Trehalose synthase PfTreS, application of trehalose synthase PfTreS, expression vector and expression strain

Abstract

The invention relates to the technical field of gene engineering and enzyme engineering, in particular to trehalose synthase PfTreS, application thereof, an expression vector and an expression strain. The invention provides a trehalose synthase PfTreS. The amino acid sequence of the trehalose synthase PfTreS is as shown in SEQ ID NO: 1. The invention also provides a preparation method of the trehalose synthase PfTreS. The enzyme can catalyze high-concentration maltose to generate trehalose, has high conversion efficiency, is simple and can be completed by only one step, so that the production cost is reduced, the risk of an additional process generated by conflicts of optimal reaction conditions during reaction of multiple enzymes is avoided, and the enzyme is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering and enzyme engineering, and in particular to a trehalose synthase PfTreS and an application thereof, an expression vector and an expression strain. Background Art

[0002] Trehalose is a non-reducing food disaccharide formed by α-1, 1-α glycosidic bonds. Many research reports indicate that trehalose can improve and delay neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Trehalose can also prevent and alleviate non-alcoholic fatty liver disease by inhibiting hexose transporters to induce autophagy, and can promote autophagy-lysosome biogenesis in macrophages to treat atherosclerosis. At the same time, studies have shown that trehalose can effectively regulate sugar metabolism and maintain blood sugar balance in diabetic patients. In addition, as a new anticancer agent, trehalose can also reduce oxidative stress, autophagy and increase cell apoptosis, thereby safely and effectively treating tumors.

[0003] At present, the enzymatic synthesis of trehalose is mainly divided into three types: 1) Phosphorylase method for synthesizing trehalose: This method mainly relies on trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, and uses UDP-glucose and phosphate-glucose as substrates. The whole reaction process includes two key steps: phosphorylation and dephosphorylation liquefaction; 2) Dual enzyme method for synthesizing trehalose: In this method, maltosyl trehalose synthase and maltosyl trehalose hydrolase are used to synthesize trehalose, and maltodextrin is used as the reaction substrate. The reaction process covers two steps: glycosidic bond isomerization and hydrolysis; 3) Unlike the first two methods, the single enzyme method only uses one enzyme-trehalose synthase, and uses maltose as the reaction substrate. The significant feature of this method is that the entire reaction process consists of only one step, that is, trehalose synthase catalyzes the isomerization of the glycosidic bond of maltose to synthesize trehalose. Compared with the phosphorylase method and the dual enzyme method, the single enzyme method for synthesizing trehalose has significant advantages. The reaction substrates and steps of the single enzyme method for synthesizing trehalose are simpler, without the need to use multiple enzymes and complex reaction conditions. In the production process, the single enzyme method only requires one enzyme, which not only reduces the production cost of using multiple enzymes, but also avoids the risk of additional processes due to conflicts in the optimal conditions between different enzymes.

[0004] However, the existing trehalose synthase is unstable and has a low conversion rate of catalyzing the production of trehalose. There is an urgent need for a stable trehalose synthase with a high conversion rate to meet the deficiencies of the existing technology. Summary of the invention

[0005] The purpose of the present invention is to provide a trehalose synthase PfTreS and its application, an expression vector and an expression strain.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a trehalose synthase PfTreS, and the amino acid sequence of the trehalose synthase PfTreS is shown in SEQ ID NO:1.

[0008] Preferably, the trehalose synthase PfTreS is derived from Pseudomonas sp. B7-1;

[0009] Pseudomonas sp. B7-1 is deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China. The deposit date is November 7, 2024. The deposit number is CCTCC NO: M20242464.

[0010] The specific activity of the trehalose synthase PfTreS is ≥11.4 U / mg.

[0011] The present invention also provides the use of the trehalose synthase PfTreS in catalyzing maltose to generate trehalose.

[0012] The present invention also provides a gene encoding trehalose synthase PfTreS, the sequence of the gene is shown in SEQ ID NO: 2;

[0013] The trehalose synthase PfTreS is the trehalose synthase PfTreS.

[0014] The present invention also provides a primer pair for amplifying the gene, and the primer pair is shown in SEQ ID NO: 3-4.

[0015] The present invention also provides an expression vector, comprising a gene encoding trehalose synthase PfTreS and an empty vector;

[0016] The gene encoding trehalose synthase PfTreS is the gene described;

[0017] The empty vector is pET-30a(+).

