A trehalose synthetase PfTreS and application thereof and an expression vector and an expression strain

Abstract

The present application relates to the technical fields of genetic engineering and enzyme engineering, and particularly relates to a trehalose synthetase PfTreS, application thereof, an expression vector and an expression strain. The present application provides a trehalose synthetase PfTreS, wherein the amino acid sequence of the trehalose synthetase PfTreS is shown as SEQ ID NO:1. The enzyme can catalyze high-concentration maltose to generate trehalose, has high conversion efficiency, and can be completed in one step, thereby reducing production cost, avoiding the risk of additional process generated due to the conflict of optimal reaction conditions in multiple enzyme reactions, and being suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical fields of genetic engineering and enzyme engineering, and particularly relates to a trehalose synthetase PfTreS and application thereof and an expression vector and an expression strain. BACKGROUND

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

[0003] At present, the enzymatic synthesis of trehalose mainly includes three methods: 1) trehalose synthesis by phosphorylase: 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 of phosphorylation and dephosphorylation; 2) double enzyme method for synthesizing trehalose: in this method, maltosyl trehalose synthetase and maltosyl trehalose hydrolase are used to synthesize trehalose, and maltodextrin is used as the reaction substrate. The reaction process covers two steps of glycosidic bond isomerization and hydrolysis; 3) different from the above two methods, single enzyme method only uses one enzyme-trehalose synthetase, and uses maltose as the reaction substrate. The significant feature of this method is that the whole reaction process only consists of one step, that is, the glycosidic bond isomerization of maltose catalyzed by trehalose synthetase, thereby synthesizing trehalose. Compared with the phosphorylase method and the double enzyme method, the single enzyme method for synthesizing trehalose has significant advantages. The reaction substrate and steps of the single enzyme method for synthesizing trehalose are simpler, and multiple enzymes and complex reaction conditions are not required. In the production process, only one enzyme is required, which not only reduces the production cost of using multiple enzymes, but also avoids the risk of additional processes due to the conflict of the optimal conditions of different enzymes.

[0004] However, the existing trehalose synthetase is unstable, and the conversion rate of catalyzing trehalose is low. There is an urgent need for a stable and high-conversion trehalose synthetase to meet the deficiencies of the prior art. SUMMARY

[0005] The present application relates to the technical fields of genetic engineering and enzyme engineering, and particularly relates to a trehalose synthetase PfTreS and application thereof and an expression vector and an expression strain.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] The application provides a trehalose synthetase PfTreS, and an amino acid sequence of the trehalose synthetase PfTreS is shown as SEQ ID NO: 1.

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

[0009] The Pseudomonas sp. B7-1 is preserved in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on November 7, 2024, and the preservation number is CCTCC NO: M20242464.

[0010] The specific activity of the trehalose synthetase PfTreS is greater than or equal to 11.4 U / mg.

[0011] The application also provides application of the trehalose synthetase PfTreS in catalyzing maltose to generate trehalose.

[0012] The application also provides a gene for encoding the trehalose synthetase PfTreS, and a sequence of the gene is shown as SEQ ID NO: 2.

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

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

[0015] The application also provides an expression vector, which comprises the gene for encoding the trehalose synthetase PfTreS and a blank carrier.

[0016] The gene for encoding the trehalose synthetase PfTreS is the gene.

[0017] The blank carrier is pET-30a (+).

[0018] The application also provides application of the expression vector in preparing a product for catalyzing maltose to generate trehalose.

[0019] The application also provides an expression strain, which comprises the gene for encoding the trehalose synthetase PfTreS and a host bacterium.

[0020] The gene for encoding the trehalose synthetase PfTreS is the gene.

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

[0022] The application also provides the use of the expression strain in the preparation of a product catalyzing maltose to generate trehalose.

[0023] The application also provides a method for catalyzing maltose to generate trehalose, comprising the following steps:

[0024] trehalose synthetase PfTreS is mixed with maltose, and the reaction is carried out for 10-1440 min;

[0025] The trehalose synthetase PfTreS is the trehalose synthetase PfTreS described above.

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

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

[0028] The temperature of the reaction is 25-50℃.

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

[0030] The application provides a trehalose synthetase PfTreS, the application and an expression vector and an expression strain.

[0031] The trehalose synthetase PfTreS has high enzyme specific activity, which can reach 11.4 U / mg, and can react at a wide pH range of 25-50℃, and can catalyze maltose with a substrate concentration of 10-40 wt% to generate trehalose with high conversion rate.

