CN109097348A - The application of alpha-galactosidase and its complex enzyme in galactomannan degradation - Google Patents
The application of alpha-galactosidase and its complex enzyme in galactomannan degradation Download PDFInfo
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
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Abstract
The present invention relates to a kind of application of alpha-galactosidase and its complex enzyme in galactomannan degradation, which has the activity of hydrolysed galactomannan.The enzyme is greatly improved palliating degradation degree to galactomannans with the alkaline ' beta '-mannase synergistic effect from Alkaliphilic bacillus N16-5, improves the conversion ratio and ratio in the product of mannobiose and manninotriose.Alpha-galactosidase Gal27A and its with 'beta '-mannase compound galactomannan degradation and mannobiose, manninotriose production in there is important application value.
Description
Technical field
The present invention relates in field of biotechnology, be related to a kind of alpha-galactosidase and its compound with 'beta '-mannase
Application in galactomannan degradation.
Background technique
Alpha-galactosidase (EC 3.2.1.23) is widely present in plant, microorganism and animal, can be catalyzed α-galactolipin
The hydrolysis of glycosidic bond.It may act on the oligosaccharide such as melibiose, raffinose, stachyose, moreover it is possible to which hydrolysis contains the more of alpha-galactoside key
Glycan.
'beta '-mannase (EC 3.2.1.78) is a kind of inscribe glycoside hydrolase, by cutting mannosan at random
Mannosan is degraded to Oligomeric manna sugar and mannose by the glycosidic bond on main chain, is most important mannosan hydrolase.
Mannosan is the most commonly used a kind of hemicellulose of distributed in nature other than xylan.Natural sweet dew is poly-
Sugar can be divided into: poly- mannose, four class of glucomannans, galactomannans and galactoglucomannan.Wherein, poly- mannose is
Linear polysaccharide is connected by forming Isosorbide-5-Nitrae-β-D-MANNOSE glycosidic bond between mannose.If galactolipin is present in poly- mannose molecules
In, and be connected by α -1,6- glycosidic bond with the mannose residue in main chain, then constitute galactomannans.Galactomannan is poly-
The complete hydrolysis of sugar needs the synergistic effect by inscribe 'beta '-mannase, beta-Mannosidase alpha-galactosidase.
In recent years, the effects of manna oligosacchride is found to have protection enteron aisle and improves immunity of organisms, is widely used in
The industries such as feed and health care product.'beta '-mannase can be improved to Chinese scholartree in alpha-galactosidase and 'beta '-mannase synergy
The degradation of the galactomannans such as bean gum, melon bean gum increases the yield of manna oligosacchride, therefore has weight in mannosan degradation
The meaning wanted.The manna oligosacchride of different polymerization degree is possible to the physiological function for having different, therefore specific aggregation degree oligosaccharides simultaneously
Production be of great significance in scientific research, had potential application in Medicines and Health Product industry.
Summary of the invention
Present invention finds a kind of protein with alpha-galactosidase activity, and are applied to and beta-mannase
Enzyme cooperates with the degradation to galactomannans.
In order to achieve this, the present invention the following technical schemes are provided:
A kind of alpha-galactosidase, amino acid sequence is as shown in sequence SEQ ID NO:1.
A kind of alpha-galactosidase, nucleotide sequence is as shown in sequence SEQ ID NO:2.
A kind of recombinant vector for the nucleotide sequence wrapping the alpha-galactosidase.
A kind of recombination bacillus coli, it includes the encoding gene of alpha-galactosidase or its recombinant vectors.
A kind of manufacturing method of alpha-galactosidase, the culture including recombination bacillus coli, the generation of alpha-galactosidase
Step, and the step of purifying the alpha-galactosidase of above-mentioned generation.
A kind of galactomannan degradation preparation, including alpha-galactosidase and amino acid sequence are SEQ ID NO:3 institute
Show alkaline ' beta '-mannase.
Amino acid sequence alpha-galactosidase as shown in SEQ ID NO:1 and amino acid sequence are SEQ ID NO:3 institute
Show alkaline ' beta '-mannase compound enzyme in galactomannan degradation, prepare mannobiose, prepare the application in manninotriose.
The present invention has cloned the alpha-galactosidase gene from Alkaliphilic bacillus N16-5, by it in Escherichia coli
Heterogenous expression is carried out, and the alpha-galactosidase of heterogenous expression is purified, as shown in Figure 1.Present invention discover that α-gala
The amino acid sequence of glycosidase is as shown in sequence 1, and the nucleotide sequence of encoding gene is as shown in sequence 2.Purify the α-obtained
Galactosidase have hydrolysed galactomannan ability, the enzyme activity to locust bean gum and guar gum be respectively 1.7U/mg and
2.0U/mg。
The alpha-galactosidase that the present invention is obtained and 'beta '-mannase synergy, can increase 'beta '-mannase pair
The degradation capability of galactomannans, the two are respectively 1.13 and 2.21 to the collaborative e-commerce of locust bean gum and guar gum.
The alpha-galactosidase that the present invention is obtained and 'beta '-mannase synergy significantly reduce degradation in the reaction
The degree of polymerization of product, and the ratio of mannobiose and manninotriose, such as Fig. 2 are improved, shown in Fig. 3.
