CN104498455B - A kind of middle temperature neutral beta glucuroide HiBgl3B of efficient degradation isoflavones and its gene and application - Google Patents
A kind of middle temperature neutral beta glucuroide HiBgl3B of efficient degradation isoflavones and its gene and application Download PDFInfo
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- CN104498455B CN104498455B CN201410717927.7A CN201410717927A CN104498455B CN 104498455 B CN104498455 B CN 104498455B CN 201410717927 A CN201410717927 A CN 201410717927A CN 104498455 B CN104498455 B CN 104498455B
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- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
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Abstract
The present invention relates to genetic engineering field, in particular it relates to a kind of middle temperature neutral beta glucuroide HiBgl3B of efficient degradation isoflavones and its gene and application.The invention provides a kind of new middle temperature neutral beta glucuroide HiBgl3B, it has the amino acid sequence as shown in SEQ ID NO.1 or 2, and present invention also offers the gene for encoding above-mentioned middle temperature neutral beta glucuroide HiBgl3B, its nucleotide sequence is as shown in SEQ ID NO.4 or 5, and recombinant vector and recombinant bacterial strain comprising the gene and its application.The maximum feature of the middle temperature neutral beta glucuroide of the present invention is its unique substrate specificity, it has recognition capability and hydrolysing activity for pNPG and genistin, daidzin, but it is inactive to natural substrate gentiobiose, cellobiose and amygdalin, with good research and application value.
Description
Technical field
The present invention relates to genetic engineering field, in particular it relates to a kind of middle temperature of efficient degradation isoflavones
Neutral beta-glucosidase HiBgl3B and its gene and application.
Background technology
Beta-glucosidase (beta-glucosidase, EC 3.2.1.21) is an important composition of cellulase system
Part, is that cellulose is thoroughly degraded to glucose specifically from single glucose residue under the hydrolysis of oligosaccharide chain reducing end
Key enzyme.Simultaneously as its can effectively hydrolysis fiber disaccharides, eliminate inhibitory action of the product accumulation to dextranase,
The hydrolysis of beta-glucosidase is also the rate-limiting step of cellulose degradation process.
Beta-glucosidase suffers from being widely applied in many fields.Industrially, as bio-ethanol is as new
The alternative energy source of safety and environmental protection is received increasing attention, and the degraded of lignocellulosic also receives much concern, and substantially increases energy work
To the demand of beta-glucosidase in industry.In food service industry, can hydrolyze the glucoside bond containing alkyl and aromatic radical and
Oligosaccharide etc., such as by the glycoside Substance P hydrolysis containing aromatic rings in food, discharges the aromatic series with strong fragrance
Compound, so as to improve flavour of food products.In feed and Medicines and Health Product industry, beta-glucosidase being capable of effectively hydrolyzing soybean
The natural glucosides such as isoflavones, realize the glucosides of inactive and have bioactivity aglycon between bioconversion, play many
Plant biological function.
Beta-glucosidase wide material sources, are found in plant, animal, microorganism.β microbe-derived now-
Glucuroide focuses primarily upon filamentous fungi, particularly aspergillus and trichoderma, becomes industrial beta-glucosidase
Main source.The beta-glucosidase HiBgl3B of the present invention derives from thermophilic fungal Humicola insolens, with good zymetology
Property, its maximum feature is its unique substrate specificity, and it has recognition capability for pNPG and genistin, daidzin
And hydrolysing activity, it is but inactive to natural substrate gentiobiose, cellobiose and amygdalin, with studying and apply valency well
Value.
The content of the invention
It is an object of the invention to provide a kind of middle middle benefit gas beta-glucosidase of efficient degradation isoflavones.
Another object of the present invention is to provide the gene for encoding above-mentioned beta-glucosidase.
It is a further object of the present invention to provide the recombinant vector comprising said gene.
It is a further object of the present invention to provide the recombinant bacterial strain comprising said gene.
It is a further object of the present invention to provide a kind of gene engineering method for preparing above-mentioned beta-glucosidase.
Another object of the present invention provides the application of above-mentioned beta-glucosidase.
