Heat-resistant alpha-galactosidase and method for enzymatically hydrolyzing raffinose

Abstract

The invention discloses heat-resistant alpha-galactosidase and a method for enzymatically hydrolyzing raffinose. The heat-resistant alpha-galactosidase has a specific amino acid sequence (SEQ ID NO. 1) and can be obtained through a genetic engineering means. The preparation method comprises the following steps: optimizing a codon, cloning into a recombinant plasmid pET20b-TstGal, transforming escherichia coli BL21 (DE3), carrying out IPTG (isopropyl-beta-d-thiogalactoside) induced expression, purifying and the like. The optimized induction conditions are as follows: the IPTG concentration is 0.1 mmol / L, the OD600 value is 1.0, the temperature is 42 DEG C, and the time is 5 hours. The obtained alpha-galactosidase is suitable for enzymatic hydrolysis of raffinose, and preferably, the enzymolysis condition is a citric acid-disodium hydrogen phosphate buffer solution system with the raffinose concentration of 0.1 mol / L, the enzyme concentration of 1.5 U / mL, the temperature of 70 DEG C and the pH value of 6.5. The alpha-galactosidase provided by the invention has heat resistance, the preparation method is efficient, enzymolysis conditions are optimized, a powerful tool enzyme is provided for hydrolysis of raffinose, and the alpha-galactosidase has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and specifically relates to the preparation of a thermostable alpha-galactosidase derived from Thermoclostridiumstercorarium subsp.thermolacticum DSM 2910 and its application in enzymatic hydrolysis of raffinose. Background Art

[0002] α-Galactosidase (EC3.2.1.22) is a glycoside hydrolase that can hydrolyze oligosaccharides and polysaccharides with galactose residues linked by α-1,6-glycosidic bonds, such as melibiose, raffinose, stachyose, verbose, galactomannan and galactoglucomannan. It is widely used in the food, feed and medical industries. This enzyme is widely found in plants, animals including humans and microorganisms. In plants, α-galactosidase has been found, especially in legume seeds. In animals, it is found in sheep pancreas, pig cells and duodenal glands in rat intestine. In microorganisms, α-galactosidase has been isolated and purified from Klebsiella pneumoniae, Fusarium moniliforme NCIM 1099 and Penicillium oxysporum APS1. In addition, α-galactosidase genes from Rhizomucor miehei, Rhizobacter racemosus, Penicillium pararubrum and Aspergillus oryzae YZ1 have been cloned, expressed, purified and characterized. Compared with animals and plants, the production of α-galactosidase using genetically engineered bacteria has the characteristics of low production cost and good stability, which has attracted the attention of many researchers.

[0003] Soybean is an important plant source of protein, but it contains anti-nutritional factors, raffinose oligosaccharides (RFOs), which cannot be digested and absorbed by monogastric animals, leading to flatulence, gastrointestinal dysfunction, and low feed utilization. In order to solve this problem, microbial α-galactosidase is usually added to soy products to hydrolyze RFOs, thereby reducing flatulence and improving the utilization of food and feed. Therefore, obtaining α-galactosidase with good enzymatic properties and efficient hydrolysis of raffinose has important application value for the food and feed industry. Summary of the invention

[0004] To solve the above problems, we disclose a thermostable α-galactosidase and a method for enzymatic hydrolysis of raffinose.

[0005] The invention provides an alpha-galactosidase, the amino acid sequence of which is shown in SEQ ID NO.1; the enzyme has an optimum reaction temperature of 70°C, an optimum reaction pH of 6.5, and good heat resistance.

[0006] The present invention provides the α-galactosidase encoding gene, which has a nucleotide sequence as shown in SEQ ID NO.2.

[0007] The invention provides a recombinant plasmid comprising a nucleotide sequence encoding the alpha-galactosidase.

[0008] The present invention provides a host cell, comprising the recombinant vector or a nucleotide sequence as shown in SEQ ID NO.2 integrated into the genome.

[0009] The invention provides a method for preparing the recombinant α-galactosidase.

[0010] In the expression conditions of the recombinant α-galactosidase of the present invention:

[0011] Induction IPTG concentration: 0.01mmol / L-5mmol / L;

[0012] Induction OD600 value: 0.2-2.0;

[0013] The induction temperature is 16-45°C;

[0014] Induction time: 18-66h.