[0032] The application can catalyze maltose to generate trehalose by only using the trehalose synthetase PfTreS, and the steps are simple, which not only reduces the cost, but also avoids the risk caused by the difference in the optimum conditions of enzymes. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 It is an SDS-PAGE detection result graph of the pure trehalose synthetase PfTreS (M is Mark, and lanes 1-2 are the trehalose synthetase PfTreS).

[0034] Figure 2 It is the enzymatic property of the trehalose synthetase PfTreS.

[0035] Figure 3 It is a standard curve of trehalose and maltose.

[0036] Figure 4 It is an HPLC graph of the trehalose synthetase PfTreS reacting with 30 wt% trehalose.

[0037] Depositing information

[0038] Pseudomonas sp. B7-1 was deposited with China Center for Type Culture Collection, Wuhan University, Wuhan, China on November 07, 2024, and was assigned accession number CCTCC NO: M20242464. DETAILED DESCRIPTION

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

[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] QHPWFQRARKAKPGSAARDFYVWSDDDQKYDGTRIIFLDTEKSNWTWDPVAGQYFW

[0045] HRFYSHQPDLNFDNPQVMKAVLSVMRYWLDMGIDGLRLDAIPYLIERDGTNNENLPE

[0046] THDVLKQIRAEIDANYPDRMLLAEANQWPEDTQLYFGDTDAEGVNGDECHMAFHFPL

[0047] MPRMYMALAQEDRFPITDILRQTPEIPANCQWAIFLRNHDELTLEMVTDKERDYLWNY

[0048] YAADRRARINLGIRRRLAPLMERDRRRIELLNSLLLSMPGTPTLYYGDEIGMGDNIYLG

[0049] DRDGVRTPMQWSIDRNGGFSRADPASLVLPPIMDPQYGYQSVNVETQAGDPHSLLNW

[0050] TRRLLTVRKQ SKAFGRGTLK MLSPANRRVL AYTREYTGPD GKHEIILCVA NVSRSAQA

[0051] VELDLSAYVGM VPVEMLGGNA FPPIGQLSFL LTLPPYGFYW GLAAENQMPS WHVEP

[0052] AQSLPDFTTL VLKKRMEELL EAPSRATLEQ AILPSWLQNR RWFAGKDAAI EQVKLAYG

[0053] VRFGDAQHPV LFSEIEVQSG GQTSRYQLPF GFIAEDQVGP ALPQQLALAR VRRVRQVGL

[0054] ITDAFSLEAF VRAVLQGMQS GTVELSDGEI RFEATPQLEK LGLGAESEVR YLSAEQSNS

[0055] SVVIGNSLVL KLIRKVASGV HPELEMSAYL TAAGFANISP LLGSVIRRGAD GEDNLLMIA

[0056] QGYLSNQGDA WEWTQNNLE RALRDELADA VSEQAQHYN ALGELKDFAG MLGQRLG

[0057] EMHQVLAAPS DNKDFAPQVS SAKDAQTSGK DVAAQVEHAL KLLKQHQGE LDAADQK

[0058] LVARLLDNKK TILAHVQELA KSAGGLRIRV HGDLHLGQVL VIKGDAYLID FEGEPARP

[0059] LAERRGKHSP YKDVSGVLRS FDYAAAMALN VHNVDNSPEE AAARRRVTER YLREARE

[0060] AFLQAYRQAA ASLDHAWQDP EGADAALALF GLEKAAYEVA YEAENRPTWL PVPLHGLYGL LTGLKPFSDL GGE.

[0061] Length: 1113 amino acids, Type: Polypeptide, Chain: Single, Geometry: Stereo.

[0062] The gene encoding trehalose synthetase 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] GCGCGGAGATCGACGCCAACCACCGGATCGCATGTTGCTGGCCGAAGCCAACCAATGGCCGGAAGACCTCAGCTGTACTTCGGCG