[0018] The present invention also provides the use of the expression vector in preparing a product that catalyzes maltose to generate trehalose.

[0019] The present invention also provides an expression strain, comprising a gene encoding trehalose synthase PfTreS and a host bacterium;

[0020] The gene encoding trehalose synthase PfTreS is the gene described;

[0021] The host bacteria is Escherichia coli BL21 (DE3).

[0022] The present invention also provides the use of the expression strain in preparing a product that catalyzes maltose to generate trehalose.

[0023] The present invention also provides a method for catalyzing maltose to produce trehalose, comprising the following steps:

[0024] The trehalose synthase PfTreS was mixed with maltose and reacted for 10 to 1440 minutes;

[0025] The trehalose synthase PfTreS is the trehalose synthase PfTreS;

[0026] The initial concentration of the trehalose synthase PfTreS is 0.6-3.26 mg / mL;

[0027] The initial concentration of maltose is 10-40wt%;

[0028] The reaction temperature is 25-50°C;

[0029] The pH of the reaction is 3-11.

[0030] The present invention provides a trehalose synthase PfTreS and its application, an expression vector and an expression strain. The scheme of the present invention has the following advantages:

[0031] The trehalose synthase PfTreS of the present invention has a high specific enzyme activity of 11.4 U / mg, a wide reaction pH range, and can react at 25-50° C. It can catalyze maltose with a substrate concentration of 10-40 wt % to produce trehalose with a high conversion rate.

[0032] The present invention can catalyze trehalose from maltose using only trehalose synthase PfTreS, and the steps are simple, which not only reduces the cost but also avoids the risks caused by the difference in optimal conditions between enzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the SDS-PAGE detection result of trehalose synthase PfTreS pure enzyme (M is Mark, lanes 1 to 2 are trehalose synthase PfTreS).

[0034] Figure 2 The enzymatic properties of trehalose synthase PfTreS.

[0035] Figure 3 The standard curves are trehalose and maltose.

[0036] Figure 4 This is the HPLC chart of the reaction of trehalose synthase PfTreS with 30 wt% trehalose.

[0037] Collection Instructions

[0038] Pseudomonas sp.B7-1 is deposited in China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. The deposit date is November 7, 2024, and the deposit number is CCTCC NO:M20242464. DETAILED DESCRIPTION

[0039] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] The amino acid sequence of trehalose synthase PfTreS is shown in SEQ ID NO: 1:

[0041] SEQ ID NO: 1:

[0042] MAKKPKAATFIKDPLWYKDAVIYQVHVKSFFDSNNDGIGDFPGLIAKLDYIAELGVNTI

[0043] WLLPFYPSPRRDDGYDIAEYRGVHSDYGTMADAKRFIAEAHKRGLRVITELVINHTSD

[0044] QHPWFQRARKAKPGSAARDFYVWSDDQKYDGTRIIFLDTEKSNWTWDPVAGQYFW

[0045] HRFYSHQPDLNFDNPQVMKAVLSVMRYWLDMGIDGLRLDAIPYLIERDGTNNENLPE

[0046] THDVLKQIRAEIDANYPDRMLLAEANQWPEDTQLYFGDTDAEGVNGDECHMAFHFPL

[0047] MPRMYMALAQEDRFPITDILRQTPEIPANCQWAIFLRNHDELTLEMVTDKERDYLWNY

[0048] YAADRRARINLGIRRRLAPLMERDRRRIELLNSLLLSMPGTPTLYYGDEIGMGDNIYLG

[0049] DRDGVRTPMQWSIDRNGGFSRADPASLVLPPIMDPQYGYQSVNVETQAGDPHSLLNW

[0050] TRRLLTVRKQSKAFGRGTLKMLSPANRRVLAYTREYTGPDGKHEIILCVANVSRSAQA

[0051] VELDLSAYVGMVPVEMLGGNAFPPIGQLSFLLTLPPYGFYWFGLAAENQMPSWHVEP

[0052] AQSLPDFTTLVLKKRMEELLEAPSRATLEQAILPSWLQNRRWFAGKDAAIEQVKLAYG

[0053] VRFGDAQHPVLFSEIEVQSGGQTSRYQLPFGFIAEDQVGPALPQQLALARVRRVRQVGL

[0054] ITDAFSLEAFVRAVLQGMQSGTVLESSDGEIRFEATPQLEKLGLGAESEEVRYLSAEQSNS

[0055] SVVIGNSLVLKLIRKVASGVHPELEMSAYLTAAGFANISPLLGSVIRRGADGEDNLLMIA

[0056] QGYLSNQGDAWEWTQNNLERALRDELADAVSEQAQHYNALGELKDFAGMLGQRLG

[0057] EMHQVLAAPSDNKDFAPQVSSAKDAQTSGKDVAAQVEHALKLLKQHQGELDAADQK

[0058] LVALLDNKKTILAHVQELAKQSAGGLRIRVHGDLHLGQVLVIKGDAYLIDFEGEPARP

[0059] LAERRGKHSPYKDVSGVLRSFDYAAAMALNVHNVDNSPEAEAARRRVTERYLREARE

[0060] AFLQAYRQAAASLDHAWQDPEGADAALALFGLEKAAYEVAYEAENRPTWLPPPLHGLYGLLTGLKPFSDLGGE.

[0061] Length: 1113 amino acids, Type: polypeptide, Chain type: single chain, Geometry: three-dimensional.

[0062] The gene encoding trehalose synthase PfTreS is shown as SEQ ID NO: 2;

[0063] SEQ ID NO: 2:

[0064] atggcgaagaaacccaaggcagccacctttatcaaggatccgctctggtacaaggacgcggttatctatcaggttcacgtcaaatccttttt

[0065] cgactccaacaacgacgggatcggcgactttcccggcctgatcgccaaactcgattacatcgccgaactcggcgtcaacaccatttggct

[0066] gttgccgttctacccctcgccacgtcgcgacgacggttacgatatcgccgaataccggggcgtccacagcgattacgggacgatggcc

[0067] gacgccaaacggttcattgccgaagcacacaagcgtggcctgcgggtgatcaccgagctggtcatcaaccacacctctgatcagcacc

[0068] cttggttccagcgtgcacgcaaggccaaacccggctcggcggcgcgggacttctatgtgtggtcggatgacgatcaaaaatacgacgg

[0069] cacccggatcattttcctcgacaccgaaaagtccaactggacctgggatccggtggcgggccaatacttctggcaccgcttctattcgcac

[0070] cagccggacctgaatttcgataacccgcaagtcatgaaagcggttctgtcggtcatgcgttactggctggacatgggcatcgacggcctg

[0071] cgcctggatgccattccgtacctgatcgagcgcgatggcaccaacaacgaaaacctcccggaaacccacgacgtcctcaagcagatcc

[0072] gcgccgagatcgacgccaactacccggatcgcatgttgctggccgaagccaaccaatggccggaagacactcagctgtacttcggcg

[0073] acaccgacgccgagggcgtgaatggtgacgagtgtcacatggcgttccacttcccgttgatgccgcgcatgtacatggcgctggcccaa

[0074] gaggatcgctttccgatcaccgacatcctgcgtcagaccccggaaatcccggccaactgccaatgggcgattttcctgcgcaaccacga

[0075] tgagctgacgctggagatggtcaccgacaaggagcgcgactacctgtggaactactacgccgctgaccgtcgtgcgcggatcaacctt

[0076] ggcatccgccgccgtctggcgccgctgatggagcgcgaccgccgccgcatcgagttgctcaacagcctgctgctgtcgatgcccggc

[0077] acgccgaccctgtattacggcgatgagatcggcatgggcgacaacatctacctcggcgaccgcgacggcgtgcgtacgccgatgcaat

[0078] ggtcgatcgaccgcaacggcggcttctctcgcgccgacccggccagtctggtgttgccgccgatcatggacccgcaatacggctatca

[0079] gtcggtcaacgtcgaaacccaggccggcgatccgcattcgctgctgaactggacccgacgcctgctgaccgtgcgcaagcaatccaa

[0080] ggcgttcggtcgtggcaccttgaagatgttgtcgccggccaaccgtcgcgtgctggcttacacccgcgaatacaccgggccggacggc

[0081] aagcacgaaatcattctgtgcgtagccaacgtgtcacgcagtgcacaagcggtggaactcgacctgtcggcttacgtcggcatggtgcc

[0082] ggtggaaatgctcggtggtaacgcgttcccgcccatcggccagttgagtttcctgctgaccctgccgccctacggtttctactggttcggc