By the alpha-galactosidase that obtains of the present invention and 'beta '-mannase synergy, mannobiose and sweet is significantly improved
The conversion ratio for revealing trisaccharide, is converted into rate and respectively reaches 14% He by substrate mannobiose and manninotriose of locust bean gum
10.8%；It is converted into rate by substrate mannobiose and manninotriose of guar gum and respectively reaches 9.4% and 5.3%.
Detailed description of the invention
The electrophoretogram of the alpha-galactosidase of Fig. 1 purifying；
In figure, M, molecular weight standard；1, alpha-galactosidase after purification.
Fig. 2 is the thin layer that alpha-galactosidase and 'beta '-mannase synergy degradation locust bean gum substrate generate product
Analysis analysis.
M1, galactolipin in figure；It M2, is from top to bottom mannose, mannobiose, manninotriose, mannotetrose, sweet dew five
Sugar；1, locust bean gum；2, product of the locust bean gum Jing Guo mannase ManA digestion；3, locust bean gum passes through alpha-galactosidase
The product of Gal27A digestion；4, locust bean gum passes through the production of mannase ManA and the common digestion of alpha-galactosidase Gal27A
Object.
Fig. 3 is the thin layer that alpha-galactosidase and 'beta '-mannase synergy degradation guar gum substrate generate product
Chromatographic analysis.
M1, galactolipin in figure；It M2, is from top to bottom mannose, mannobiose, manninotriose, mannotetrose, sweet dew five
Sugar；1, guar gum；2, product of the guar gum Jing Guo mannase ManA digestion；3, guar gum passes through alpha-galactoside
The product of enzyme Gal27A digestion；4, guar gum is by mannase ManA and the common digestion of alpha-galactosidase Gal27A
Product.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Test method in following embodiments is unless otherwise specified
Conventional method.Material as used in the following examples, reagent, instrument etc., are commercially available unless otherwise specified.
Quantitative test in following embodiment is respectively provided with and repeats to test three times, and results are averaged.
PET28a plasmid, bacillus coli DH 5 alpha, e. coli bl21 (DE3) in following examples are commercially available.
The solvent of LB culture medium in following examples is water, and each component and its concentration are respectively as follows: 0.5% (mass percent
Concentration) yeast extract, 1% (mass percent concentration) peptone, 1% (mass percent concentration) sodium chloride.IPTG is dense eventually
Degree is 50 μ g/ml.
Embodiment 1
The preparation of alpha-galactosidase Gal27A
1. the clone of alpha-galactosidase gene gal27A
According to the sequence design upstream primer gal27A-F:5 '-of alpha-galactosidase gene gal27A
GCGGATCCATGACTTTAAAACCAA-3 ' and downstream primer gal27A-R:5 '-
GGCGTCGACCTACTCTTTATTTTTTGAC-3 ', with the gene of Alkaliphilic bacillus bacillus N16-5 (CGMCC No.0369)
Group is template, carries out sequence amplification using round pcr, obtains target gene.
2. the preparation of recombinant plasmid and recombinant bacterium
DNA fragmentation between the BamHI of pET28a plasmid and SalI identification sequence is replaced with into gene shown in sequence 1, is obtained
To recombinant plasmid, which is named into pET28a-Gal27A.
Protein shown in pET28a-Gal27A energy expressed sequence 1, is named as Gal27A for the protein, Gal27A's
Expression is started by T7 promoter.
Recombinant plasmid pET28a-Gal27A is transformed into bacillus coli DH 5 alpha competent cell, be coated on containing card that
The LB plate of mycin, screening obtain the bacterial strain containing recombinant plasmid, and picking Colony Culture is in liquid LB containing kanamycin
In culture medium, collect thallus, extract plasmid, double digestion verifying, the positive recombinant plasmid after verifying is sequenced, sequencing result with
Former sequence alignment.
It compares correct positive recombinant plasmid to be transformed into the competence of e. coli bl21 (DE3), screening obtains the positive
Clone, picking obtain Colony Culture in fluid nutrient medium containing kanamycin, and recombinant bacterium is stored in glycerol, and -80 DEG C
It saves.
3. the expression and purifying of alpha-galactosidase Gal27A
After above-mentioned recombinant bacterial strain takes out from -80 DEG C, first it is inoculated in LB liquid medium containing kanamycin and carries out
Activation, is forwarded to shaking flask after activated, cultivates in 37 DEG C, 220rpm to OD600Reach 0.6, IPTG inducer be added, in 30 DEG C,
220rpm cultivates 6h, is then centrifuged for collecting thallus, (buffer is the lemon by 20mM to citrate-phosphate disodium hydrogen buffer
The disodium hydrogen phosphate aqueous solution of aqueous acid and 20mM preparation) cleaning three times after, blown with the citrate buffer solution of certain volume outstanding
Thallus carries out ultrasonication on ice, is centrifuged after ultrasound, collects supernatant.
The supernatant being collected into utilizes the method purified pool liquid of Ni column affinity chromatography, elutes foreign protein using 80mM imidazoles,
Destination protein is eluted using 500mM imidazoles aqueous solution.
The Gal27A of purifying is subjected to SDS- polyacrylamide gel electrophoresis, as shown in Figure 1.
Embodiment 2
The enzyme activity determination of alpha-galactosidase Gal27A
Using paranitrophenol-α-galactopyranose as substrate, alpha-galactosidase Gal27A enzyme activity in Gal27A solution is detected.