The present invention is isolated from humicola lanuginosa (Humicola insolens) a kind of new to have unique substrate specificity
Middle middle benefit gas beta-glucosidase HiBgl3B, and construct the recombinant yeast for being capable of high efficient expression this beta-glucosidase
Strain.
The invention provides a kind of middle middle benefit gas beta-glucosidase HiBgl3B, its amino bases sequence such as SEQ ID NO.1
It is shown.
SEQ ID NO.1：
MGHHTATVCLWLALGSLTPVSFARVVEPRDPVPQGYHAASYYPAPHGGWVSSWREAYEKAYALVSQMTL
AEKVNITSGVGIYMGPCVGNTGSVDRLGFPQLCLQDSALGVASADNVTAFPAGITTGATWDKQLMYARGVAIGKEFR
GKGANIHLGPSVGPLGRKPLGGRNWEGFGSDPVLQGKAAALHIRGVQEQGIIATIKHLVGNEQEMYRMYHIFQEGYS
ANIDDRTLHELYLWPFAEAVRAGVGAAMTAYNAVNGSACSQNSYLINGILKDELGFQGLVMSDWLSHISGVGSALAG
LDLNMPGDTNIPLFGNSLWQYELTRAVLNGSVPVDRLNDMATRVVATWYKFGQDKNHPRPNFSSNTRSRDGPLYPGA
LFSPSGQVNWFVNVQEDHYLVARQVAQDAITLLKNNDSLLPLDAGDLTGGKLSVFGTDAQVNPDGPNSCLARACNKG
TLGMGWGSGIADYPYMDDPIGAIRKRVPDVKFYNTDSFPWFFGTPENDEVAMVFISSDSGENTLTVEGNHGDRDSAK
LRAWHDGDKLVQKVAEKFKNVIVVVHTVGPLDLEPWIELPSVKAVLFAHLPGQEAGESLTNVLFGDVSPSGHLPYSI
TRKASDLPDSIANLKGFTWGQVQDTYSEGLYIDYRYLQKHSIQPRFAFGHGLSYTNFSFTNATIRAITTPLSVTPPA
PPATRPASVVAKYSTDIPPASEAYEPAGFSRIWRYLYPWLSKSDADAAHAIGTSKSKTYPYPPGYSTVQRASFPPAG
GGEGGNPALWDVAYEVTVRVTNTGKRPGKASAQLYLQFPEGIEYDTPVLQLRDFEKTKELQPGESQELKLTLTRKDV
SVWDVRRQNWVVPTAIDDKKGFTAWVGEASDKLKVACYTGEGRCVEGAKQPV
Wherein, the enzyme includes 891 amino acid, and 23 amino acid of N-terminal are signal peptide sequence
“MGHHTATVCLWLALGSLTPVSFA”(SEQ ID NO.3)。
Therefore, ripe beta-glucosidase HiBgl3B theoretical molecular is 94.58kDa, and its amino acid sequence is such as
Shown in SEQ ID NO.2：
RVVEPRDPVPQGYHAASYYPAPHGGWVSSWREAYEKAYALVSQMTLAEKVNITSGVGIYMGPCVGNTGS
VDRLGFPQLCLQDSALGVASADNVTAFPAGITTGATWDKQLMYARGVAIGKEFRGKGANIHLGPSVGPLGRKPLGGR
NWEGFGSDPVLQGKAAALHIRGVQEQGIIATIKHLVGNEQEMYRMYHIFQEGYSANIDDRTLHELYLWPFAEAVRAG
VGAAMTAYNAVNGSACSQNSYLINGILKDELGFQGLVMSDWLSHISGVGSALAGLDLNMPGDTNIPLFGNSLWQYEL
TRAVLNGSVPVDRLNDMATRVVATWYKFGQDKNHPRPNFSSNTRSRDGPLYPGALFSPSGQVNWFVNVQEDHYLVAR
QVAQDAITLLKNNDSLLPLDAGDLTGGKLSVFGTDAQVNPDGPNSCLARACNKGTLGMGWGSGIADYPYMDDPIGAI
RKRVPDVKFYNTDSFPWFFGTPENDEVAMVFISSDSGENTLTVEGNHGDRDSAKLRAWHDGDKLVQKVAEKFKNVIV
VVHTVGPLDLEPWIELPSVKAVLFAHLPGQEAGESLTNVLFGDVSPSGHLPYSITRKASDLPDSIANLKGFTWGQVQ
DTYSEGLYIDYRYLQKHSIQPRFAFGHGLSYTNFSFTNATIRAITTPLSVTPPAPPATRPASVVAKYSTDIPPASEA
YEPAGFSRIWRYLYPWLSKSDADAAHAIGTSKSKTYPYPPGYSTVQRASFPPAGGGEGGNPALWDVAYEVTVRVTNT
GKRPGKASAQLYLQFPEGIEYDTPVLQLRDFEKTKELQPGESQELKLTLTRKDVSVWDVRRQNWVVPTAIDDKKGFT
AWVGEASDKLKVACYTGEGRCVEGAKQPV
The invention provides encode above-mentioned beta-glucosidase gene Hibgl3B.Specifically, the cDNA sequence of the gene is such as
Shown in SEQ ID NO.4：
atgggtcatcacactgccaccgtatgcctctggctcgccctgggctccttgacgcccgtctcctttgcccgcgttgt