[0083] ctggcggcggaaaaccagatgccgagctggcacgtggaacctgcgcagagcctgccggacttcaccacgctggtgttgaaaaaacgc

[0084] atggaagaactgctcgaagcgccgtcccgggccaccctcgagcaggcgatcctgccgagctggctgcagaaccgccgctggttcgcc

[0085] ggcaaggacgcggccatcgaacaggtgaaactggcctacggcgtgcgcttcggcgatgcgcagcatccggtgttgttcagcgaaatc

[0086] gaagtgcagagcggcgggcaaaccagccggtatcaactgccgttcggcttcattgccgaagatcaggtcggcccggcattgccgcag

[0087] caattggccttggcccgtgtgcgccgggttcgccaggttgggctgatcaccgatgctttcagtcttgaagcatttgttcgcgcggtgctgca

[0088] aggcatgcagagcggcacggtgctggaatccagtgatggcgagatccgtttcgaggcgacgccgcaactggaaaaactcggcctcgg

[0089] cgcggaatccgaagtgcgctatctgtccgccgagcaatccaacagttcagtggtgatcggcaacagtctggtacttaagctgatccgaaa

[0090] agtcgcttctggcgtacacccggaactggagatgagcgcttacctgaccgcagccggatttgccaatatctcaccgctgctgggttcggt

[0091] gattcgccgcggcgccgatggtgaagacaatctattgatgattgcacagggctatctgagcaatcagggcgacgcctgggaatggacg

[0092] cagaacaacctcgaacgggcgctgcgcgatgaactggccgacgctgtttccgaacaggcgcagcactacaacgccctgggtgaactg

[0093] aaggatttcgccggcatgctcggccagcgtctgggggaaatgcatcaggtactggcagcgccgagcgacaacaaggacttcgcgccg

[0094] caagtcagttcggccaaggatgcgcagacgagcggcaaggatgtcgcggcgcaggtcgagcatgcgctgaagctgctcaaacagca

[0095] tcagggcgaactagacgcggcggatcagaagctggtcgctcgtttgctcgacaacaagaaaaccatccttgcccacgtgcaggaactg

[0096] gccaagcaatccgccggcggtctgcggatccgcgtccacggcgatctgcatttggggcaggtgctggtaatcaagggcgatgcctatct

[0097] gatcgacttcgaaggtgagccggcgcggccactggccgagcgccgaggcaagcacagtccttacaaggacgtcagcggagtgctgc

[0098] gctccttcgactatgccgcggccatggcgctgaatgtgcacaacgtcgacaacagccccgaggccgaagccgcgcgtcggcgggtca

[0099] cggagcgttacctgcgtgaagcccgggaagcctttctccaggcatatcgtcaggcagcagctagtcttgatcatgcctggcaagatcctg

[0100] aaggtgccgacgccgcactggcgttgttcggtctggagaaagcggcctacgaagtggcctatgaagccgaaaatcgccccacctggctgcccgtgccgctgcacggtctgtatgggttattgacggggctcaaacccttttccgatcttggtggagag.

[0101] Length: 3339 base pairs, Type: DNA, Stranding: double-stranded, Geometry: linear.

[0102] In the present invention, the gene encoding trehalose synthase PfTreS also includes a sequence obtained by deletion, nonsense, insertion, or missense mutation based on SEQ ID NO: 2.

[0103] Example 1

[0104] (1) DNA extraction

[0105] The genome of Pseudomonas B7-1 was extracted according to the genome extraction method of Gram-positive bacteria in the bacterial DNA extraction kit FastPurebacteria DNAisolationmini kit of Vazyme of Nanjing Novi Medical Technology Co., Ltd. Pseudomonas sp. B7-1 was deposited in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with a deposit date of November 7, 2024 and a deposit number of CCTCC NO: M20242464.

[0106] (2) Primer design

[0107] Upstream and downstream PCR primers were designed according to the genome of Pseudomonas sp. B7-1. EcoRⅠ and Not I restriction sites were designed for subsequent vector ligation.

[0108] The upstream primer was 5′-CCGGAATTCATGGCGAAGAAACCCAAGG-3′ (SEQ ID NO: 3);

[0109] The downstream primer was 5'-AAGGAAAAAAGCGGCCGCCTCTCCACCAAGATCG G-3' (SEQ ID NO: 4).