Alpha -galactosidase enzyme definition living: it is an enzyme activity that degradation of substrates, which discharges enzyme amount required for 1 μm of ol paranitrophenol, in per minute
Unit (U).
(1) drafting of paranitrophenol standard curve:
139mg paranitrophenol is dissolved in 1mL methanol solution, adds water to be settled to 100ml and is configured to final concentration of 10mM's
Solution.The different volume of paranitrophenol solution is drawn respectively, is settled to the citrate-phosphate disodium hydrogen buffer that pH is 6.0
Various concentration, respectively 0, the standard of 0.02,0.04,0.06,0.08,0.1,0.15,0.2,0.25,0.3,0.35 and 0.4mM
Gradient solution.The light absorption value of each normal gradients solution is measured under 405nm wavelength condition.Using light absorption value as abscissa, p-nitrophenyl
Phenol concentration is ordinate, draws standard curve.
(2) enzyme activity determination
Pnitrophenylα Dgalactospyranoside is dissolved in 20mM citrate-phosphate disodium hydrogen (pH 6) buffer, obtains to nitre
Base phenol-α-D- galactopyranose concentration is the pnitrophenylα Dgalactospyranoside solution of 20mM.
It is 7.0 citrate-phosphate disodium hydrogens buffering by 50 μ l pnitrophenylα Dgalactospyranoside solution and 50 μ l pH
Then liquid is added the resulting enzyme solution after purification of step 2 and is sufficiently mixed, in 30 DEG C of water bath with thermostatic control 10min, obtain in 37 DEG C of preheating 3min
To mixed liquor；100 μ l aqueous sodium carbonates (concentration of sodium carbonate is 1M) is added into mixed liquor, shaken well terminates reaction,
Measure absorbance OD405Value.Amount by the paranitrophenol generated with paranitrophenol curve control, calculating enzyme digestion reaction.
The experimental results showed that α-gala of the Gal27A to pnitrophenylα Dgalactospyranoside under the conditions of 7,37 DEG C of pH
Glycosidase enzyme activity is 1.08U/mg.
Embodiment 3
Alpha-galactosidase Gal27A and 'beta '-mannase ManA synergistic effect degradation galactomannans
1. the preparation of locust bean gum and guar gum substrate
0.5% locust bean gum (guar gum): take 0.5g locust bean gum (guar gum) in 100ml buffer, boiling water bath adds
Hot 10min, it is cooling after 12000rpm be centrifuged 5min, take supernatant, be stored in 4 DEG C it is spare.
2. the drafting of reduced sugar mark song
180mg reduced sugar is dissolved in the buffer of citrate-phosphate disodium hydrogen of 10ml, final concentration of 10mM is configured to
Solution.The different volume of paranitrophenol solution, the citrate-phosphate disodium hydrogen buffer constant volume for being 6.0 with pH are drawn respectively
To various concentration, the standard ladder of respectively 0,0.1,0.2,0.3,0.4,0.5,0.6,0.8,1.0,1.4,1.8,2.0 and 2.4mM
Solution is spent, final volume is 1ml.It is separately added into the DNS reagent of 1ml, reaction solution is cooled down after boiling water bath 5min rapidly, is surveyed at 540nm
Determine light absorption value.Using light absorption value as abscissa, concentration of reduced sugar is ordinate, draws standard curve.
3. 3 experimental groups of experimental setup test 1, experiment 2, experiment 3, every group of three parallel tests, concrete operation step
It is as follows:
The operating procedure of experimental group 1 is as follows: 800 μ l, 0.5% locust bean gum substrate taken, under the conditions of 37 DEG C, preheats 3min,
The citrate-phosphate disodium hydrogen buffer of 0.05U/mL Gal27A enzyme solution 100 μ l and 100 μ l is added, reacts 15min.
The operating procedure of experimental group 2 is as follows: the Gal27A enzyme solution in experimental group 1 is substituted for 0.25U/ml ManA enzyme solution,
Other steps are constant.
The operating procedure of experimental group 3 is as follows: 0.5% locust bean gum substrate of 800ul taken, under the conditions of 37 DEG C, preheats 3min,
100 μ l and 0.25U/ml ManA enzyme solution of 0.05U/ml Gal27A enzyme solution, 100 μ l is added, reacts 15min.
Calculate collaboration rate, collaboration rate=A/ (AA+AC)
The reduced sugar production quantity after reacting in substrate solution, unit mM is added in Gal27A and ManA collaboration in A-experimental group；
ACGal27A is individually added into the reduced sugar production quantity after reacting in substrate solution, unit mM in experimental group；
AAManA is individually added into the reduced sugar production quantity after reacting in substrate solution, unit mM in experimental group；
The experimental results showed that the common reaction treatment locust bean gum collaboration rate of Gal27A and ManA is 1.13.
4. 0.5% locust bean gum of the substrate of step 3 according to the method described above, is substituted for 0.5% guar gum, 0.05U/ is measured
Synergistic effect of ml Gal27A and the 0.25U/ml ManA to 0.5% guar gum substrate.
The experimental results showed that collaborative e-commerce when Gal27A and ManA collective effect is 2.21.