cgagccccgcgatcctgttcctcaagggtatcatgctgcttcctactaccccgcgccccatggcggttgggtcagct
cgtggcgcgaggcctacgaaaaagcctatgcactggtgtcgcagatgacgctggctgagaaggtgaacatcacatcg
ggcgttggcatttatatgggaccctgtgtaggaaataccgggagtgtggatcgtctcggcttcccccagctctgcct
ccaagacagcgctctcggcgtcgcctccgccgacaatgtcacggcttttcccgctggcattaccaccggtgccacct
gggacaagcagttgatgtatgcccgaggcgtcgcgatcggcaaggagttccgcggcaagggcgccaatattcacttg
ggtccttcggttgggcccctcggccgcaagcccttgggcggccggaactgggagggctttggctccgacccggtgct
ccagggcaaggccgctgctctgcacatccgcggcgttcaggagcagggcattattgccaccattaagcaccttgtcg
gcaatgagcaggagatgtaccgcatgtaccacatttttcaggagggttacagcgccaacattgacgaccgtactctg
catgagctctacctctggcccttcgcggaggcagttcgtgctggagtgggagccgccatgacggcctacaatgccgt
caatggctccgcttgttcccagaacagttatctcatcaatggcattctcaaagacgaactcggcttccagggattgg
tcatgtcggactggctcagtcacatctcaggcgtgggctcggctctggccggtcttgacctcaacatgccgggcgat
acaaacattcccttgtttggtaacagtctgtggcagtacgagctgactcgtgccgtcctgaacggctccgtgcctgt
agacagactgaacgacatggccacgcgcgttgtggccacctggtacaagtttgggcaggataagaaccacccacggc
ccaacttctcatccaacactcgcagccgtgacgggcccctgtaccccggcgcccttttttctcccagcggtcaggtg
aattggttcgtcaacgtccaggaggatcactatcttgtcgcccgccaggtggctcaagatgccatcacgctgctcaa
gaataacgacagcctcttgcctctggacgctggggatcttactggcggcaagctcagcgtcttcggcactgacgctc
aagtcaaccccgatgggcccaactcctgcctagcacgggcttgtaacaaaggcactcttggcatgggttggggctcg
ggtatcgcggactatccgtacatggacgatcccatcggagccatccgcaagcgcgtccccgacgtcaagttctacaa
cacggacagctttccgtggttctttggcacgccggagaatgacgaggttgctatggtgttcatcagctccgactcgg
gagagaatacgctgacagtcgagggcaaccatggcgaccgcgactcggccaagctgagggcatggcacgacggtgac
aagctcgtgcagaaggtggctgagaaattcaagaacgtaatcgtcgtcgtgcatacggttggtcccctggaccttga
gccatggattgaacttccctcggtcaaagccgtcctctttgcccaccttcccggccaagaagccggcgagtctctga
ccaacgtcctcttcggcgacgtctcgccgagtggccacctcccctactccatcactcgcaaggcctccgatctgccc
gacagcatcgccaacctgaagggtttcacctggggccaagtccaagacacctactccgaagggctctacatcgacta
ccgctacctgcaaaagcactcgatccagccccgcttcgccttcggccacggcttgagctacaccaacttctccttca
ccaatgccaccatccgcgccatcactacccccctatccgtcaccccgccagccccgccagccaccaggcccgcctca
gtcgtcgctaaatactccaccgacatcccgcccgccagcgaagcctacgagcctgcaggcttctccaggatctggcg
ctacctctacccctggctgtccaaatccgacgccgacgccgcccacgctatcggcacgagcaagtccaaaacctacc
cctaccctcccggctattccaccgtgcagcgcgcgtctttccctcccgctggcggcggcgagggcggcaaccccgcg
ctctgggacgtggcatacgaggtgacggtgcgcgtcaccaacacgggcaagaggcccgggaaagcctcggcgcagct
gtatctgcagttcccggaggggatcgagtacgatacccccgtactgcagctccgggatttcgagaagaccaaggagc
tccagccgggcgagagccaggagctgaagctgacattgacgaggaaggatgtcagcgtgtgggacgtgaggaggcag
aactgggttgttccgacggcgattgacgacaagaaggggttcacggcgtgggtgggcgaggcgagcgataagttgaa
ggtggcgtgctacacgggtgaggggaggtgtgtggagggggcgaagcagccggtttga
Beta-glucosidase gene Hibgl3B, DNA complete sequence analysis knot has been cloned in the method separation of PCR-based of the present invention