[0072] GCGCGGAGATCGACGCCAACCACCGGATCGCATGTTGCTGGCCGAAGCCAACCAATGGCCGGAAGACCTCAGCTGTACTTCGGCG

[0073] GCGCGGAGATCGACGCCAACCACCGGATCGCATGTTGCTGGCCGAAGCCAACCAATGGCCGGAAGACCTCAGCTGTACTTCGGCG

[0074] GCGCGGAGATCGACGCCAACCACCGGATCGCATGTTGCTGGCCGAAGCCAACCAATGGCCGGAAGACCTCAGCTGTACTTCGGCG

[0075] GCGCGGAGATCGACGCCAACCACCGGATCGCATGTTGCTGGCCGAAGCCAACCAATGGCCGGAAGACCTCAGCTGTACTTCGGCG

[0076] GCGCGGAGATCGACGCCAACCACCGGATCGCATGTTGCTGGCCGAAGCCAACCAATGGCCGGAAGACCTCAGCTGTACTTCGGCG

[0077] GCGCGGAGATCGACGCCAACCACCGGATCGCATGTTGCTGGCCGAAGCCAACCAATGGCCGGAAGACCTCAGCTGTACTTCGGCG

[0078] GCGCGGAGATCGACGCCAACCACCGGATCGCATGTTGCTGGCCGAAGCCAACCAATGGCCGGAAGACCTCAGCTGTACTTCGGCG

[0079] GCGCGGAGATCGACGCCAACCACCGGATCGCATGTTGCTGGCCGAAGCCAACCAATGGCCGGAAGACCTCAGCTGTACTTCGGCG

[0080] ggcgttcggt cgtggcacct tgaagatgtt gtcgccggcc aaccgtcgcg tgctggctta cacccgcgaa tacaccgggc cgacggc

[0081] aagcacgaaat cattctgtgc gtagccaacg tgtcacgcag tgcacaagcg gtggaactcg acctgtcggc ttacgtcggc atggtgcc

[0082] ggtggaaatg ctcggtggta acgcgttccc gcccatcggc cagttgagtt tcctgctgac cctgccgccc tacggtttct actggttcgg c

[0083] ctggcggcgg aaaaccagat gccgagctgg cacgtggaac ctgcgcagag cctgccggac ttcaccacgc tggtgttgaa aaacgc

[0084] atggaagaac tgctcgaagc gccgtcccgg gccaccctcg agcaggcgat cctgccgagc tggtgcagaa ccgccgctgg ttcgcc

[0085] ggcaaggacg cgccatcgaa caggtgaaac tggtcctacg gcgtgcgctt cggcgatgcg agcatccggt gttgttcagc gaaatc

[0086] gaagtgcaga gcggcgggca accagccggt atcaactgcc gttcggcttc attgccgaag atcaggtcgg cccggcattg ccgcag

[0087] caattggcct tggcccgtgt gcgccgggtt cgccaggttg ggctgatcac cgatgctttc agtcttgaag catttgttcg cggtgctgca

[0088] aggcatgcag agcggcacgg tgctggaatc cagtgatggc gagatccgtt tcgaggcgac gccgcaactg gaaaaactcg gcctcgg

[0089] cgcggaatcc gaagtgcgct atctgtccgc cgagcaatcc aacagttcag tggtgatcgg caacagtctg gtacttaagc tgatccgaaa

[0090] agtcgcttct ggcgtacacc cggaactgga gatgagcgct tacctgaccg cagccggatt tgccaatatc tcaccgctgc tgggttcggt

[0091] gattcgccgc ggcgccgatg gtgaagacaa tctattgatg attgcacagg gctatctgag caatcagggc gacgcctggg aatggacg

[0092] cagaacaacc tcgaacgggc gctgcgcgat gaactggccg acgctgtttc cgaacaggcg cagcactaca acgccctggg tgaactg

[0093] aaggatttcg ccggcatgct cggccagcgt ctgggggaaa tgcatcaggt actggcagcg ccgagcgaca aaggacttcg ccgc

[0094] caagtcagtt cggccaagga tgcgcagacg agcggcaagg atgtcgcggc gcaggtcgag catgcgctga agctgctcaa acagca

[0095] tcagggcgaactagacgcggcggatcagaagctggtcgctcgtttgctcgacaacaagaaaaccatccttgcccacgtgcaggaactg

[0096] gccaagcaatccgccggcggtctgcggatccgcgtccacggcgatctgcatttggggcaggtgctggtaatcaagggcgatgcctatct

[0097] gatcgacttcgaaggtgagccggcgcggccactggccgagcgccgaggcaagcacagtccttacaaggacgtcagcggagtgctgc

[0098] gctccttcgactatgccgcggccatggcgctgaatgtgcacaacgtcgacaacagccccgaggccgaagccgcgcgtcggcgggtca

[0099] cggagcgttacctgcgtgaagcccgggaagcctttctccaggcatatcgtcaggcagcagctagtcttgatcatgcctggcaagatcctg

[0100] aaggtgccgacgccgcactggcgttgttcggtctggagaaagcggcctacgaagtggcctatgaagccgaaaatcgccccacctggctgcccgtgccgctgcacggtctgtatgggttattgacggggctcaaacccttttccgatcttggtggagag。

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

[0102] In the present application, the gene encoding trehalose synthetase PfTreS also includes the sequences obtained after deletion, nonsense, insertion, missense mutation on the basis of SEQ ID NO: 2.