5. 3 experimental groups of experimental setup test 1, experiment 2, experiment 3, every group of three parallel tests, concrete operation step
It is as follows:
The operating procedure of experimental group 1 is as follows: 800 μ l, 0.5% locust bean gum substrate taken, under the conditions of 37 DEG C, preheats 3min,
The 200 μ l of citrate-phosphate disodium hydrogen buffer containing 0.2U Gal27A is added, reacts 1 hour
The operating procedure of experimental group 2 is as follows: the Gal27A in experimental group 1 being substituted for 10U ManA, other steps are constant.
The operating procedure of experimental group 3 is as follows: 0.5% locust bean gum substrate of 800ul taken, under the conditions of 37 DEG C, preheats 3min,
It is added and contains 200 μ l of 0.2U Gal27A and 10U ManA citrate-phosphate disodium hydrogen buffer, react 1 hour.
Product component is analyzed using thin layer chromatography, as shown in Figure 2.Use ion chromatography analysis mannobiose and sweet dew
The content of trisaccharide, respectively 0.56g/L and 43.3g/L, calculating conversion ratio is 14% and 10.8%.
6. 0.5% locust bean gum of the substrate of step 5 according to the method described above, is substituted for 0.5% guar gum, analysis product at
Point, as shown in Figure 3.Use the content of ion chromatography analysis mannobiose and manninotriose, respectively 0.37g/L and 0.21g/
L, calculating conversion ratio is 9.4% and 5.3%.
Sequence 1: alpha-galactoside enzyme amino acid sequence
MTLKPKRYAEGLAKTPPMGWNSFNTFGCEPTEELIKQSADVMVKSGLLEAGYRYINIDDGWMADERDSA
GNLVPDPQKFPNGMKPVTDYIHEKGLLAGTYLGCGQKTYGEKPGSLGYEERDAQLIADQGFDLLKYDYRELPGDPIG
RGVKEDYVTMRNALMKTGRDMVFSICEHGKSHPETWAQEIGHMWRTTPDIKDSFDEDINWGWSINHIIDETHALHHY
AGPGGWNDPDMLVVGINGLNDWLGPGCTYNEYKSHFSLWCLLAAPLLIGCDIRKMSEETKTILLNKEMIAINQDPLG
IQGHLLKKEHGIDYWVKPLANNDIAIGLLNRFNEPKEAVLSLSDLLEDGNYLMKDVWTNERKELQSEWISKTLKSHE
CAVFRLMSKNKE
Sequence 2 is alpha-galactosidase nucleotide sequence
ATGACTTTAAAACCAAAACGATATGCTGAAGGATTGGCTAAAACTCCCCCGATGGGATGGAACTCATTT
AATACTTTCGGGTGTGAACCAACGGAAGAGTTGATAAAACAGAGTGCAGATGTCATGGTAAAATCAGGTCTTTTAGA
AGCAGGATACCGCTATATCAACATTGATGACGGGTGGATGGCAGACGAGAGAGATAGTGCTGGTAATCTAGTTCCTG
ATCCTCAAAAATTTCCGAACGGCATGAAACCCGTCACTGATTATATCCATGAAAAAGGACTTTTAGCAGGCACCTAT
TTAGGCTGTGGCCAAAAAACATACGGAGAAAAACCAGGAAGCTTAGGATATGAAGAAAGAGATGCACAATTAATTGC
TGACCAAGGGTTTGATTTACTGAAGTATGATTACCGTGAACTCCCAGGTGATCCAATAGGAAGAGGGGTGAAAGAAG
ATTACGTGACGATGAGGAACGCCTTGATGAAAACTGGTAGAGACATGGTCTTCAGTATTTGTGAACATGGGAAGTCA
CATCCAGAAACGTGGGCTCAGGAAATTGGTCATATGTGGCGTACAACCCCAGATATTAAAGACAGCTTTGATGAGGA
TATAAACTGGGGCTGGTCAATTAATCATATTATAGATGAGACCCATGCACTTCATCACTACGCAGGGCCAGGTGGTT
GGAACGATCCAGATATGTTAGTCGTGGGGATTAATGGATTAAACGATTGGCTTGGACCCGGCTGTACGTATAATGAA
TATAAATCACATTTTAGCTTGTGGTGTCTCCTCGCAGCCCCTCTCCTTATTGGTTGTGATATTCGAAAAATGAGTGA
GGAAACCAAAACGATTTTACTAAATAAAGAGATGATTGCCATTAATCAAGATCCATTAGGTATACAAGGTCATCTTC
TTAAAAAAGAACATGGGATTGATTATTGGGTAAAACCTCTTGCGAATAATGACATAGCTATTGGCTTGCTAAATCGC
TTTAATGAGCCTAAAGAGGCTGTTCTTAGCCTGAGTGATCTCCTCGAAGACGGCAACTATTTAATGAAAGATGTATG
GACAAATGAACGGAAGGAGCTACAGTCAGAATGGATAAGTAAAACATTGAAGAGCCATGAATGTGCGGTTTTTAGGC
TTATGTCAAAAAATAAAGAG
ManA amino acid sequence
MKKKLSQIYHLIICTLIISVGIMGITTSPSEASSGFYVDGNTLYDANGQPFVMKGINHGHAWYKDTAST
AIPAIAEQGANTIRIVLSDGGQWEKDDIDTVREVIELAEQNKMVAVVEVHDATGRDSRSDLDRAVDYWIEMKDALIG