Fruit shows, removes the beta-glucosidase gene Hibgl3B cDNA total lengths 2676bp after introne.Wherein, the base of signal peptide
Sequence is：
atgggtcatcacactgccaccgtatgcctctggctcgccctgggctccttgacgcccgtctcctttgcc
(SEQ IDNO.6)。
Ripe beta-glucosidase gene HiBgl3B gene order is as shown in SEQ ID NO.5.
SEQ ID NO.5
cgcgttgtcgagccccgcgatcctgttcctcaagggtatcatgctgcttcctactaccccgcgccccatggcggttg
ggtcagctcgtggcgcgaggcctacgaaaaagcctatgcactggtgtcgcagatgacgctggctgagaaggtgaaca
tcacatcgggcgttggcatttatatgggaccctgtgtaggaaataccgggagtgtggatcgtctcggcttcccccag
ctctgcctccaagacagcgctctcggcgtcgcctccgccgacaatgtcacggcttttcccgctggcattaccaccgg
tgccacctgggacaagcagttgatgtatgcccgaggcgtcgcgatcggcaaggagttccgcggcaagggcgccaata
ttcacttgggtccttcggttgggcccctcggccgcaagcccttgggcggccggaactgggagggctttggctccgac
ccggtgctccagggcaaggccgctgctctgcacatccgcggcgttcaggagcagggcattattgccaccattaagca
ccttgtcggcaatgagcaggagatgtaccgcatgtaccacatttttcaggagggttacagcgccaacattgacgacc
gtactctgcatgagctctacctctggcccttcgcggaggcagttcgtgctggagtgggagccgccatgacggcctac
aatgccgtcaatggctccgcttgttcccagaacagttatctcatcaatggcattctcaaagacgaactcggcttcca
gggattggtcatgtcggactggctcagtcacatctcaggcgtgggctcggctctggccggtcttgacctcaacatgc
cgggcgatacaaacattcccttgtttggtaacagtctgtggcagtacgagctgactcgtgccgtcctgaacggctcc
gtgcctgtagacagactgaacgacatggccacgcgcgttgtggccacctggtacaagtttgggcaggataagaacca
cccacggcccaacttctcatccaacactcgcagccgtgacgggcccctgtaccccggcgcccttttttctcccagcg
gtcaggtgaattggttcgtcaacgtccaggaggatcactatcttgtcgcccgccaggtggctcaagatgccatcacg
ctgctcaagaataacgacagcctcttgcctctggacgctggggatcttactggcggcaagctcagcgtcttcggcac
tgacgctcaagtcaaccccgatgggcccaactcctgcctagcacgggcttgtaacaaaggcactcttggcatgggtt
ggggctcgggtatcgcggactatccgtacatggacgatcccatcggagccatccgcaagcgcgtccccgacgtcaag
ttctacaacacggacagctttccgtggttctttggcacgccggagaatgacgaggttgctatggtgttcatcagctc
cgactcgggagagaatacgctgacagtcgagggcaaccatggcgaccgcgactcggccaagctgagggcatggcacg
acggtgacaagctcgtgcagaaggtggctgagaaattcaagaacgtaatcgtcgtcgtgcatacggttggtcccctg
gaccttgagccatggattgaacttccctcggtcaaagccgtcctctttgcccaccttcccggccaagaagccggcga
gtctctgaccaacgtcctcttcggcgacgtctcgccgagtggccacctcccctactccatcactcgcaaggcctccg
atctgcccgacagcatcgccaacctgaagggtttcacctggggccaagtccaagacacctactccgaagggctctac
atcgactaccgctacctgcaaaagcactcgatccagccccgcttcgccttcggccacggcttgagctacaccaactt
ctccttcaccaatgccaccatccgcgccatcactacccccctatccgtcaccccgccagccccgccagccaccaggc
ccgcctcagtcgtcgctaaatactccaccgacatcccgcccgccagcgaagcctacgagcctgcaggcttctccagg
atctggcgctacctctacccctggctgtccaaatccgacgccgacgccgcccacgctatcggcacgagcaagtccaa
aacctacccctaccctcccggctattccaccgtgcagcgcgcgtctttccctcccgctggcggcggcgagggcggca
accccgcgctctgggacgtggcatacgaggtgacggtgcgcgtcaccaacacgggcaagaggcccgggaaagcctcg