[0015] In some specific embodiments, the induction temperature is 42°C;

[0016] The induction IPTG concentration was: 0.1mmol / L;

[0017] The induced OD600 value was: 1.0;

[0018] Induction time: 5h.

[0019] The present invention provides the use of the α-galactosidase in hydrolyzing raffinose.

[0020] In the method provided by the present invention, the buffer for enzymatic hydrolysis is a citric acid-disodium hydrogen phosphate buffer.

[0021] The citric acid-disodium hydrogen phosphate buffer solution is 10mmol / L-200mmol / L, and the pH is 5.0-8.0;

[0022] In some specific embodiments, the buffer is a citric acid-disodium hydrogen phosphate buffer with a concentration of 100 mmol / L and a pH of 6.5.

[0023] In the enzymatic hydrolysis system of the present invention:

[0024] The concentration of raffinose is 0.01mol / L-1mol / L;

[0025] The concentration of α-galactosidase is 0.1U / mL-10U / mL.

[0026] In some specific embodiments, the concentration of raffinose is 0.1 mol / L;

[0027] The concentration of α-galactosidase was 1.5 U / mL.

[0028] In the present invention, the conditions for enzymolysis are:

[0029] React at 50°C-75°C.

[0030] In some specific embodiments, the enzymatic hydrolysis condition is: reacting at 70°C.

[0031] Compared with the prior art, the present invention has the following outstanding beneficial effects:

[0032] The present invention obtains a heat-resistant α-galactosidase that can efficiently hydrolyze raffinose:

[0033] (1) The α-galactosidase of the present invention has the advantages of high optimum temperature and good heat resistance;

[0034] (2) The α-galactosidase of the present invention can efficiently hydrolyze raffinose, with the highest hydrolysis rate being 99.8%. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The circuit diagram of the enzymatic hydrolysis of raffinose of the present invention is shown;

[0036] Figure 2 This is a graph showing the PCR detection results of the α-galactosidase TstGal gene described in Example 1, wherein lane M is DNAMarker (purchased from Beijing Quanshijin, catalog number BM141-01), and lane 1 is the PCR product;

[0037] Figure 3 This is the result of double digestion verification of α-galactosidase TstGal described in Example 2, wherein lane M is DNAMarker (purchased from Beijing Quanshijin, catalog number BM141-01), and lanes 1 and 2 are positive recombinant plasmids;

[0038] Figure 4 The result diagram of the change of enzyme yield at different time of induced expression of α-galactosidase TstGal described in Example 3 is shown;

[0039] Figure 5The SDS-PAGE diagram of the purification process of α-galactosidase TstGal described in Example 4 is shown; wherein lane M is a protein Marker (purchased from Beijing Quanshijin, item number DR201-01), lane 1 is the whole cell protein after the cells containing plasmid pET20b are broken, lane 2 is the supernatant protein after the cells containing plasmid pET20b are broken, lane 3 is the whole cell protein after the cells containing plasmid pET20b-TstGal strain are induced to express at 42°C, lane 4 is the supernatant protein after the cells containing plasmid pET20b-TstGal strain are induced to express at 42°C, and lane 5 is the α-galactosidase TstGal separated using a Ni-NTA protein purification column;

[0040] Figure 6 Graphs showing the qualitative determination results of α-galactosidase TstGal described in Example 5; wherein Graph A is a graph showing the determination results of the optimum reaction pH, Graph B is a graph showing the determination results of the optimum reaction temperature, Graph C is a graph showing the determination results of the temperature stability, and Graph D is a graph showing the determination results of the pH stability;

[0041] Figure 7 The figure shows the hydrolysis changes of raffinose at different times by α-galactosidase TstGal described in Example 6. DETAILED DESCRIPTION

[0042] The present invention provides a method for hydrolyzing raffinose by a thermostable α-galactosidase and an enzymatic method, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0043] The present invention provides a thermostable α-galactosidase, whose amino acid sequence is shown in SEQ ID NO.1 and is named TstGal.

[0044] The present invention also provides a DNA molecule fragment encoding the α-galactosidase of the present invention. Due to the degeneracy of codons, there may be many nucleotide sequences that can encode the α-galactosidase of the present invention.

[0045] In some embodiments, the present invention provides a DNA molecule that can encode the α-galactosidase TstGal, and its nucleotide sequence is shown in SEQ ID NO.2.

