A trehalose synthase mutant and its preparation and application

Abstract

The present invention belongs to the fields of genetic engineering and enzyme engineering technology and discloses a trehalose synthase mutant. The trehalose synthase mutant is obtained by subjecting one of the amino acids 160, 192, 216, 228, or 234 of the parent trehalose synthase to a point mutation. Specifically, the mutant is obtained by designing site-directed mutagenesis primers, performing site-directed mutagenesis using a vector carrying the trehalose synthase gene as a template, constructing a plasmid vector containing the mutant, and then transforming the vector into a host cell. The trehalose synthase mutant is used in the production of trehalose. The trehalose synthase mutant of the present invention contains a trehalose synthase with improved thermal stability and has good industrial prospects for its use in trehalose production.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a trehalose synthase mutant and its preparation and application. Background Art

[0002] Trehalose is composed of two glucose molecules connected by an α-1,1 glycosidic bond. It is a non-reducing disaccharide with a sweetness of about 45% of sucrose. It is known as the "sugar of life" and is widely present in various organisms, including bacteria, yeast, fungi and algae, as well as some insects, invertebrates and plants. It is especially abundant in yeast. Fermented foods such as bread and beer, as well as shrimp, also contain trehalose.

[0003] Trehalose's non-reducing properties, high safety, structural stability, and nonspecific protective effects on biomembranes, proteins, and other biomacromolecules have given it broad application prospects and attracted increasing attention from researchers. The US FDA designated trehalose as a safe food in 2000, followed by the European Regulation System in 2001. my country's Ministry of Health officially approved trehalose as a new resource food in 2005. Currently, trehalose is widely used in the food, cosmetics, and pharmaceutical industries.

[0004] Trehalose production methods include yeast extraction, dual-enzyme methods, and single-enzyme methods. Early trehalose extraction from yeast resulted in low extraction yields and very low production efficiency, resulting in a trehalose price as high as $700 / kg. This process has now been eliminated. In 1995, Hayashihara Biochemical Co., Ltd. in Japan invented a dual-enzyme method (maltooligosaccharyl trehalose synthase and maltooligosaccharyl trehalose hydrolase) to produce trehalose. This process also requires the addition of pullulanase and cyclodextrin glycosyltransferase, resulting in a low conversion temperature, high bacterial contamination, and extremely unstable production. The single-enzyme method uses maltose as a substrate and utilizes trehalose synthase to convert trehalose into trehalose in a single step. This process allows for continuous conversion and is simple. However, the single-enzyme method has the following drawbacks: The conversion rate of trehalose synthase is as high as 71-80% at 30-50°C. However, this rate decreases with increasing temperature, dropping to 66% at 60°C. As those skilled in the art know, enzyme conversion at temperatures below 50°C is prone to bacterial contamination, leading to unstable production and decreased yields. Therefore, molecular biological modification of trehalose synthase has been conducted to increase its trehalose conversion rate under high-temperature conditions, which is beneficial for improving the efficiency and stability of trehalose production by the single-enzyme method. Summary of the Invention

[0005] The first technical problem to be solved by the present invention is to provide a trehalose synthase mutant obtained by performing a point mutation on one of the amino acids 160, 192, 216, 228 or 234 of the trehalose synthase parent;

[0006] The second technical problem to be solved by the present invention is to provide a method for preparing a trehalose synthase mutant, designing mutation primers for site-directed mutagenesis, performing site-directed mutagenesis using a vector carrying the trehalose synthase gene as a template and constructing a plasmid vector containing the mutant, which is then transformed into a host cell to obtain;

[0007] The third technical problem to be solved by the present invention is to provide an application of a trehalose synthase mutant in trehalose production.

[0008] In order to solve the above technical problems, the specific technical solution of the present invention is:

[0009] A trehalose synthase mutant is obtained by subjecting the trehalose synthase having the amino acid sequence of SEQ NO. 1 (NCBI accession number BAA19934.1) to a point mutation at amino acid position 160, 192, 216, 228 or 234, respectively. In the mutant R160L, amino acid position 160 is changed from arginine (Arg) to isoleucine (Leu); in the mutant D192A, amino acid position 192 is changed from aspartic acid (Asp) to alanine (Ala); in the mutant T216N, amino acid position 216 is changed from threonine (Thr) to asparagine (Asn); in the mutant E228V, amino acid position 228 is changed from glutamic acid (Glu) to valine (Val); and in the mutant G234D, amino acid position 234 is changed from glutamic acid (Gla) to aspartic acid (Asp).