gcgcagctgtatctgcagttcccggaggggatcgagtacgatacccccgtactgcagctccgggatttcgagaagac
caaggagctccagccgggcgagagccaggagctgaagctgacattgacgaggaaggatgtcagcgtgtgggacgtga
ggaggcagaactgggttgttccgacggcgattgacgacaagaaggggttcacggcgtgggtgggcgaggcgagcgat
aagttgaaggtggcgtgctacacgggtgaggggaggtgtgtggagggggcgaagcagccggtttga
Maturation protein theoretical molecular is 94.58kDa, by the ripe coded sequences of beta-glucosidase gene Hibgl3B and
The amino acid sequence derived carries out BLAST comparisons, and it is a kind of new beta-glucosidase to determine HiBgl3B.
The invention provides the recombinant vector for including above-mentioned beta-glucosidase gene Hibgl3B, pPIC- is elected as
Hibgl3B.The beta-glucosidase gene of the present invention is inserted between the suitable restriction enzyme site of expression vector, makes it
Nucleotide sequence is exercisable to be connected with expression regulation sequence.As the present invention a most preferred embodiment, preferably
For by the beta-glucosidase gene of the present invention be inserted into EcoR I and Not I restriction enzyme sites on plasmid pPIC9 it
Between, the nucleotide sequence is located at the downstream of AOX1 promoters and is regulated and controled by it, obtain expression of recombinant yeast plasmid pPIC9-
Hibgl3B。
Present invention also offers the recombinant bacterial strain for including above-mentioned beta-glucosidase gene Hibgl3B, preferably described bacterial strain
For Escherichia coli, saccharomycete, preferably recombinant bacterial strain Hibgl3B.
Present invention also offers a kind of method for preparing beta-glucosidase gene HiBgl3B, comprise the following steps：
1) host cell is converted with above-mentioned recombinant vector, obtains recombinant bacterial strain；
2) recombinant bacterial strain, induction restructuring beta-glucosidase gene HiBgl3B expression are cultivated；
3) reclaim and purify expressed beta-glucosidase gene HiBgl3B.
Wherein, preferably described host cell be Pichia pastoris, beer yeast cells or many types of inferior yeast cells, preferably
Expression of recombinant yeast plasmid is converted into Pichia pastoris (Pichia pastoris) GS115, recombinant bacterial strain GS115/ is obtained
Hibgl3B。
Present invention also offers above-mentioned beta-glucosidase HiBgl3B application.To the degradation experiment of isoflavones, table
Bright beta-glucosidase HiBgl3B can efficient degradation isoflavones, be translated into three kinds of active aglycones, degradation rate is up to 90%
More than.In the case where adding the beta-glucosidase of equal enzyme unit, HiBgl3B effects are substantially much better than other originated from fungus
Beta-glucosidase, its special substrate selective make its be more suitable for degrade isoflavones.