KEDTVIINIANEWYGSWDGAAWADGYIDVIPKLRDAGLTHTLMVDAAGWGQYPQSIHDYGQDVFNADPLKNTIFSIH
MYEYAGGDANTVRSNIDRVIDQDLALVIGEFGHRHTDGDVDEDTILSYSEETGTGWLAWSWKGNSAEWDYLDLSEDW
AGNHLTDWGNRIVHGANGLQETSKPSTVFTDDNGGAPEPPTTTTLYDFEGSTQGWHGSNVMGGPWSVTEWGASGNYS
LKGDVNLSSNSSHELYSEQSRNLHGYSQLNATVRHANWGNPGNGMNARLYVKTGSDYTWYSGPFTRINSSNSGTTLS
FDLNNIENSHHVREIGVQFSAADNSSGQTALYVDNVTLR
ManA gene order
ATGAAAAAAAAGTTATCACAGATTTATCATTTAATTATTTGCACACTTATAATAAGTGTGGGAATAATG
GGGATTACAACGTCCCCATCAGAAGCAAGTTCAGGCTTTTATGTTGATGGCAATACGTTATATGACGCAAACGGGCA
ACCATTTGTCATGAAAGGCATTAACCATGGACATGCTTGGTATAAAGACACCGCTTCAACAGCTATTCCTGCCATTG
CAGAGCAAGGCGCGAACACGATACGTATTGTTTTATCAGATGGCGGTCAATGGGAAAAAGACGACATTGACACCGTT
CGTGAAGTTATTGAGCTTGCGGAGCAAAATAAAATGGTGGCTGTCGTTGAAGTTCATGATGCCACGGGCCGTGATTC
ACGCAGTGATTTAGATCGGGCAGTCGATTATTGGATAGAGATGAAAGATGCACTTATCGGCAAAGAGGATACTGTCA
TTATTAACATTGCAAACGAATGGTATGGCAGTTGGGATGGCGCCGCTTGGGCTGATGGCTACATTGATGTCATTCCG
AAGCTTCGCGATGCCGGCTTAACACACACCTTAATGGTTGATGCAGCAGGATGGGGGCAATATCCGCAATCTATTCA
TGATTACGGACAAGATGTGTTTAATGCAGATCCGTTAAAAAATACGATATTCTCCATCCATATGTATGAGTATGCTG
GTGGTGATGCTAACACTGTTAGATCAAATATTGATAGAGTCATAGATCAAGACCTTGCTCTCGTAATAGGTGAGTTC
GGTCATAGACACACTGATGGCGATGTTGATGAAGATACAATCCTTAGTTATTCTGAAGAAACTGGCACAGGATGGCT
CGCTTGGTCTTGGAAAGGCAACAGTGCCGAATGGGATTATTTAGACCTTTCAGAAGATTGGGCTGGTAACCATTTAA
CTGATTGGGGAAATAGGATTGTCCACGGGGCAAATGGCTTGCAGGAAACCTCCAAACCATCCACCGTATTTACAGAT
GATAACGGTGGTGCCCCTGAACCGCCAACTACTACTACCTTGTATGACTTTGAAGGAAGCACACAAGGGTGGCATGG
AAGCAACGTGATGGGTGGCCCTTGGTCCGTAACAGAATGGGGTGCGTCAGGCAACTACTCTTTAAAGGGCGATGTCA
ATTTAAGCTCAAATTCTTCACATGAACTGTATAGTGAACAAAGTCGTAATCTACACGGATACTCTCAGCTAAATGCA
ACCGTTCGCCATGCCAATTGGGGAAATCCCGGTAATGGCATGAATGCAAGACTTTACGTGAAAACGGGCTCTGATTA
TACATGGTATAGCGGTCCTTTTACACGTATCAATAGCTCCAACTCAGGTACAACGTTATCTTTTGATTTAAACAACA
TCGAAAATAGTCATCATGTTAGGGAAATAGGTGTGCAATTTTCAGCTGCAGATAATAGCAGCGGTCAAACTGCTCTA
TACGTTGATAATGTTACTTTAAGA
Sequence table
<110>University Of Science and Technology Of Tianjin
<120>application of alpha-galactosidase and its complex enzyme in galactomannan degradation
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 389
<212> PRT
<213>alpha-galactoside enzyme amino acid sequence (Unknown)
<400> 1
Met Thr Leu Lys Pro Lys Arg Tyr Ala Glu Gly Leu Ala Lys Thr Pro
1 5 10 15
Pro Met Gly Trp Asn Ser Phe Asn Thr Phe Gly Cys Glu Pro Thr Glu
20 25 30
Glu Leu Ile Lys Gln Ser Ala Asp Val Met Val Lys Ser Gly Leu Leu
35 40 45
Glu Ala Gly Tyr Arg Tyr Ile Asn Ile Asp Asp Gly Trp Met Ala Asp
50 55 60
Glu Arg Asp Ser Ala Gly Asn Leu Val Pro Asp Pro Gln Lys Phe Pro
65 70 75 80
Asn Gly Met Lys Pro Val Thr Asp Tyr Ile His Glu Lys Gly Leu Leu
85 90 95
Ala Gly Thr Tyr Leu Gly Cys Gly Gln Lys Thr Tyr Gly Glu Lys Pro
100 105 110
Gly Ser Leu Gly Tyr Glu Glu Arg Asp Ala Gln Leu Ile Ala Asp Gln
115 120 125
Gly Phe Asp Leu Leu Lys Tyr Asp Tyr Arg Glu Leu Pro Gly Asp Pro
130 135 140
Ile Gly Arg Gly Val Lys Glu Asp Tyr Val Thr Met Arg Asn Ala Leu
145 150 155 160
Met Lys Thr Gly Arg Asp Met Val Phe Ser Ile Cys Glu His Gly Lys
165 170 175
Ser His Pro Glu Thr Trp Ala Gln Glu Ile Gly His Met Trp Arg Thr
180 185 190
Thr Pro Asp Ile Lys Asp Ser Phe Asp Glu Asp Ile Asn Trp Gly Trp
195 200 205
Ser Ile Asn His Ile Ile Asp Glu Thr His Ala Leu His His Tyr Ala
210 215 220
Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Val Gly Ile Asn