[0110] (3) Gene sequence determination

[0111] The above primers were used for PCR amplification to obtain an amplified product. The amplified product was sequenced by entrusting Sangon Biotech (Shanghai) Co., Ltd. to obtain the gene sequence of trehalose synthase PftreS.

[0112] (4) Gene sequence analysis

[0113] The gene sequence was analyzed using Snapegene 4.1.8 software. The gene of trehalose synthase PftreS consists of 3339 nucleotides, and the sequence is shown in SEQ ID NO: 2. SEQ ID NO: 2 was submitted to the blast website for sequence alignment with the NCBI library. The alignment results showed that the SEQ ID NO: 2 gene sequence had the highest similarity with the base sequence at positions 3221202 to 3224540 in the Pseudomonasfluorescens DR397 genome (Sequence ID: CP048408.1), reaching 97.15%.

[0114] (5) Amino acid sequence analysis

[0115] The gene of trehalose synthase PftreS encodes a protein containing 1113 amino acids, and the amino acid sequence is shown in SEQ ID NO: 1. The theoretical molecular weight of the protein is predicted to be 125 kDa by Snapegene 4.1.8 software. The amino acid sequence of the obtained trehalose synthase PftreS was submitted to the blast website and the NCBI library for amino acid sequence alignment. The alignment results showed that the amino acid sequence of SEQ ID NO: 1 had the highest similarity with maltose α-D-glucosyltransferase from Pseudomonas (Sequence ID: WP_064596283.1) and maltose α-D-glucosyltransferase from Pseudomonasfluorescens (Sequence ID: WP_163975072.1), both of which were 99.64%, and 1109 of the 1113 amino acids were identical.

[0116] (6) Cloning and expression of trehalose synthase PftreS gene

[0117] The amplified trehalose synthase PftreS gene was digested with restriction endonucleases EcoRI and Not I, and then connected to the expression vector pET30a(+) digested with EcoRI and Not I. The ligation product was then transformed into Escherichia coli BL21(DE3) to obtain a recombinant Escherichia coli BL21(DE3) strain containing plasmid pET30a(+)-PftreS.

[0118] The recombinant E. coli BL21 (DE3) strain containing plasmid pET30a (+) -PftreS was inoculated into 5 mL of LBK medium containing kanamycin (50 μg / mL), and cultured at 37°C for 12 hours. 5 mL of bacterial solution was added to 800 mL of LBK medium containing kanamycin (50 μg / mL) for culture. When OD600 was 0.6, IPTG was added to make the final concentration 1.0 mM, and induced at 16°C for 24 hours. Centrifuge at 6000 rpm for 12 minutes, collect the cells, resuspend the cells with 20 mL of pH 7.0 200 mM PBS, and ultrasonically disrupt the cells for 30 minutes. Centrifuge at 12000 rpm for 10 minutes, and the supernatant is the crude enzyme solution containing trehalose synthase PftreS.

[0119] (7) Purification of Trehalose Synthase PftreS

[0120] The crude enzyme solution was chromatographed on a nickel ion metal chelate column and gradient eluted with an elution buffer containing 20-500 mM imidazole. First, a binding buffer was prepared: a final concentration of 0.5 M NaCl, 10 mM imidazole, and 20 mM phosphate buffer were fully mixed and adjusted to pH 7.4; an elution buffer: a final concentration of 0.5 M NaCl, 20-500mM imidazole and 20mM phosphate buffer are fully mixed and the pH value is adjusted to 7.4, and filtered through a 0.22μm filter membrane; 5mL of nickel ion filler is loaded into a 10mL empty gravity column and allowed to stand at 4°C overnight to prepare a gravity column, and the gravity column is washed with 5 times the column volume of ultrapure water after membrane sonication, and then the gravity column is washed with 5 times the column volume of binding buffer; the crude enzyme solution is filtered through a 0.22μm filter and mixed with the binding buffer for loading, so that it is fully mixed with the nickel filler; the elution buffer containing 20-500mM imidazole is used for elution in turn, and the eluate corresponding to the peak is collected; 3 times the column volume is washed with 500mM elution buffer, the gravity column is washed with ultrapure water, and finally the gravity column is washed with 20% ethanol and then sealed; the elution product obtained by collecting the elution solution with different imidazole concentration gradient is verified by SDS-PAGE, so as to determine the optimal imidazole elution concentration. The results of SDS-PAGE of pure trehalose synthase PfTreS are shown in Figure 1 The concentration of pure enzyme protein was determined using the Coomassie Brilliant Blue method.