[0046] The present invention provides a recombinant plasmid containing the DNA molecule of the alpha-galactosidase of the present invention, wherein the recombinant plasmid is pET20b-TstGal.

[0047] The host bacteria for transformation and expression of the present invention are Escherichia coli expression strains, including BL21 and JM109 series strains. In a preferred embodiment, the host cell is BL21 (DE3) strain.

[0048] The induced expression of the preparation method of α-galactosidase of the present invention is specifically to culture the recombinant bacteria containing the TstGal gene in LB, collect the bacteria by centrifugation after the enzyme production stage is completed, then crush the cells with an ultra-high pressure crusher, centrifuge and take the supernatant, and purify it with a Ni-NTA protein purification column to obtain the desired target enzyme protein.

[0049] The present invention also provides a method for hydrolyzing raffinose by enzymatic method, specifically, the α-galactosidase of the present invention can hydrolyze raffinose under the conditions of pH 6.5 and 70°C.

[0050] The following are the sequences SEQ ID NO. 1-4 in the examples of the present invention.

[0051] Sequence 1: Amino acid sequence of α-galactosidase TstGal from Thermoclostridium stercorarium subsp.thermolacticum DSM 2910

[0052] 1 MPIIFHEESG EFHLYNKSIS YIIKILPNKQ LGNLYFGKVI KDRKSSFSHLF QKKARPLSAY

[0053] 61 VFEDDPAFSL QHTMQEYPSY GTTDFRSPAF EIKQKNGSRI SCFEYKKHEI FAGKNGLEGL

[0054] 121 PATYVEEPAD ADTLEITLYD ELIKTELVLS YTIFNNYSAI ARSARFINKG EETIVLERAM

[0055] 181 SASIDFIDSD FEMIQLSGAW SRERHIKTRK LQQGIQGVYS MRGISSAEHN PFIALKRPNT

[0056] 241 DENSGEVYGF SLIYSGNHLE QVEVDTHNMT RVMLGIHPDT FEWPLEKGEF FQTPEAIIVY

[0057] 301 SDEGLNKMSQ TFHKLFSKHL ISRQWRDKPR PVLINSWEAT GANFTEEKLL RMAEIAKELG

[0058] 361 IELFVLDDGW FGNRDNDKAG LGDWNIVNRK KLPDGIEGLA DKIESMGMKF GLWFEPESVN

[0059] 421 KDSNLYRKHP DWIIATPGRR TSASRNQYVL DFSRKEVVDY IYSVMEKVLS SAKISYVKWD

[0060] 481 MNRYITECYS LGAEAGSQGT VFHRYILGVY NLFSRLTERF PYILFEACSS GGARFDPGIL

[0061] 541 YYAPQVWTSD NTDAIERLKIQYGTSLLYPI STMGAHVSEV PNQQVGRITP IETRANVAYF

[0062] 601 GVFGYELDLT LLTEEEKEKI KKQITFYKFH RELFLKGTFY RILSPFDGNE TSWIVVSEDK

[0063] 661 SEAIAAYYKT LNYANKGWKR LKLVGLDRDK KYIIDNDNER YYYGDELMNV GIVLKDEELC

[0064] 721 ANGNDFSSII FYLKSI

[0065] Sequence 2: The nucleotide sequence of α-galactosidase TstGal from Thermoclostridium stercorarium subsp. thermolacticum DSM 2910