Brief description of the drawings
The optimal pH of Fig. 1 beta-glucosidases.
Fig. 2 recombinates the pH stability of beta-glucosidase.
Fig. 3 recombinates the optimum temperature of beta-glucosidase.
Fig. 4 recombinates the heat endurance of beta-glucosidase.
Embodiment
Test material and reagent
1st, bacterial strain and carrier：The present invention is isolated from humicola lanuginosa (Humicola insolens Y1CGMCC 4573)
A kind of new beta-glucosidase HiBgl3B.Yeast expression vector pPIC9 and bacterial strain GS115 is purchased from Invitrogen
Company.
2nd, enzyme and other biochemical reagents：Restriction endonuclease is purchased from TaKaRa companies, and ligase is purchased from Invitrogen companies.Birch
Wooden xylan is purchased from Sigma companies, and other is all domestic reagent (can be commercially available from common biochemical Reagent Company).
3rd, culture medium：
(1) humicola lanuginosa (Humicola insolens Y1CGMCC 4573) culture medium is potato juice culture medium：
1000mL potato juices, 10g glucose, 25g agar, pH is natural.
(2) Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH are natural).
(3) BMGY culture mediums：1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 1% is sweet
Oily (V/V).
(4) BMMY culture mediums：Divided by 0.5% methanol replace glycerine, remaining composition is identical with BMGY, and pH is natural.
Explanation：Do not make the experimental methods of molecular biology illustrated, equal reference in following examples《Molecular Cloning: A Laboratory
Guide》Listed specific method is carried out in the book of (third edition) J. Pehanorm Brookers one, or according to kit and product description
Carry out.
The humicola lanuginosa of embodiment 1 (Humicola insolens) beta-glucosidase enzyme coding gene bgl3B clone
Design gene-specific primer：
PF:GGGGAATTCCGCGTTGTCGAGCCCCGCGATC
PR:GGGGCGGCCGCTCAAACCGGCTGCTTCGCCCCCTC
Humicola lanuginosa Humicola insolens Y1CGMCC 4573RNA are extracted, reverse transcription obtains cDNA.With Humicola
Insolens Y1CGMCC 4573cDNA are that template enters performing PCR amplification.PCR response parameters are：94 DEG C of denaturation 5min；Then 94
DEG C denaturation 30sec, 60 DEG C annealing 30sec, 72 DEG C extension 2min30s, 35 circulation after 72 DEG C insulation 10min.Obtain one about
2600bp fragments, be connected the sequencing of Bioisystech Co., Ltd of Song Rui Boxings section after the fragment is reclaimed with pEASY-T3 carriers.
Sequencing result is beta-glucosidase gene Hibgl3B genes 2607bp (removal signal peptide), encodes 868 amino
Acid and a terminator codon.The theoretical molecular for predicting the maturation protein of the coded by said gene is 94.58kDa.
The preparation of the recombined xylanase of embodiment 2
Expression vector pPIC9 is subjected to double digestion (EcoR I+Not I), while by encoding beta-glucosidase Bgl3B's
Gene bgl3B double digestions (EcoR I+Not I), the genetic fragment for cutting out encoding mature beta-glucosidase (does not include signal peptide
Sequence) it is connected with expression vector pPIC9, obtain and contain humicola lanuginosa (Humicola insolens Y1CGMCC 4573) β-grape
Glycosidase genes Hibgl3B recombinant plasmid pPIC-Hibgl3B simultaneously converts Pichia pastoris GS115, obtains recombinant yeast pichia pastoris bacterium
Strain GS115/Hibgl3B.
The expression vector containing signal peptide sequence is built in the same way, and converts Pichia pastoris.
The GS115 bacterial strains containing recombinant plasmid are taken, are inoculated in 400mL BMGY nutrient solutions, 30 DEG C of 250rpm shaken cultivations
After 48h, thalline is collected by centrifugation.Then it is resuspended in 200mL BMMY culture mediums, 30 DEG C of 250rpm shaken cultivations.Induce after 48h, from
The heart collects supernatant.Determine the vigor of beta-glucosidase.SDS-PAGE results show that restructuring beta-glucosidase is in Pichia pastoris
In expressed.