[0121] (8) Method for determining the enzyme activity of trehalose synthase PftreS

[0122] The trehalose synthase PftreS in the determination method is all trehalose synthase PfTreS pure enzyme.

[0123] 125 μL of 0.636 mg / mL pure trehalose synthase PfTreS enzyme was added to pH=6.575 μL of 10wt% maltose substrate and reacted at 30°C for 10 min. The reaction was terminated in a boiling water bath for 5 min, and the reaction solution was centrifuged at 10000 rpm for 1 min, and the supernatant was taken. After the supernatant was cooled, 5 μL was taken and diluted with 195 μL of water. The dilution was added with 200 μL of DNS solution and boiled in a water bath for 5 min for color development. After the solution was cooled, 200 μL was taken and measured at 540 nm. The unit U of trehalose synthase activity is defined as the amount of enzyme required to generate 1 μmol of trehalose per minute. The final test showed that the specific activity (enzyme activity) of trehalose synthase PfTreS was 17.36 U / mg.

[0124] Determination of optimal enzyme pH: Prepare pH 5.5-850mM sodium citrate buffer and pH 8.550mM Tris-HCl buffer. Prepare 10wt% maltose substrate of different pH. Add 40μL 1.9mg / mL trehalose synthase PfTreS to 160μL 10wt% maltose substrate, and set the reaction conditions to 35℃ for 10min. The control group used inactivated trehalose synthase PfTreS for the reaction. The maximum conversion rate in the reaction group was set to 100%. The final results are as follows Figure 2 As shown in a. As can be seen from the figure, the optimum pH of trehalose synthase PfTreS is 6.5, the enzyme activity is 70% in the pH range of 5-6.5, the enzyme activity is 60% in the pH range of 7-8, and the enzyme activity drops sharply to 29% at pH = 8.5.

[0125] Optimum temperature determination: Prepare 10wt% maltose substrate with pH 6.5. Add 40μL 1.9mg / mL trehalose synthase PfTreS to 160μL 10wt% maltose substrate, and set the reaction conditions to 20-45℃ for 10min. The control group uses inactivated trehalose synthase PfTreS for reaction. Set the maximum conversion rate in the reaction group to 100%. The test results are as follows: Figure 2 As shown in b, it can be seen from the figure that the optimum temperature of trehalose synthase PfTreS is 30°C, and the enzyme activity is maintained at more than 80% at 20-45°C.

[0126] pH stability determination: The pH stability determination range is 3-11, and pH 3-8 sodium citrate buffer, pH 8.5-9 Tris-HCl buffer, pH 10-11 glycine-sodium hydroxide buffer and trehalose synthase PfTreS are used for incubation at 4°C for 24 hours, and all buffers are 50mM. Subsequently, 40μL 0.4mg / mL trehalose synthase PfTreS is added to pH=6.5160μL 10wt% maltose substrate, reacted at 30°C for 10min, and the remaining enzyme activity is determined. The enzyme activity of trehalose synthase PfTreS that was not placed in a pH buffer and was placed at 4°C for 24h was set to 100%. The control group was determined using inactivated trehalose synthase PfTreS that was placed at different pH and 4°C for 24h. The test results are as follows Figure 2 As shown in c, it can be seen from the figure that the enzyme has good pH stability. When the pH is lower than 5, the enzyme is completely inactivated. At pH 5-8, the remaining enzyme activity is maintained at more than 80%. When the pH is higher than 8, the enzyme activity decreases. At pH 11, it is still active, with an enzyme activity of 11%.

[0127] Temperature stability determination: The temperature was set at 25-50°C. 1.9 mg / mL trehalose synthase PfTreS was placed at different temperatures for 30 min. Then 40 μL of trehalose synthase PfTreS was added to 160 μL of 10 wt% maltose substrate at pH = 6.5, reacted at 30°C for 10 min, and the remaining enzyme activity was determined. The enzyme activity of the unincubated trehalose synthase PfTreS was set as 100%. The results are shown in Figure 2. Figure 2 As shown in Figure d, it can be seen from the figure that at 25°C, the enzyme activity decreased significantly, and 18% of the enzyme activity was lost after standing at 25°C.