225 230 235 240
Gly Leu Asn Asp Trp Leu Gly Pro Gly Cys Thr Tyr Asn Glu Tyr Lys
245 250 255
Ser His Phe Ser Leu Trp Cys Leu Leu Ala Ala Pro Leu Leu Ile Gly
260 265 270
Cys Asp Ile Arg Lys Met Ser Glu Glu Thr Lys Thr Ile Leu Leu Asn
275 280 285
Lys Glu Met Ile Ala Ile Asn Gln Asp Pro Leu Gly Ile Gln Gly His
290 295 300
Leu Leu Lys Lys Glu His Gly Ile Asp Tyr Trp Val Lys Pro Leu Ala
305 310 315 320
Asn Asn Asp Ile Ala Ile Gly Leu Leu Asn Arg Phe Asn Glu Pro Lys
325 330 335
Glu Ala Val Leu Ser Leu Ser Asp Leu Leu Glu Asp Gly Asn Tyr Leu
340 345 350
Met Lys Asp Val Trp Thr Asn Glu Arg Lys Glu Leu Gln Ser Glu Trp
355 360 365
Ile Ser Lys Thr Leu Lys Ser His Glu Cys Ala Val Phe Arg Leu Met
370 375 380
Ser Lys Asn Lys Glu
385
<210> 2
<211> 1167
<212> DNA
<213>alpha-galactosidase nucleotide sequence (Unknown)
<400> 2
atgactttaa aaccaaaacg atatgctgaa ggattggcta aaactccccc gatgggatgg 60
aactcattta atactttcgg gtgtgaacca acggaagagt tgataaaaca gagtgcagat 120
gtcatggtaa aatcaggtct tttagaagca ggataccgct atatcaacat tgatgacggg 180
tggatggcag acgagagaga tagtgctggt aatctagttc ctgatcctca aaaatttccg 240
aacggcatga aacccgtcac tgattatatc catgaaaaag gacttttagc aggcacctat 300
ttaggctgtg gccaaaaaac atacggagaa aaaccaggaa gcttaggata tgaagaaaga 360
gatgcacaat taattgctga ccaagggttt gatttactga agtatgatta ccgtgaactc 420
ccaggtgatc caataggaag aggggtgaaa gaagattacg tgacgatgag gaacgccttg 480
atgaaaactg gtagagacat ggtcttcagt atttgtgaac atgggaagtc acatccagaa 540
acgtgggctc aggaaattgg tcatatgtgg cgtacaaccc cagatattaa agacagcttt 600
gatgaggata taaactgggg ctggtcaatt aatcatatta tagatgagac ccatgcactt 660
catcactacg cagggccagg tggttggaac gatccagata tgttagtcgt ggggattaat 720
ggattaaacg attggcttgg acccggctgt acgtataatg aatataaatc acattttagc 780
ttgtggtgtc tcctcgcagc ccctctcctt attggttgtg atattcgaaa aatgagtgag 840
gaaaccaaaa cgattttact aaataaagag atgattgcca ttaatcaaga tccattaggt 900
atacaaggtc atcttcttaa aaaagaacat gggattgatt attgggtaaa acctcttgcg 960
aataatgaca tagctattgg cttgctaaat cgctttaatg agcctaaaga ggctgttctt 1020
agcctgagtg atctcctcga agacggcaac tatttaatga aagatgtatg gacaaatgaa 1080
cggaaggagc tacagtcaga atggataagt aaaacattga agagccatga atgtgcggtt 1140
tttaggctta tgtcaaaaaa taaagag 1167
<210> 3
<211> 493
<212> PRT
<213>ManA amino acid sequence (Unknown)
<400> 3
Met Lys Lys Lys Leu Ser Gln Ile Tyr His Leu Ile Ile Cys Thr Leu
1 5 10 15
Ile Ile Ser Val Gly Ile Met Gly Ile Thr Thr Ser Pro Ser Glu Ala
20 25 30
Ser Ser Gly Phe Tyr Val Asp Gly Asn Thr Leu Tyr Asp Ala Asn Gly
35 40 45
Gln Pro Phe Val Met Lys Gly Ile Asn His Gly His Ala Trp Tyr Lys
50 55 60
Asp Thr Ala Ser Thr Ala Ile Pro Ala Ile Ala Glu Gln Gly Ala Asn
65 70 75 80
Thr Ile Arg Ile Val Leu Ser Asp Gly Gly Gln Trp Glu Lys Asp Asp
85 90 95
Ile Asp Thr Val Arg Glu Val Ile Glu Leu Ala Glu Gln Asn Lys Met
100 105 110
Val Ala Val Val Glu Val His Asp Ala Thr Gly Arg Asp Ser Arg Ser
115 120 125
Asp Leu Asp Arg Ala Val Asp Tyr Trp Ile Glu Met Lys Asp Ala Leu
130 135 140
Ile Gly Lys Glu Asp Thr Val Ile Ile Asn Ile Ala Asn Glu Trp Tyr
145 150 155 160
Gly Ser Trp Asp Gly Ala Ala Trp Ala Asp Gly Tyr Ile Asp Val Ile
165 170 175
Pro Lys Leu Arg Asp Ala Gly Leu Thr His Thr Leu Met Val Asp Ala
180 185 190
Ala Gly Trp Gly Gln Tyr Pro Gln Ser Ile His Asp Tyr Gly Gln Asp
195 200 205