[0103] Example 1

[0104] (1) Extraction of DNA

[0105] The genome of Pseudomonas B7-1 was extracted according to the method for extracting the genome of gram-positive bacteria in the bacterial DNA extraction kit FastPure bacteria DNA isolation mini kit of Nanjing Vazyme Medical Technology Co., Ltd. Pseudomonas Pseudomonas sp. B7-1 was preserved in China Center for Type Culture Collection, Wuhan University, Wuhan, China, on November 7, 2024, and the preservation number was CCTCC NO: M20242464.

[0106] (2) Design of primers

[0107] According to the genome of Pseudomonas B7-1, the upstream and downstream PCR primers were designed, and EcoR I and Not I enzyme cutting sites were designed for subsequent vector connection.

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

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

[0110] (3) Gene sequence determination

[0111] The above primers were used for PCR amplification to obtain the amplification product. The amplification product was sequenced by Shengong Bioengineering (Shanghai) Co., Ltd., and the gene sequence of trehalose synthetase PftreS was obtained.

[0112] (4) Gene sequence analysis

[0113] The gene sequence was analyzed by Snapegene 4.1.8 software. The gene of trehalose synthetase PftreS consisted of 3339 nucleotides, and the sequence was shown in SEQ ID NO: 2. SEQ ID NO: 2 was submitted to the blast website and the NCBI library for sequence alignment, and the alignment result showed that the gene sequence of SEQ ID NO: 2 had the highest similarity with the base sequence of 3221202-3224540 in Pseudomonas fluorescens DR397 genome (Sequence ID: CP048408.1), reaching 97.15%.

[0114] (5) Amino acid sequence analysis

[0115] The gene of trehalose synthetase 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 by Snapegene 4.1.8 software to be 125 kDa. The obtained amino acid sequence of trehalose synthetase PftreS is submitted to the blast website and the NCBI library for amino acid sequence alignment, and the alignment result shows that the amino acid sequence of SEQ ID NO: 1 has the highest similarity with maltose alpha-D-glucosyltransferase from Pseudomonas (Sequence ID: WP_064596283.1) and maltose alpha-D-glucosyltransferase from Pseudomonas fluorescens (Sequence ID: WP_163975072.1), both of which are 99.64%, and 1109 of the 1113 amino acids are identical.

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

[0117] The amplified gene of trehalose synthetase PftreS is digested with restriction enzymes EcoR I and Not I, and then ligated with the expression vector pET30a(+) digested with EcoR I and Not I. The ligation product is then transformed into E. coli BL21(DE3) to obtain a recombinant E. coli BL21(DE3) strain containing plasmid pET30a(+)-PftreS.

[0118] The recombinant E. coli BL21(DE3) strain containing plasmid pET30a(+)-PftreS is inoculated into 5 mL of LBK medium containing kanamycin (50 μg / mL) and cultured at 37°C for 12 h. 5 mL of bacterial solution is added to 800 mL of LBK medium containing kanamycin (50 μg / mL) and cultured. When the OD600 is 0.6, IPTG is added to a final concentration of 1.0 mM, and induced at 16°C for 24 h. The bacterial cells are collected by centrifugation at 6000 rpm for 12 min, resuspended in 20 mL of pH 7.0 200 mM PBS, and broken by ultrasonic wave for 30 min. The supernatant is centrifuged at 12000 rpm for 10 min to obtain the crude enzyme solution containing trehalose synthetase PftreS.