[0066] 1 ATGCCAATTA TTTTTCATGA AGAATCTGGT GAATTTCATC TTTATAATAA AAGCATCAGC

[0067] 61 TATATATT AAATATTGCC TTATATAACAG CTTGGAAACC TTTATTTCGG CASETATA

[0068] 121 AAAGACAGAA AGTCATTTTC ACATTTATTC CAGAAAAAAG CCAGACCCTT ATCGGCTTAT

[0069] 181 GTTTTTGAAG ATGATCCGGC TTTTTCACTT CAGCATACCA TGCAAAGATA TCCGTCATAT

[0070] 241 GGGACTACTG ATTTTAGAAG CCCTGCTTTT GAGATAAAGC AGAAAAAATGG GAGATAGAAG

[0071] 301 TCCTGTTTTG AATATAAAAA ACATGAAATT TTTGCAGGCA AGAATGGCTT AGAAGGCCTT

[0072] 361 CCGGCGACTT ATGTGGAAGA ACCAGCCGAT GCAGATACCC TTGAAATAAC GCTGTATGAT

[0073] 421 CONNECTIONS AAACAGACT GGTTTTGAGC CHAINCHAPTERCONCURRENT TTCTGCGATA

[0074] 481 GCCAGAAGCG CAAGGTTCAT AACAAAAGGAGAAAACGA TTGTGCTGGA AAGGGCAATG

[0075] 541 AGCGCAAGCA TTGATTTTAT TGACAGCGAT TTTGAGATGATCCAGCTTTC CGGCGCATGG

[0076] 601 TCACGGGAAC GGCATTATTAA AACGAGAAAA CTTCAGCAGGGCATACAGGG GGTTTACAGT

[0077] 661 ATGCCGGTA TTAGCAGTGC GGAACATAAT CCGTTTATTGCCCTAAAAAG ACCAAATACG

[0078] 721 GATGAAAACA GCGGCGAAGT ATATGGTTTC AGCTTAATACAGCGGCAA CCATCTTGAA

[0079] 781 CAGGTTGAGG TAGATACCCA CAACATGACA AGAGTTATGCTGGGGTATTCA TCCGGATACT

[0080] 841 TTTGAGTGGC CGTTGGAAAA AGGAATTT TTTCAGACACCTGAAGCAAT AATTGTTTAT

[0081] 901 TCAGACGAGG GGCTCAATAA GATGAGTCAG ACATTCCATAAGCTGTTCAG CAAACATCTT

[0082] 961 ATAAGCAGAC AATGGCGTGA CAAACCAGTTG GGAAGCTACT

[0083] 1021 GGCGCAATT TTACTGAGGA AAAGCTTTTA AGAATGGCAGAAATAGCAAA GGAACTTGGA

[0084] 1081 ATCGAACTGT TTGTGCTTGA TGACGGATGG TTTGGAAACCGAGACAACGA TAAAGCAGGG

[0085] 1141 CTGGGAGATT GGAATATTGT TAACAGGAAA AAACTTCCCGATGGTATTGA AGGCCTGGCT

[0086] 1201 GACAAAATTG AATCAATGGG ATTCAATTC GGGCTTTGGTTTGAGCCGGA ATCAGTAAAT

[0087] 1261 AAGGACAGCA ACTTATACAG AAAACACCCT GATTGGATAATTGCGACCCC GGGAAGAAGG

[0088] 1321 ACATCAGCTT CACGTAACCA GTACGTTTTG GATTTCTCGAGAAAAGAAGT GGTTGATTAT

[0089] 1381 ATTTATTCCG TAATGGAAAA AGTTCTGAGC AGTGCAAAAATATCCTATGT AAAATGGGAT

[0090] 1441 ATGAACAGGT ATATAACTGA GTGTTATTCT TTGGGGGCGGAAGCCGGCAG CCAAGGAACG

[0091] 1501 GTTTTTCACC GGTATATTCT GGGAGTATAT AATTTATTTTCAAGATTGAC AGAGAGGTTT

[0092] 1561 CCATATATAC TTTTTGAAGC ATGCTCAAGC GGTGGGGCACGGTTTGATCC CGGAATACTG

[0093] 1621 TATTATGCTC CGCAAGTATG GACAAGCGAT AATACTGATGCAATTGAGAG ACTGAAGATT

[0094] 1681 CAGTATGGTA CTTCTCTGCT CTATCCAATA AGTACTATGGGAGCTCATGT TTCAGAAGTT

[0095] 1741 CCCAATCAGC AAGTAGGAAG AATTACGCCT ATTGAAACAAGAGCCAATGT AGCTTATTTC

[0096] 1801 GGAGTGTTTG GTTATGAACT TGATTTGACT TTATTAACAGAAGAAGAAAA AGAAAAGATA

[0097] 1861 AAAAAACAGATTACTTTTTTA TAAATTCCAC AGAGAACTTTTTTCTTAAAGG AAATTTTAC

[0098] 1921 CGTATATTAA GCCCGTTTGA CGGAAATGAA ACGTCCTGGATAGTTGTATC AGAAGATAAA

[0099] 1981 TCAGAAGCTA TTGCAGCGTA TTACAAGACT CTGAATTATGCTAACAAAGG CTGGAAAAGG

[0100] 2041 CTTAAACTTG TAGGGCTTGA CAGAGATAAG AAATATATTATTGACAACGA CAATGAAAGG

[0101] 2101 TATTATTACG GTGATGAACT TATGAATGTC GGTATTGTATTAAAGGATGA AGAATTATGT

[0102] 2161 GCAAACGGAA ATGACTTTTC GTCCATTATC TTTTATTTGAAAAGTATCTA A

[0103] Sequence 3: 5'-CATG CCATGG GAATTAAATTTTTTGAGGATCGG-3'