Val Phe Asn Ala Asp Pro Leu Lys Asn Thr Ile Phe Ser Ile His Met
210 215 220
Tyr Glu Tyr Ala Gly Gly Asp Ala Asn Thr Val Arg Ser Asn Ile Asp
225 230 235 240
Arg Val Ile Asp Gln Asp Leu Ala Leu Val Ile Gly Glu Phe Gly His
245 250 255
Arg His Thr Asp Gly Asp Val Asp Glu Asp Thr Ile Leu Ser Tyr Ser
260 265 270
Glu Glu Thr Gly Thr Gly Trp Leu Ala Trp Ser Trp Lys Gly Asn Ser
275 280 285
Ala Glu Trp Asp Tyr Leu Asp Leu Ser Glu Asp Trp Ala Gly Asn His
290 295 300
Leu Thr Asp Trp Gly Asn Arg Ile Val His Gly Ala Asn Gly Leu Gln
305 310 315 320
Glu Thr Ser Lys Pro Ser Thr Val Phe Thr Asp Asp Asn Gly Gly Ala
325 330 335
Pro Glu Pro Pro Thr Thr Thr Thr Leu Tyr Asp Phe Glu Gly Ser Thr
340 345 350
Gln Gly Trp His Gly Ser Asn Val Met Gly Gly Pro Trp Ser Val Thr
355 360 365
Glu Trp Gly Ala Ser Gly Asn Tyr Ser Leu Lys Gly Asp Val Asn Leu
370 375 380
Ser Ser Asn Ser Ser His Glu Leu Tyr Ser Glu Gln Ser Arg Asn Leu
385 390 395 400
His Gly Tyr Ser Gln Leu Asn Ala Thr Val Arg His Ala Asn Trp Gly
405 410 415
Asn Pro Gly Asn Gly Met Asn Ala Arg Leu Tyr Val Lys Thr Gly Ser
420 425 430
Asp Tyr Thr Trp Tyr Ser Gly Pro Phe Thr Arg Ile Asn Ser Ser Asn
435 440 445
Ser Gly Thr Thr Leu Ser Phe Asp Leu Asn Asn Ile Glu Asn Ser His
450 455 460
His Val Arg Glu Ile Gly Val Gln Phe Ser Ala Ala Asp Asn Ser Ser
465 470 475 480
Gly Gln Thr Ala Leu Tyr Val Asp Asn Val Thr Leu Arg
485 490
<210> 4
<211> 1479
<212> DNA
<213>ManA gene order (Unknown)
<400> 4
atgaaaaaaa agttatcaca gatttatcat ttaattattt gcacacttat aataagtgtg 60
ggaataatgg ggattacaac gtccccatca gaagcaagtt caggctttta tgttgatggc 120
aatacgttat atgacgcaaa cgggcaacca tttgtcatga aaggcattaa ccatggacat 180
gcttggtata aagacaccgc ttcaacagct attcctgcca ttgcagagca aggcgcgaac 240
acgatacgta ttgttttatc agatggcggt caatgggaaa aagacgacat tgacaccgtt 300
cgtgaagtta ttgagcttgc ggagcaaaat aaaatggtgg ctgtcgttga agttcatgat 360
gccacgggcc gtgattcacg cagtgattta gatcgggcag tcgattattg gatagagatg 420
aaagatgcac ttatcggcaa agaggatact gtcattatta acattgcaaa cgaatggtat 480
ggcagttggg atggcgccgc ttgggctgat ggctacattg atgtcattcc gaagcttcgc 540
gatgccggct taacacacac cttaatggtt gatgcagcag gatgggggca atatccgcaa 600
tctattcatg attacggaca agatgtgttt aatgcagatc cgttaaaaaa tacgatattc 660
tccatccata tgtatgagta tgctggtggt gatgctaaca ctgttagatc aaatattgat 720
agagtcatag atcaagacct tgctctcgta ataggtgagt tcggtcatag acacactgat 780
ggcgatgttg atgaagatac aatccttagt tattctgaag aaactggcac aggatggctc 840
gcttggtctt ggaaaggcaa cagtgccgaa tgggattatt tagacctttc agaagattgg 900
gctggtaacc atttaactga ttggggaaat aggattgtcc acggggcaaa tggcttgcag 960
gaaacctcca aaccatccac cgtatttaca gatgataacg gtggtgcccc tgaaccgcca 1020
actactacta ccttgtatga ctttgaagga agcacacaag ggtggcatgg aagcaacgtg 1080
atgggtggcc cttggtccgt aacagaatgg ggtgcgtcag gcaactactc tttaaagggc 1140
gatgtcaatt taagctcaaa ttcttcacat gaactgtata gtgaacaaag tcgtaatcta 1200
cacggatact ctcagctaaa tgcaaccgtt cgccatgcca attggggaaa tcccggtaat 1260
ggcatgaatg caagacttta cgtgaaaacg ggctctgatt atacatggta tagcggtcct 1320
tttacacgta tcaatagctc caactcaggt acaacgttat cttttgattt aaacaacatc 1380
gaaaatagtc atcatgttag ggaaataggt gtgcaatttt cagctgcaga taatagcagc 1440
ggtcaaactg ctctatacgt tgataatgtt actttaaga 1479
Claims (7)
1. a kind of alpha-galactosidase, it is characterised in that: amino acid sequence is as shown in sequence SEQ ID NO:1.