[0128] In summary, 125 μL of 0.636 mg / mL trehalose synthase PfTreS was added to 75 μL of 10 wt% maltose substrate, and the reaction was carried out for 10 min at the optimal reaction conditions of pH=6.5 and 35°C. The specific activity of trehalose synthase PfTreS obtained by this method was 17.36 U / mg.

[0129] Example 2

[0130] 15 μL 3.26 mg / mL pure trehalose synthase PfTreS enzyme was added to pH=7160 μL 30 wt% and 40 wt% maltose substrates, and pH=7 PBS buffer was added to 200 μL. The reaction was carried out at 25°C pH=7 for 24 hours. The reaction was terminated in a boiling water bath for 5 minutes, and the reaction solution was centrifuged at 10000 rpm for 1 minute. The supernatant was diluted 300 times with water and then tested by HPLC to calculate the trehalose yield, trehalose yield = (trehalose concentration / initial maltose concentration) × 100%. HPLC conditions were as follows: amino column for HPLC (model Hypersil-NH2, 5 μm, column size 4.6 mm×250 mm, Dalian Yilite Analytical Instrument Co., Ltd.), acetonitrile / water = 75 / 25 (v / v), 1 mL / min, injection volume 10 μL, column temperature 30°C, detection using ELSD detector, drift tube temperature 80°C, air pressure 350 kPa, gain 1. HPLC was used to prepare standard curves for trehalose and maltose. The standard curves are as follows: Figure 3 The HPLC test results of the reaction liquid of trehalose synthase PftreS with 30wt% maltose as substrate at 25℃ for 24h are shown in Figure 4 The trehalose content was calculated by peak area, and the conversion rate of trehalose synthase PftreS was 66.31% when 30wt% and 40wt% maltose were used as substrates at 25℃ and pH=7 for 24h.

[0131] As can be seen from the above embodiments, the present invention provides a trehalose synthase PfTreS and its application, an expression vector and an expression strain. The present invention obtains a trehalose synthase from Pseudomonas, which is a maltose α-D-glucosyltransferase. The enzyme can catalyze high-concentration maltose into trehalose, and the catalytic process is only one step, the operation is simple, the cost is saved, and the conversion rate is high.

[0132] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A trehalose synthase PfTreS, characterized in that The amino acid sequence of the trehalose synthase PfTreS is shown in SEQ ID NO:

1.

2. The trehalose synthase PfTreS according to claim 1, characterized in that The trehalose synthase PfTreS is derived from Pseudomonas sp. B7-1; Pseudomonas sp. B7-1 is deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China. The deposit date is November 7, 2024. The deposit number is CCTCC NO: M20242464. The specific activity of the trehalose synthase PfTreS is ≥11.4 U / mg.

3. Use of the trehalose synthase PfTreS according to claim 1 or 2 in catalyzing the production of trehalose from maltose.

4. A gene encoding trehalose synthase PfTreS, characterized in that: The sequence of the gene is shown in SEQ ID NO: 2; The trehalose synthase PfTreS is the trehalose synthase PfTreS according to claim 1 or 2.

5. A primer pair for amplifying the gene according to claim 4, characterized in that: The primer pair is shown in SEQ ID NO: 3-4.

6. An expression vector, characterized in that: It includes a gene encoding trehalose synthase PfTreS and an empty vector; The gene encoding trehalose synthase PfTreS is the gene according to claim 4; The empty vector is pET-30a(+).

7. Use of the expression vector according to claim 6 in preparing a product that catalyzes maltose to produce trehalose.

8. An expression strain, characterized in that including a gene encoding trehalose synthase PfTreS and a host bacterium; The gene encoding trehalose synthase PfTreS is the gene according to claim 4; The host bacteria is Escherichia coli BL21 (DE3).

9. Use of the expression strain according to claim 8 in preparing a product that catalyzes maltose to produce trehalose.

10. A method for catalyzing maltose to produce trehalose, characterized in that: The steps include: The trehalose synthase PfTreS was mixed with maltose and reacted for 10 to 1440 minutes; The trehalose synthase PfTreS is the trehalose synthase PfTreS according to claim 1 or 2; The initial concentration of the trehalose synthase PfTreS is 0.6-3.26 mg / mL; The initial concentration of maltose is 10-40wt%; The reaction temperature is 25-50°C; The pH of the reaction is 3-11.

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