[0104] Sequence 4: 5'-CCG CTCGAG CTTTTCTTTCACCAGATGTATGAG-3'.

[0105] In order to further understand the present invention, the present invention is described in detail below in conjunction with embodiments.

[0106] Example 1

[0107] Construction and identification of recombinant plasmid pET20b-TstGal.

[0108] Primers were designed according to the codon-optimized TstGal gene of Escherichia coli and synthesized by Suzhou Genewise Biotechnology Co., Ltd. The primer sequences are as follows:

[0109] TstGal-F:CATG CCATGGGAATTAAATTTTTTGAGGATCGG, the underline indicates the Nco I site (SEQ ID NO. 3).

[0110] TstGal-R:CCG CTCGAG CTTTTCTTTCACCAGATGTATGAG, the underline indicates the Xho I site (SEQ ID NO. 4).

[0111] The DSM 2910 genome (purchased from the German Microbial Culture Collection) was used as a template for PCR amplification. The amplification conditions were 94°C, 3 min; 30 cycles (94°C, 5 s; 57°C, 15 s; 72°C, 43 s); 72°C, 10 min; reaction stop, 4°C incubation. The PCR product was detected by agarose gel electrophoresis. The results are as follows: Figure 2 As shown. Figure 2 As a result, a bright and single target band was obtained.

[0112] Then, the PCR amplification product was purified using a gel recovery kit to obtain the DNA molecule of α-galactosidase TstGal. The obtained DNA molecule of α-galactosidase TstGal and pET20b were double-digested with BamH I and EcoR I, respectively, and recovered with a gel recovery kit, respectively. After concentration, they were connected at 16°C overnight, and the connected products were transformed into Escherichia coli DH5α competent cells, and positive transformants were screened by ampicillin resistance plates. The positive transformants were inoculated into LB liquid culture medium (LB culture medium: tryptone 10g / L, yeast extract 5g / L, NaCl 5g / L) containing ampicillin (50mg / mL), cultured overnight at 37°C, and the recombinant plasmid was extracted using a plasmid extraction kit. Double enzyme digestion verification was performed using BamH I and EcoR I. The results are as follows. Figure 3 As shown. Figure 3 The results showed that there were two bands after double enzyme digestion, and the double enzyme digestion was verified to be correct.

[0113] The correct recombinant plasmid was verified by double enzyme digestion and sequence analysis was performed; clones with correct sequences were selected to extract plasmids, and recombinant plasmid pET20b-TstGal containing the DNA molecule of α-galactosidase TstGal was obtained, and its nucleotide sequence is shown in ISEQ ID NO.2.

[0114] Example 2

[0115] α-galactosidase TstGal enzyme activity determination method.

[0116] Using 4-nitrophenyl-α-D-pyranogalactoside (pNP-Gal) as substrate, the p-nitrophenol obtained by hydrolysis reacts with sodium carbonate to develop a color reaction, and the absorbance of the product is measured at a wavelength of 405nm. The 100μL reaction system includes 75μL 100mM buffer with optimal pH, 20μL 5mM substrate, and 5μL diluted enzyme solution is added after preheating. The reaction is carried out at the optimal temperature for 10min, and then 0.3mL 1M NaCO3 is added to terminate the reaction. After mixing, the microplate reader is used to measure at 405nm. At the same time, a control with enzyme solution but no substrate and a control with substrate but no enzyme solution are performed.

[0117] One unit of enzyme activity (U) is defined as the amount of enzyme required to hydrolyze and release 1 μmol of p-nitrophenol at 70°C and pH 6.5.

[0118] Example 3

[0119] Inducible expression of recombinant α-galactosidase TstGal.