2. a kind of alpha-galactosidase, it is characterised in that: nucleotide sequence is as shown in sequence SEQ ID NO:2.
3. a kind of recombinant vector of the nucleotide sequence comprising alpha-galactosidase described in claim 2.
4. a kind of recombination bacillus coli, it includes the encoding gene of alpha-galactosidase described in claim 2 or claims 3
The recombinant vector.
5. a kind of manufacturing method of alpha-galactosidase described in claim 1, including recombination large intestine bar as claimed in claim 3
The step of alpha-galactosidase of the culture of bacterium, the generation step of alpha-galactosidase, and the above-mentioned generation of purifying.
6. a kind of galactomannan degradation preparation, it is characterised in that: including α-described in claim 1 or claim 2 half
Lactoside enzyme and amino acid sequence are alkaline ' beta '-mannase shown in SEQ ID NO:3.
7. amino acid sequence alpha-galactosidase as shown in SEQ ID NO:1 and amino acid sequence are shown in SEQ ID NO:3
Alkaline ' beta '-mannase compound enzyme in galactomannan degradation, prepare mannobiose, prepare the application in manninotriose.
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Cited By (8)
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| CN109055333A (en) * | 2018-07-26 | 2018-12-21 | 天津科技大学 | A kind of application of glycoside hydrolase and its complex enzyme in galactomannan degradation |
| CN109917032A (en) * | 2019-03-13 | 2019-06-21 | 杭州老爸评测科技有限公司 | A kind of quantitative detecting method turning Bt albumen in Bt protein food |
| CN110656098A (en) * | 2019-09-11 | 2020-01-07 | 天津科技大学 | Novel acetyl esterase, preparation and application thereof in promoting degradation of beta-mannase on acetylated mannan |
| CN110669805A (en) * | 2019-09-27 | 2020-01-10 | 天津科技大学 | Method for producing galactose, mannose and mannan oligosaccharide by hydrolyzing locust bean gum through compounding of mannanase and galactosidase |
| CN110747242A (en) * | 2019-09-27 | 2020-02-04 | 天津科技大学 | Method for producing galactose, mannose and mannan oligosaccharide by hydrolyzing guar gum through compounding of mannanase and galactosidase |
| CN112322678A (en) * | 2020-10-22 | 2021-02-05 | 天津科技大学 | Method for synergistically hydrolyzing guar gum by using mannase and galactosidase |
| CN112391425A (en) * | 2020-10-22 | 2021-02-23 | 天津科技大学 | Method for synergistically hydrolyzing locust bean gum |
| CN113209202A (en) * | 2021-05-20 | 2021-08-06 | 曾文好 | Bacteriostatic gynecological lotion and preparation method thereof |
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| CN109055333A (en) * | 2018-07-26 | 2018-12-21 | 天津科技大学 | A kind of application of glycoside hydrolase and its complex enzyme in galactomannan degradation |
| CN109917032A (en) * | 2019-03-13 | 2019-06-21 | 杭州老爸评测科技有限公司 | A kind of quantitative detecting method turning Bt albumen in Bt protein food |
| CN109917032B (en) * | 2019-03-13 | 2021-08-24 | 杭州老爸评测科技有限公司 | Quantitative detection method for Bt protein in Bt protein-transgenic food |
| CN110656098A (en) * | 2019-09-11 | 2020-01-07 | 天津科技大学 | Novel acetyl esterase, preparation and application thereof in promoting degradation of beta-mannase on acetylated mannan |
| CN110656098B (en) * | 2019-09-11 | 2022-10-28 | 天津科技大学 | Application of acetyl esterase in promoting degradation of beta-mannase on acetylated mannan |
| CN110669805A (en) * | 2019-09-27 | 2020-01-10 | 天津科技大学 | Method for producing galactose, mannose and mannan oligosaccharide by hydrolyzing locust bean gum through compounding of mannanase and galactosidase |
| CN110747242A (en) * | 2019-09-27 | 2020-02-04 | 天津科技大学 | Method for producing galactose, mannose and mannan oligosaccharide by hydrolyzing guar gum through compounding of mannanase and galactosidase |
| CN112322678A (en) * | 2020-10-22 | 2021-02-05 | 天津科技大学 | Method for synergistically hydrolyzing guar gum by using mannase and galactosidase |
| CN112391425A (en) * | 2020-10-22 | 2021-02-23 | 天津科技大学 | Method for synergistically hydrolyzing locust bean gum |
| CN113209202A (en) * | 2021-05-20 | 2021-08-06 | 曾文好 | Bacteriostatic gynecological lotion and preparation method thereof |
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