[0010] A method for preparing a trehalose synthase mutant comprises the following steps:

[0011] a. Based on the amino acid sequence of the trehalose synthase parent (NCBI accession number BAA19934.1), amino acids 160, 192, 216, 228, or 234 were identified as mutation sites, and site-directed mutagenesis primers were designed:

[0012] The site-directed mutagenesis primers for introducing the R160L mutation are:

[0013] Forward primer: 5'-AGGCCTACTACTGGCAC CTC TTCTACTGGCA-3' (underlined mutated bases)

[0014] Reverse primer: 5'-GGCTGGTGCCAGTAGAA GAGGCGGTGCCAGT-3' (mutated bases are underlined);

[0015] The site-directed mutagenesis primers for introducing the D192A mutation are:

[0016] Forward primer: 5'-GGGCCGACCTGGGGGTG GCC GGCTTCCGCCT-3' (the underlined base is the mutant)

[0017] Reverse primer: 5'-GCGTCCAGGCGGAAGCC GGC CACCCCCAGGT-3′ (mutated bases are underlined);

[0018] The site-directed mutagenesis primers for introducing the T216N mutation are:

[0019] Forward primer: 5'-GCGAGAACCTCCCCGAG AAC ATTGAGGCGGT-3' (the underlined base is the mutant)

[0020] Reverse primer: 5'-CGCTTCACCGCCTCAAT GTT CTCGGGGAGGT-3′ (mutated bases are underlined);

[0021] The site-directed mutagenesis primers for introducing the E228V mutation are:

[0022] Forward primer: 5'-GCCTGAGGAAGGCCCTG GTG GAGCGCTACGG-3' (the underlined base is the mutant)

[0023] Reverse primer: 5'-CCGGGGCCGTAGCGCTC CAC CAGGGCCTTCC-3′ (mutated bases are underlined);

[0024] The site-directed mutagenesis primers for introducing the G234D mutation are:

[0025] Forward primer: 5'-AGGAGCGCTACGGCCCC GTC AAGATCCTCCT-3' (underlined mutated bases)

[0026] Reverse primer: 5'-TCGGCGAGGAGGATCTT GAC GGGGCCGTAGC-3' (the mutated base is underlined);

[0027] b. Using the expression vector TreS / pET-24a(+) as a template, PCR amplification was performed using the primers in step a, and the PCR products were digested with DpnⅠ and transferred into plasmid vectors;

[0028] c. The plasmid vectors obtained in step b were transformed into host cells to obtain trehalose synthase mutants, namely mutant R160L, mutant D192A, mutant T216N, mutant E228V and mutant G234D.

[0029] Preferably, the expression vector TreS / pET-24a(+) in step b is a plasmid TreS / pMD18T containing a trehalose synthase gene and a pET24a(+) plasmid, which are double-digested with NdeI and EcoRI, respectively. The trehalose synthase gene fragment is recovered by gel tapping of the digested product and then ligated to the cut pET24a(+) plasmid using T4 ligase.

[0030] Preferably, the plasmid vector in step b is any one of the PUC series, PET series or PGEX series.

[0031] Furthermore, the plasmid vector in step b is a PET series plasmid vector.

[0032] Preferably, the host cell in step c is a bacterial cell or a fungal cell.

[0033] Furthermore, in step c, the host cell is a Gram-positive bacterial cell or a Gram-negative bacterial cell.

[0034] Furthermore, the host cell in step c is BL21 (DE3).

[0035] Application of a trehalose synthase mutant: Application of the trehalose synthase mutant in trehalose production.

[0036] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0037] The present invention introduces point mutations into the trehalose synthase gene and obtains mutants containing trehalose synthases with improved thermal stability through transformation and expression. These mutants are used to produce trehalose. At 60°C, the trehalose conversion rates of the mutants R160L, D192A, T216N, E228V, and G234D reached 76.6%, 71.5%, 69.4%, 79.3%, and 76.1%, respectively, while the conversion rate of the wild-type trehalose synthase was only 66.3%. Therefore, the invention has good prospects for industrialization. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the embodiments.