Embodiment 3 recombinates the activity analysis of beta-glucosidase
The measure of activity of beta-glucosidase：The product that measure enzyme hydrolysis substrate pNPG is generated under 405nm is to nitro
The amount of phenol (pNP).
Reactions steps：125 μ l 2mM pNPG substrates and 125 μ l buffer solutions are mixed, and add the enzyme liquid that 250 μ l suitably dilute,
10min is reacted in 60 DEG C, 1.5mL 1M Na is added2CO3Terminating reaction, uses spectrophotometric determination OD405Value.
The definition of enzyme-activity unit：1 activity of beta-glucosidase unit (U) is defined as under the given reaction conditions, every point
Clock decomposes the enzyme amount needed for substrate pNPG 1 μm of ol p-nitrophenol (pNP) of generation.
The property of the restructuring beta-glucosidase of embodiment 4 is determined
Determine the property that embodiment 2 obtains restructuring beta-glucosidase HiBgl3B
1st, restructuring beta-glucosidase HiBgl3B optimal pH and the assay method of pH stability are as follows：
The restructuring beta-glucosidase HiBgl3B that embodiment 2 is purified carries out enzymatic reaction to determine under different pH
Its optimal pH.Substrate pNPG carries out β-grape with different pH 0.1mol/L citrate-phosphate disodium hydrogen buffer solutions 50 DEG C
Glucosides enzyme activity determination.As a result (Fig. 1) shows, beta-glucosidase HiBgl3B optimal pH is 6.0.Beta-glucosidase in
37 DEG C of processing 60min in above-mentioned various different pH buffer solution, then enzymatic activity is determined at 50 DEG C in pH6.0 buffer solution systems,
With the pH stability of studying enzyme.As a result (Fig. 2) shows beta-glucosidase HiBgl3B pH tolerances.
2nd, beta-glucosidase HiBgl3B optimum temperature and thermal stability determination method are as follows：
The optimum temperature of beta-glucosidase is determined as in citrate-phosphate disodium hydrogen buffer solution (pH6.0) buffer solution
Enzymatic reaction is carried out under system and different temperatures.When temperature tolerance is determined as beta-glucosidase and handles different at different temperatures
Between, then carry out under optimum temperature enzyme assay.Enzyme reaction optimum temperature measurement result (Fig. 3) shows that its optimum temperature is 50
℃.The heat endurance experiment of enzyme shows (Fig. 4) that beta-glucosidase HiBgl3B has good heat endurance, warm at 50 DEG C
1h is educated, enzyme activity can be kept not reduce.
3rd, beta-glucosidase HiBgl3B enzyme kinetics assay method is as follows：
It is substrate with the pNPG of various concentrations, in citrate-phosphate disodium hydrogen buffer solution (pH4.5) buffer solution system,
Enzymatic activity is determined at 50 DEG C, its K at 50 DEG C is calculatedmValue.K when after measured, using pNPG as substratemIt is worth for 1.51mM, most
Big reaction speed VmaxFor 25.53 μm of ol/minmg.
4th, influence of the different metal ions chemical reagent to beta-glucosidase HiBgl3B enzyme activity is determined as follows：
The different metal ions and chemical reagent of various concentrations are added in enzymatic reaction system, it are studied to enzymatic activity
Influence, the various final concentration of 5mmol/L of material.Enzymatic activity is determined under the conditions of 50 DEG C, pH6.0.As a result show, it is most of from
Son and chemical reagent do not influence on the vigor for recombinating beta-glucosidase, but SDS and Ag+ seriously suppress its enzyme activity, respectively
Show under optimum condition 5% and 24% enzyme activity.
5th, restructuring beta-glucosidase HiBgl3B substrate specificity
To substrates such as a variety of polysaccharide, natural glucosides and synthetic pNPG, enzyme activity is determined under optimum condition, β-Portugal is studied
Polyglycoside enzyme HiBgl3B substrate specificity.As a result it is as shown in table 2.Compared to most of beta-glucosidases, humicola lanuginosa
The HiBgl3B in (Humicola insolens) source has very unique substrate specificity, and it can not hydrolyze such hydrolase
Natural substrate cellobiose, gentiobiose and amygdalin etc..Found through amino acid sequence analysis, HiBgl3B is at multiple and bottom
The crucial aromatic amino acid residue site that+1 glucose molecule of thing has intermolecular interaction is mutated, so guessing
The change for surveying its substrate specificity is due to that substrate can not be combined correctly.
