according to claim 8, characterized in that It is used in the industrial production of amylase.

10. The use according to claim 8, characterized in that It is used in the production of yeast extract.

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