[0039] The detection methods involved in the following embodiments are as follows:

[0040] The trehalose HPLC detection conditions were as follows: differential refractive index detector, NH2 column (APS-2HYPERSIL, Thermo Scientific), mobile phase (water:acetonitrile = 1:4), flow rate: 0.8 mL / min, column temperature: 40°C;

[0041] Trehalase activity assay: Mix 5 mL of appropriately diluted enzyme solution with 5 mL of 20% (w / v) maltose solution prepared in 20 mmol / L pH 6.0 phosphate buffer. Preheat for 10 minutes, add the enzyme solution, and react at 30°C for 30 minutes. Quickly remove from the reaction solution and incubate in a boiling water bath for 15 minutes to inactivate the enzyme and terminate the reaction. Detect the content of each component in the reaction solution by HPLC.

[0042] Definition of enzyme activity unit: Under the above reaction conditions, the amount of enzyme required to produce 1 μmol of trehalose in 1 min is defined as 1 enzyme activity unit.

[0043] Example 1: Construction of TreS / pET24a(+) plasmid

[0044] The pET24a(+) plasmid and the plasmid TreS / pMD 18T containing the trehalose synthase gene (TreS gene) were double-digested with NdeI and EcoRI, respectively. The trehalose synthase gene fragment was recovered by gel tapping of the digested product, and then ligated to the cut pET24a(+) plasmid using T4 ligase. The ligation product was transformed into E. coli JM109 competent cells and cultured at 37°C for 8 h. Transformants were selected and shake-cultured in LB medium containing 30 mg / L kanamycin liquid. The plasmid was extracted and verified by enzyme digestion to obtain the expression plasmid TreS / pET24a(+).

[0045] Example 2: Expression of wild-type trehalose synthase

[0046] The plasmid TreS / pET24a(+) obtained in Example 1 was transformed into E. coli BL21(DE3) host cells and plated onto LB plates containing 30 mg / L kanamycin. Cultured at 37°C for 8 hours, this was designated TreS / pET24a(+) / E. coli BL21(DE3). A single colony was picked and transferred to liquid LB medium containing 30 mg / L kanamycin, cultured overnight at 37°C, and stored in a glycerol tube.

[0047] TreS / pET24a / BL21(DE3) was inoculated from a preserved glycerol tube in LB liquid medium (containing 100 mg / L kanamycin) and grown for 8 hours. The seed was then inoculated into TB liquid fermentation medium (containing 100 mg / L kanamycin) at a 5% inoculum. E. coli was cultured at 37°C for 2 hours and induced with the addition of IPTG (isopropylthio-β-D-galactopyranoside) at a final concentration of 0.01 mmol / L. Fermentation was continued on a shaker at 25°C for 24 hours. After this, 25 mL of the fermentation broth was centrifuged at 4°C and 12,000 rpm for 10 minutes. The supernatant was discarded, and the bacterial pellet was collected. The pellet was resuspended in 20 mmol / L Na2HPO4-NaH2PO4 buffer, pH 6.0, and mixed thoroughly. The cell walls of the cells were disrupted using an ultrasonic cell disruptor (operating conditions: ψ6 probe, 10-minute operation, 2-second on / 3-second off, 20% power), to obtain a crude enzyme solution.

[0048] Example 3: Preparation of trehalose synthase mutants

[0049] Based on the gene sequence of trehalose synthase from Thermus thermophilus ATCC33923, primers for introducing R160L, D192A, T216N, E228V, and G234D mutations were designed and synthesized, respectively, to perform site-directed mutagenesis on the maltooligosaccharide-trehalose synthase gene TreS:

[0050] The site-directed mutagenesis primers for introducing the R160L mutation are:

[0051] Forward primer: 5'-AGGCCTACTACTGGCAC CTC TTCTACTGGCA-3' (underlined mutated bases)

[0052] Reverse primer: 5'-GGCTGGTGCCAGTAGAA GAG GCGGTGCCAGT-3' (the underlined base is the mutant)

[0053] The site-directed mutagenesis primers for introducing the D192A mutation are:

[0054] Forward primer: 5'-GGGCCGACCTGGGGGTG GCC GGCTTCCGCCT-3' (the underlined base is the mutant)

[0055] Reverse primer: 5'-GCGTCCAGGCGGAAGCC GGC CACCCCCAGGT-3' (underlined bases are mutated)

[0056] The site-directed mutagenesis primers for introducing the T216N mutation are:

[0057] Forward primer: 5'-GCGAGAACCTCCCCGAG AAC ATTGAGGCGGT-3' (the underlined base is the mutant)

[0058] Reverse primer: 5'-CGCTTCACCGCCTCAAT GTT CTCGGGGAGGT-3' (the underlined base is the mutant)

[0059] The site-directed mutagenesis primers for introducing the E228V mutation are:

[0060] Forward primer: 5'-GCCTGAGGAAGGCCCTG GTG GAGCGCTACGG-3' (the underlined base is the mutant)

[0061] Reverse primer: 5'-CCGGGGCCGTAGCGCTC CAC CAGGGCCTTCC-3' (the underlined base is the mutant)

[0062] The site-directed mutagenesis primers for introducing the G234D mutation are:

[0063] Forward primer: 5'-AGGAGCGCTACGGCCCC GTC AAGATCCTCCT-3' (underlined mutated bases)

[0064] Reverse primer: 5'-TCGGCGAGGAGGATCTT GAC GGGGCCGTAGC-3' (the underlined base is the mutant)

[0065] Using the expression vector TreS / pET-24a(+) carrying the wild-type trehalose synthase gene obtained in Example 1 as a template, the genes encoding the above-mentioned site-directed mutants were PCR amplified using rapid PCR technology: the PCR reaction system was: 10 μL of 5×PS buffer, 4 μL of dNTPs Mix (2.5 mM), 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 1 μL of template DNA, 0.5 μL of PrimerStar HS (5 U / μL), and double-distilled water was added to 50 μL.

[0066] PCR amplification conditions were as follows: initial denaturation at 94°C for 4 min, followed by 30 cycles (98°C for 10 s, 60°C for 5 s, and 72°C for 8.5 min), extension at 72°C for 10 min, and incubation at 4°C. PCR products were detected by 1% agarose gel electrophoresis.

[0067] Strain construction: The PCR product was digested with restriction endonuclease DpnⅠ and ligated to a plasmid that was also cut by DpnⅠ. The competent E. coli JM109 cells were then transformed. The competent cells were cultured overnight in LB solid medium (containing 30 μg / mL kanamycin). After the clones were cultured in LB liquid medium (containing 30 μg / mL kanamycin), the plasmids were extracted. All mutant plasmids were sequenced correctly. The mutant plasmids were transformed into the expression host E. coli BL21 (DE3) competent cells, and finally the competent cells capable of expressing the gene were obtained. Recombinant strains of mutant R160L, mutant D192A, mutant T216N, mutant E228V, and mutant G234D .

[0068] Example 4: Expression of Trehalose Synthase Mutants

[0069] Will Mutant R160L, mutant D192A, mutant T216N, mutant E228V, mutant G234D Each mutant was inoculated into LB liquid medium (containing 100 mg / L kanamycin) and grown for 8 hours. The mutants were then inoculated into TB liquid fermentation medium (containing 100 mg / L kanamycin) at a 5% inoculum for culture. After incubation at 37°C for 2 hours, IPTG (isopropylthio-β-D-galactopyranoside) was added to induce the culture. The culture was further cultured and fermented on a shaker at 25°C for 24 hours. After that, 25 ml of the fermentation broth was centrifuged at 4°C and 12,000 rpm for 10 minutes. The supernatant was discarded, and the cells were collected. The cell pellet was resuspended in 20 mmol / L pH 6.0 Na2HPO4-NaH2PO4 buffer and mixed. The cell walls of the cells were then disrupted using an ultrasonic cell disruptor (operating conditions: ψ6 probe, 10-minute operation, 2-second on, 3-second off, 20% power), to obtain a crude enzyme solution.

[0070] Experimental example: Transformation of trehalose production capacity

[0071] 200 mL of a 20% (w / v) maltose solution prepared in 50 mmol / L pH 6.0 phosphate buffer was added to a stoppered flask. 0.5 mL of crude enzyme solution of the wild-type trehalose synthase obtained in Example 2, and 0.5 mL of crude enzyme solution of mutants R160L, D192A, T216N, E228V, and G234D obtained in Example 4 (the amount added was determined based on the fermentation enzyme activity, at a rate of 2.5 U / g maltose) were added. The mixture was placed in a shaking water bath and allowed to react for 48 hours. After the reaction, samples were taken and boiled for 10 minutes to inactivate the enzyme. The trehalose content was then determined by HPLC. The test results are shown in Table 1.

[0072] Table 1 Conversion rate of trehalose produced by wild enzyme and mutant under different temperature conditions (%)

[0073]

[0074] The results are shown in Table 1. Compared with the wild-type enzyme, the mutant showed a significantly higher trehalose conversion rate at 60°C. Therefore, the mutant can achieve an increased conversion rate for trehalose production under high-temperature conditions. Furthermore, because the production process is less susceptible to bacterial contamination under high-temperature conditions, it has good prospects for industrial application.