[0119] (7) Purification of trehalose synthetase PftreS

[0120] The crude enzyme solution was subjected to nickel ion metal chelate column chromatography and gradient elution was performed using elution buffer containing 20-500 mM imidazole. First, the binding buffer was prepared: 0.5 M NaCl, 10 mM imidazole and 20 mM phosphate buffer were thoroughly mixed and the pH was adjusted to 7.4; the elution buffer: 0.5 M NaCl, 20-500 mM imidazole and 20 mM phosphate buffer were thoroughly mixed and the pH was adjusted to 7.4, and 0.22 μm filter membrane was used for filtration; 5 mL of nickel ion filler was packed into a 10 mL gravity column, which was allowed to stand overnight at 4°C to prepare the gravity column, and the gravity column was washed with 5 times the column volume of ultrapure water after ultrasonic treatment of the membrane, and then the gravity column was washed with 5 times the column volume of the binding buffer; the crude enzyme solution was filtered with a 0.22 μm filter and mixed with the binding buffer for loading, and the nickel filler was thoroughly mixed; the elution buffer containing 20-500 mM imidazole was used for elution, and the eluate corresponding to the peak was collected; 3 times the column volume of 500 mM elution buffer was used to wash the gravity column, and the gravity column was washed with ultrapure water and finally with 20% ethanol, and then the column was sealed; the elution products obtained from the gradient elution of different imidazole concentrations were subjected to SDS-PAGE to determine the optimal imidazole elution concentration. The results of the SDS-PAGE detection of the purified trehalose synthase PfTreS are shown in Figure 2. Figure 1 The concentration of the purified enzyme protein was determined by the Coomassie brilliant blue method.

[0121] (8) Enzyme activity determination method of trehalose synthase PftreS

[0122] In the determination method, the trehalose synthase PftreS is the purified trehalose synthase PfTreS.

[0123] 125 μL of 0.636 mg / mL purified trehalose synthase PfTreS was added to 575 μL of 10 wt% maltose substrate at pH 6.6, and the reaction was carried out at 30°C for 10 min. The reaction was terminated by boiling in a water bath for 5 min, and the reaction solution was centrifuged at 10000 rpm for 1 min. After cooling, 5 μL of the supernatant was diluted with 195 μL of water. The diluted solution was added with 200 μL of DNS solution, and color development was carried out by boiling in a water bath for 5 min. After cooling, 200 μL of the solution was measured at 540 nm. The trehalose synthase enzyme activity unit U was defined as the amount of enzyme required to produce 1 μmol of trehalose per minute. The final detection showed that the specific activity (enzyme activity) of the trehalose synthase PfTreS was 17.36 U / mg.

[0124] Enzyme optimum pH determination: pH 5.5-8.5 mM sodium citrate buffer and pH 8.5 50 mM Tris-HCl buffer were prepared. 10 wt% maltose substrate was prepared at different pH. 40 μΐ^of 1.9 mg / mL trehalose synthase PfTreS was added into 160 μΐ^of 10 wt% maltose substrate. The reaction condition was set as 35 °C for 10 min. The control group used inactivated trehalose synthase PfTreS for reaction. The maximum conversion rate in the reaction group was set as 100%. The final results are shown in Figure 2 a. It can be seen from the figure that 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 sharply decreases to 29% at pH 8.5.

[0125] Optimum temperature determination: 10 wt% maltose substrate was prepared at pH 6.5. 40 μΐ^of 1.9 mg / mL trehalose synthase PfTreS was added into 160 μΐ^of 10 wt% maltose substrate. The reaction condition was set as 20-45 °C for 10 min. The control group used inactivated trehalose synthase PfTreS for reaction. The maximum conversion rate in the reaction group was set as 100%. The detection results are shown in Figure 2 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 above 80% in the temperature range of 20-45 °C.

[0126] pH stability determination: The pH stability determination range is 3-11, and 50 mM pH 3-8 sodium citrate buffer, pH 8.5-9 Tris-HCl buffer, pH 10-11 glycine-sodium hydroxide buffer and trehalose synthase PfTreS were used for 4 °C incubation for 24 h. Then 40 μΐ^of 0.4 mg / mL trehalose synthase PfTreS was added into 160 μΐ^of 10 wt% maltose substrate at pH 6.5, and the reaction was carried out at 30 °C for 10 min. The residual enzyme activity was determined. The enzyme activity of trehalose synthase PfTreS without being placed in pH buffer and incubated at 4 °C for 24 h was set as 100%. The control group used inactivated trehalose synthase PfTreS incubated at different pH at 4 °C for 24 h for determination. The detection results are shown in Figure 2 c. It can be seen from the figure that the pH stability of the enzyme is good, the enzyme is completely inactivated at pH below 5, the residual enzyme activity is maintained above 80% at pH 5-8, the enzyme activity decreases at pH above 8, and the enzyme activity is 11% at pH 11.