[0120] The recombinant plasmid pET20b-TstGal was transformed into competent cells of Escherichia coli strain BL21 (DE3) by heat shock transformation method and plated on LB plates (LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 5g / L, agar 15g / L) after culturing at 37°C overnight, pick the transformant into 50mL LB medium (50mg / mL ampicillin) and culture at 37°C with shaking at 180rpm until OD600 is 1.0, add isopropyl β-D-thiogalactopyranoside (IPTG) inducer with a final concentration of 0.1mmol / L, and culture at 42°C for 0h, 1h, 2h, 3h, 4h, 5h, and 6h, respectively. Centrifuge the culture solution at 12,000g for 8min at 4°C in a high-speed refrigerated centrifuge to collect the bacteria, add 50mL of 1×PBS buffer for resuspending and washing, and centrifuge at 12,000g for 8min at 4°C in a high-speed refrigerated centrifuge to collect the washed bacteria. Add 50 mL of 1× PBS buffer to the washed cells, resuspend, and disrupt them three times with a French high-pressure cell disruptor. Centrifuge at 12,000 g for 8 min at 4°C in a high-speed refrigerated centrifuge, collect the crude enzyme solution in the supernatant, and measure its enzyme activity. Figure 4 As shown. Figure 4 The results show that the recombinant α-galactosidase TstGal of the present invention obtains the maximum enzyme yield when the expression is induced for 5 hours, and the maximum enzyme yield is 23.8 U / mL.

[0121] Example 4

[0122] Purification of α-galactosidase TstGal.

[0123] Since the recombinant plasmid pET20b-TstGal contains a His tag, it can be purified by Ni-NTA purification column to obtain purified recombinant α-galactosidase TstGal. Specific operation process:

[0124] A. Preparation of crude enzyme solution

[0125] (1) Take the crude enzyme solution and filter it through a 0.22 μm microporous membrane.

[0126] B. Treatment of columns

[0127] (1) Wash the column with 4 mL of pure water to remove ethanol.

[0128] (2) Equilibrate the medium with 8 mL of 1× binding buffer to ensure that the composition and pH of the solution in the medium are consistent with those of the sample.

[0129] C. Loading

[0130] (1) The sample is centrifuged and filtered (0.22 μm) before loading, and the flow rate is controlled at about 6 drops per minute.

[0131] (2) Use 4 mL of 1× washing solution to wash away non-specifically adsorbed proteins.

[0132] (3) Elute with 8 mL of 1× eluent at a low flow rate and collect the eluate.

[0133] (4) Wash the medium with 8 mL of purified water and remove the eluent in the medium.

[0134] (5) Wash the washing medium with 8 mL of 20% ethanol and store at 4-8°C. Purified recombinant α-galactosidase TstGal was obtained through this process. The purity of the purified recombinant α-galactosidase TstGal was identified by SDS-PAGE method. The results are as follows: Figure 5 shown.

[0135] Depend on Figure 5 The results showed that the expression level of TstGal gene was high in dormitory bacteria BL21 (DE3), and after the target band was purified by HisTag tag, the purity of recombinant α-galactosidase TstGal in its eluate was high, reaching the electrophoresis purity level.

[0136] Example 5

[0137] Qualitative assay of recombinant α-galactosidase TstGal.

[0138] 5.1 Optimal reaction pH

[0139] The enzyme activity was measured at 70°C in a citric acid-sodium hydrogen phosphate buffer solution with a pH range of 5.0-8.0. The results are as follows: Figure 6 As shown in A.

[0140] Depend on Figure 6 A The results show that the optimal reaction pH of the recombinant α-galactosidase TstGal of the present invention is 6.5.

[0141] 5.2 Determination of the optimal reaction temperature

[0142] The enzyme activity was measured at 50-80°C at 5°C intervals in a citric acid-sodium hydrogen phosphate buffer solution at pH 6.5. The results are as follows: Figure 6 As shown in B.

[0143] Depend on Figure 6 B. The results show that the optimum reaction temperature of the recombinant α-galactosidase TstGal of the present invention is 70°C, which is a medium-temperature enzyme.

[0144] 5.3 Temperature stability

[0145] At pH 6.5, the enzyme was kept at 60°C and 65°C, and the enzyme activity was measured at regular intervals, with the enzyme activity without incubation being 100%. The results are as follows: Figure 6 As shown in C.