[0075] It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

Claims

1. A trehalose synthase mutant, characterized in that: The trehalose synthase having the amino acid sequence of SEQ NO. 1 in the sequence table is obtained by subjecting the amino acid at position 160, 192, 216, 228 or 234 to a point mutation. In the mutant R160L, the amino acid at position 160 is changed from arginine to isoleucine; in the mutant D192A, the amino acid at position 192 is changed from aspartic acid to alanine; in the mutant T216N, the amino acid at position 216 is changed from threonine to asparagine; in the mutant E228V, the amino acid at position 228 is changed from glutamic acid to valine; and in the mutant G234D, the amino acid at position 234 is changed from glutamic acid to aspartic acid.

2. The method for preparing a trehalose synthase mutant according to claim 1, wherein: The following steps are involved: a. Based on the amino acid sequence of trehalose synthase in the sequence table SEQ NO.1, amino acids 160, 192, 216, 228 or 234 were determined as mutation sites, and primers for site-directed mutagenesis were designed: The site-directed mutagenesis primers for introducing the R160L mutation are: Forward primer: 5'-AGGCCTACTACTGGCAC CTC TTCTACTGGCA-3' (underlined mutated bases) Reverse primer: 5'-GGCTGGTGCCAGTAGAA GAG GCGGTGCCAGT-3' (mutated bases are underlined); The site-directed mutagenesis primers for introducing the D192A mutation are: Forward primer: 5'-GGGCCGACCTGGGGGTG GCC GGCTTCCGCCT-3' (the underlined base is the mutant) Reverse primer: 5'-GCGTCCAGGCGGAAGCC GGC CACCCCCAGGT-3′ (mutated bases are underlined); The site-directed mutagenesis primers for introducing the T216N mutation are: Forward primer: 5'-GCGAGAACCTCCCCGAG AAC ATTGAGGCGGT-3' (the underlined base is the mutant) Reverse primer: 5'-CGCTTCACCGCCTCAAT GTT CTCGGGGAGGT-3′ (mutated bases are underlined); The site-directed mutagenesis primers for introducing the E228V mutation are: Forward primer: 5'-GCCTGAGGAAGGCCCTG GTG GAGCGCTACGG-3' (the underlined base is the mutant) Reverse primer: 5'-CCGGGGCCGTAGCGCTC CAC CAGGGCCTTCC-3′ (mutated bases are underlined); The site-directed mutagenesis primers for introducing the G234D mutation are: Forward primer: 5'-AGGAGCGCTACGGCCCC GTC AAGATCCTCCT-3' (underlined mutated bases) Reverse primer: 5'-TCGGCGAGGAGGATCTT GAC GGGGCCGTAGC-3' (the mutated base is underlined); b. Using the expression vector TreS / pET-24a(+) as a template, PCR amplification was performed using the primers in step a, and the PCR products were digested with DpnⅠ and transferred into plasmid vectors; c. The plasmid vectors obtained in step b were transformed into host cells to obtain trehalose synthase mutants, namely mutant R160L, mutant D192A, mutant T216N, mutant E228V and mutant G234D.

3. The method for preparing a trehalose synthase mutant according to claim 2, wherein: The expression vector TreS / pET-24a(+) in step b is obtained by subjecting the plasmid TreS / pMD18T containing the trehalose synthase gene and the pET24a(+) plasmid to double digestion with NdeI and EcoRI, respectively, and recovering the trehalose synthase gene fragment by cutting the digestion product, which is then ligated to the cut pET24a(+) plasmid using T4 ligase.

4. The method for preparing a trehalose synthase mutant according to claim 2, wherein: The plasmid vector in step b is any one of the PUC series, PET series or PGEX series.

5. The method for preparing a trehalose synthase mutant according to claim 4, wherein: The plasmid vector in step b is a PET series plasmid vector.

6. The method for preparing a trehalose synthase mutant according to claim 2, wherein: In step c, the host cell is a bacterial cell or a fungal cell.

7. The method for preparing a trehalose synthase mutant according to claim 6, wherein: In the step c, the host cell is a Gram-positive bacterial cell or a Gram-negative bacterial cell.

8. The method for preparing a trehalose synthase mutant according to claim 7, wherein: The host cell in step c is BL21 (DE3).

9. The use of the trehalose synthase mutant according to claim 1, characterized in that: Application of the trehalose synthase mutant in trehalose production.

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