[0127] Temperature stability assay: The temperature was set from 25 to 50 °C. 1.9 mg / mL trehalose synthase PfTreS was incubated at different temperatures for 30 min. Then 40 μL trehalose synthase PfTreS was added to 160 μL 10 wt% maltose substrate at pH = 6.5, and the reaction was carried out at 30 °C for 10 min, and the residual enzyme activity was determined. The enzyme activity of trehalose synthase PfTreS without incubation was set as 100%. The results are shown in Figure d, from which it can be seen that the enzyme activity decreased significantly at 25 °C, and 18% of the enzyme activity was lost after standing at 25 °C. Figure 2

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

[0129] Example 2

[0130] 15 μL 3.26 mg / mL trehalose synthase PfTreS pure enzyme was added to 160 μL 30 wt% and 40 wt% maltose substrates at pH = 7, respectively, and pH = 7 PBS buffer was added to 200 μL. The reaction was carried out at 25 °C and pH = 7 for 24 h. The reaction was terminated by boiling in a water bath for 5 min, and the reaction solution was centrifuged at 10000 rpm for 1 min. The supernatant was diluted 300 times with water, and then HPLC detection was carried out to calculate the yield of trehalose. The yield of trehalose = (trehalose concentration / initial maltose concentration) x 100%. The HPLC conditions were as follows: HPLC was carried out on an amino column (model Hypersil-NH2, 5 μm, column size 4.6 mm x 250 mm, Dalian Elite Analytical Instruments Co., Ltd.), acetonitrile / water = 75 / 25 (v / v), 1 mL / min, injection volume 10 μL, column temperature 30 °C, and ELSD detector was used for detection, drift tube temperature 80 °C, air pressure 350 kpa, and gain 1. The standard curve of trehalose and maltose was prepared by HPLC, as shown in Figure Figure 3 The detection results of the reaction solution of trehalose synthase PftreS with 30 wt% maltose as substrate and reacted at 25 °C for 24 h are shown in Figure Figure 4 The content of generated trehalose was calculated by peak area, and the conversion rate of trehalose synthase PftreS with 30 wt% and 40 wt% maltose as substrate and reacted at 25 °C and pH = 7 for 24 h was 66.31%.

[0131] ​From the above examples, the application provides a trehalose synthetase PfTreS and its application and an expression vector and an expression strain. The application obtains a trehalose synthetase from Pseudomonas, and the enzyme is a maltose alpha-D-glucosyltransferase. The enzyme can catalyze high-concentration maltose into trehalose in one step, and the operation is simple, the cost is saved, and the conversion rate is high.

[0132] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

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; The trehalose synthase PfTreS is derived from Pseudomonas. Pseudomonas sp. B7-1; Pseudomonas Pseudomonas sp. B7-1 is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on November 7, 2024, with accession number CCTCC NO:M20242464. The specific activity of the trehalose synthase PfTreS is ≥11.4 U / mg.

2. The application of the trehalose synthase PfTreS according to claim 1 in catalyzing the production of trehalose from maltose.

3. 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 described in claim 1.

4. An expression carrier, characterized in that, This includes the gene encoding the trehalose synthase PfTreS and the empty vector. The gene encoding trehalose synthase PfTreS is the gene described in claim 3; The empty carrier is pET-30a(+).

5. The application of the expression vector according to claim 4 in the preparation of a product that catalyzes the conversion of maltose to trehalose.

6. An expression strain, characterized in that, This includes the gene encoding the trehalose synthase PfTreS and the host bacteria; The gene encoding trehalose synthase PfTreS is the gene described in claim 3; The host bacterium is Escherichia coli BL21(DE3).

7. The use of the expression strain according to claim 6 in the preparation of a product that catalyzes the conversion of maltose to trehalose.

8. A method for catalyzing the production of trehalose from maltose, characterized in that, Includes the following steps: Mix trehalose synthase PfTreS with maltose and react for 10-1440 min. The trehalose synthase PfTreS is the trehalose synthase PfTreS as described in claim 1; The initial concentration of the trehalose synthase PfTreS was 0.6–3.26 mg / mL; The initial concentration of maltose is 10-40 wt%; The reaction temperature is 25~50℃; The pH of the reaction is 5-11.

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