[0146] Depend on Figure 6 C The results show that the recombinant α-galactosidase TstGal of the present invention can still maintain more than 45% of the enzyme activity after being incubated at 60°C for 8 hours, and has excellent thermal stability. It still has more than 40% of the residual enzyme activity after being incubated at 40°C for 3 hours, and has a certain degree of heat resistance.

[0147] 5.4 pH stability

[0148] The purified recombinant α-galactosidase TstGal was treated at different pH values ​​(5.0-7.5, 100 mmol / L citric acid-sodium hydrogen phosphate buffer) at 60°C for 4 h. The results were compared with those of the unheated enzyme. Figure 6 As shown in D.

[0149] Depend on Figure 6 D The results show that the recombinant α-galactosidase TstGal of the present invention still has more than 80% residual enzyme activity in the pH range of 5.0-7.5, and has good pH stability.

[0150] Example 6

[0151] α-Galactosidase TstGal hydrolyzes raffinose.

[0152] The concentration of raffinose was 0.1 mol / L, the conversion conditions were 60°C, 100 mmol / L citric acid-sodium hydrogen phosphate buffer at pH 6.5, 1.0 U / mL recombinant α-galactosidase TstGal, and samples were taken at different time points (0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h) and detected by HPLC. The results are shown in Figure 7 shown.

[0153] Depend on Figure 7 The results show that the hydrolysis rate of raffinose by the recombinant α-galactosidase TstGal of the present invention reaches the maximum at 10 h, and the maximum hydrolysis rate is 99.8% ( Figure 7 ).

[0154] The above examples show that the present invention has obtained a heat-resistant α-galactosidase that can efficiently hydrolyze raffinose, and the hydrolysis efficiency is high. Experiments show that the α-galactosidase provided by the present invention can hydrolyze raffinose, and the maximum hydrolysis rate is 99.8%.

[0155] The above are only a few preferred embodiments of the present invention, which are described in a more specific and detailed manner, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A thermostable α-galactosidase, characterized in that The amino acid sequence of the α-galactosidase is shown in SEQ ID NO.

1.

2. A biomaterial, characterized in that: Contains any one or more of the following substances 1)-3): 1) Gene: the gene encodes the amino acid sequence of α-galactosidase as claimed in claim 1; 2) Recombinant plasmid: containing the gene described in 1); 3) Host cell: containing the gene described in 1) or the recombinant plasmid described in 2).

3. A gene encoding the amino acid sequence of α-galactosidase according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. The method for preparing α-galactosidase according to claim 1, characterized in that: The following steps are involved: S1 performs codon optimization based on the amino acid sequence to synthesize a nucleic acid sequence suitable for expression in Escherichia coli; S2 clones the above nucleic acid sequence into a recombinant plasmid; S3 transforms the recombinant plasmid into an E. coli expression strain for IPTG-induced expression; S4: washing and crushing the fermented bacteria, collecting the supernatant by centrifugation to obtain a crude enzyme solution; S5 purifies the crude enzyme solution by nickel column to obtain α-galactosidase.

5. The method for preparing α-galactosidase according to claim 4, characterized in that: The recombinant plasmid is pET20b-TstGal; The expression strains include BL21 and JM109 series strains, preferably BL21 (DE3) strains.

6. The method for preparing α-galactosidase according to claim 5, characterized in that: In the induced expression, The induction IPTG concentration is: 0.01mmol / L-5mmol / L, preferably 0.1mmol / L; The induction OD600 value is: 0.2-2.0, preferably 1.0; The induction temperature is 16-45°C, preferably 42°C; The induction time is: 18-66 h, preferably 5 h.

7. Use of the α-galactosidase according to claim 1 in enzymatic hydrolysis of raffinose.

8. The use according to claim 7, characterized in that In the enzymatic hydrolysis system: The concentration of raffinose is 0.01 mol / L-1 mol / L, preferably 0.1 mol / L; The concentration of α-galactosidase is 0.1 U / mL-10 U / mL, preferably 1.5 U / mL; The temperature of enzymatic hydrolysis is 50°C-75°C, preferably 70°C.

9. The use according to claim 7, characterized in that: The enzymatic hydrolysis system contains a citric acid-disodium hydrogen phosphate buffer having a concentration of 10 mmol / L-200 mmol / L and a pH of 5.0-8.0; preferably, the concentration is 100 mmol / L and the pH is 6.5.

Images