The beta-glucosidase HiBgl3B substrate specificities of table 1. are analyzed
The restructuring beta-glucosidase encoding gene Hibgl3B degraded isoflavones effect experiments of embodiment 5
Isoflavones includes daidzein, 3 kinds of isoflavones of genistein and Daidezin, and they and grape
The natural glucosides that sugar, acetyl glucosamine and malonyl glucose are constituted with 9 kinds of isoflavone glucosides of β-glycosidic bond formation.β-grape
Glycosidase can effectively cut off β-glycosidic bond between isoflavone genin and glucose, so that soybean isoflavone glycoside be turned
The active aglycone with bioactivity is turned to, is extracted for isoflavones medicine and animal health has good application value.
The bean cake powder that 10g crosses 40 mesh sieves is weighed, 30ml water is added and stirs, adjusts pH to appropriate pH, adds by every gram of dregs of beans
Plus 3U beta-glucosidases, after 37 DEG C of waters shaking tables hydrolysis 1h, 100ml absolute ethyl alcohols are added, 70 DEG C of extraction 2h obtain soybean different
Flavone extractive, carries out HPLC quantitative analyses.Beta-glucosidase selects HiBgl3B and 3 families of two other originated from fungus
Beta-glucosidase, to compare.
The beta-glucosidase HiBgl3B hydrolyzed soy bean isoflavones of table 2.
It can be seen that, HiBgl3B can efficient degradation isoflavones, be translated into three kinds of active aglycones, degradation rate is up to 90%
More than.In the case where adding the beta-glucosidase of equal enzyme unit, HiBgl3B effects are substantially much better than other originated from fungus
Beta-glucosidase, its possible cause is HiBgl3B to+1 glucose molecule of substrate combines related crucial ammonia multiple
Base acid Residue positions are all mutated, so that very unique substrate specificity is shown, no disaccharides substrate active, but right+1
Position aglucon is higher than other beta-glucosidases, this special substrate selection for the isoflavones substrate enzyme activity of phenyl ring
Property make its be more suitable for degrade isoflavones.
Claims (8)
1. a kind of middle middle benefit gas beta-glucosidase HiBgl3B of efficient degradation isoflavones, it is characterised in that its amino acid
Sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2.
2. a kind of middle middle benefit gas beta-glucosidase gene Hibgl3B of efficient degradation isoflavones, it is characterised in that coding
Beta-glucosidase HiBgl3B described in claim 1.
3. beta-glucosidase gene Hibgl3B as claimed in claim 2, it is characterised in that its base sequence such as SEQ ID
Shown in NO.4 or SEQ ID NO.5.
4. include the recombinant vector of beta-glucosidase gene Hibgl3B described in claim 2.
5. the recombinant vector pPIC-Hibgl3B of beta-glucosidase gene Hibgl3B described in claim 2 is included, wherein, will
Beta-glucosidase gene of the nucleotide sequence as shown in SEQ ID NO.5 is inserted on plasmid pPIC9, obtains recombinant vector
pPIC-Hibgl3B。
6. include the recombinant bacterial strain of beta-glucosidase gene Hibgl3B described in claim 2.
7. a kind of Prepare restructuring beta-glucosidase HiBgl3B method, it is characterised in that comprise the following steps：
1) with the recombinant vector conversion host cell of claim 4, recombinant bacterial strain is obtained；
2) recombinant bacterial strain, induction restructuring beta-glucosidase HiBgl3B expression are cultivated；
3) reclaim and purify expressed beta-glucosidase HiBgl3B.
8. applications of the beta-glucosidase HiBgl3B described in claim 1 for isoflavones of degrading.
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| 黑曲霉β-葡萄糖苷酶水解大豆异黄酮糖苷研究;孙艳梅等;《东北农业大学学报》;20071231;第38卷(第1期);9-12 * |
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