CN109355274A - The beta-glucosidase that a kind of pair of trypsase stomach function regulating protease resistant improves - Google Patents
The beta-glucosidase that a kind of pair of trypsase stomach function regulating protease resistant improves Download PDFInfo
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Abstract
The beta-glucosidase improved the invention discloses a kind of pair of trypsase stomach function regulating protease resistant and its application.The present invention is transformed by critical amino acid residues of the protein engineering to wild type beta-glucosidase intramolecular, provides a kind of β-glucosidase mutants for obtaining and improving to trypsase stomach function regulating protease resistant.The mutant beta-glucosidase (mBGL1) of the present invention for acting on cellobiose and short chain cell-oligosaccharide, the resistance of trypsase and pepsin is improved than wild type BGL1, other zymologic properties and wild-type enzyme are almost the same.
Description
Technical field
The present invention relates to beta-glucosidase, it is related specifically to the β-grape improved to trypsase stomach function regulating protease resistant
Glycosidase.
Background technique
Beta-glucosidase (β-D-Glucosidase, EC3.2.1.21, abbreviation BGL1) is also known as glucoside hydrolysis
Enzyme, its energy hydrolysis fiber disaccharides and short chain cell-oligosaccharide generate glucose, it is poly- to inscribe Portugal to release cellobiose and cell-oligosaccharide
The feedback inhibition of carbohydrase and cellobiohydrolase can also digest the latent aromatic substance in fruit and tealeaves, play enhancing
The effect of fragrance.Beta-glucosidase belongs to hydrolase, is one of important composition ingredient of cellulase system.
Beta-glucosidase the glycogen degradation of the mankind and animal, plant, microorganism carbohydate metabolism in terms of have it is important
Physiological function.In recent years, important application prospect: beta-glucosidase hydrolyzable fruits and vegetables is shown in the food industry
In such as single note enol glucosides of flavor precursors, release the substance with strong natural scents, it has also become food industry
In important special flavour improvement agent.The conversion of bioactive substance is then used for when preparing functional food, as added β-in soymilk
Glucuroide or inoculation produce the microorganism of beta-glucosidase, can be lower by biological effectiveness in soymilk and soymilk powder
Isoflavone glucoside compound Efficient Conversion is the isoflavone of high activity.Another important use of beta-glucosidase is effect
In cellulose bioconversion, the effective use of this huge renewable resource of cellulose is to solution environmental pollution, food shortages, energy
Source crisis has Great significance.In feed industry, for beta-glucosidase often by as a kind of feed addictive, it can be with
The rich fibrous cell wall of enzymatic hydrolysis, releases the nutriments such as the protein for including into the cell, while again by fiber degradation
For the reduced sugar absorbed convenient for animal digestion, to improve efficiency of feed utilization.But in practical applications or there are many insufficient,
Such as stability is not good enough, this will affect the validity of beta-glucosidase and increases application cost.Therefore to glucoside
Enzyme, which is transformed, to be had a very important significance.
Over nearly 18 years, the patent in relation to beta-glucosidase has nearly 190, these patents focus primarily upon discovery and sieve
It selects the bacterial strain of the beta-glucosidase of the production different characteristic from separate sources or is obtained by gene recombination method and produce different spies
The beta-glucosidase of sign bacterial strain (as production beta-glucosidase have it is thermophilic/heat-resisting (ZL201610060528.7,
ZL201310049606.X, ZL201510203453.9, ZL201510203484.4, ZL201110214130.1), high temperature it is neutral
(ZL201410713835.1), medium temperature neutrality (ZL201410717927.7, ZL201110334815.X), thermo philic alkali
(ZL200510116748.9), thermophilic acid resistance (ZL200910218167.4, ZL201410753327.6,
ZL201510460386.9), acid (ZL201410718610.5), mesophilic resistance to ethyl alcohol (ZL201010577713.6,
ZL201410235451.3), high expression (ZL201010170523.2, ZL201410037538.X, ZL201410037437.2,
ZL201010565278.5), resistance to high sugar (ZL201210540712.3, ZL201210541293.5, ZL201710698739.8,
ZL 201710366423.9, ZL201610095165.0, ZL201310224173.7, ZL201010248207.2), resistance to sugar it is resistance to
Sour (ZL201710839935.2), the resistance to sugar of high yield acidproof (ZL201710839898.5), resistance to high sugar are alkaline-resisting
(ZL201410668699.9), the mesophilic resistance to ethyl alcohol (ZL201110417104.9) of high yield, high vigor (ZL201110102978.5),
High catalytic efficiency (ZL201610407183.8, ZL201610061066.0, ZL201610060603.X,
ZL201610061593.1), height endurability (ZL201510175655.7), continue efficient secretion (ZL201310187648.X,
The bacterial strain of one of features such as ZL201510626711.4)), and the method that screening produces beta-glucosidase bacterial strain
(ZL201510903621.5、ZL201210127162.2、ZL201710279045.0、ZL201410075528.5、
ZL200710114837.9), beta-glucosidase enzyme purification method (ZL200710043571.3), β-glucosidase activity measurement
Method (ZL200910096109.9), beta-glucosidase enzyme immobilization method (ZL201510920113.8,
ZL201710579688.7、ZL201710689763.5、ZL201610348992.6、ZL201610899746.X、
ZL201310712037.2、ZL201210449928.9、ZL201210052838.6、ZL201110293398.9、
ZL200810035950.2, ZL201810109984.5), beta-glucosidase preparation method (ZL201710581264.4,
ZL201510026295.4、ZL201410510836.6、ZL201110374822.2、ZL200910153807.8、
ZL200910092695.X, ZL201810034473.1), building produces beta-glucosidase gene engineering bacteria method
(ZL201410126924.6、ZL 201410699758.9、ZL201610285023.0、ZL201610838951.5、
ZL201510172770.9、ZL201510658194.9、ZL201410801030.2、ZL201410801031.7、
ZL201210017857.5、ZL201110221185.5、ZL201110301931.1、ZL201110034926.9、
ZL201110132525.7、ZL201110266535.X、ZL200910092527.0、ZL200610025503.X、
ZL201110120297.1, ZL200910029055.4, ZL200910159235.4) and above-mentioned gained beta-glucosidase
Encoding gene and application etc..By retrieving opinion of the Chinese Dissertations Database discovery in recent years in relation to β-glucosidase activity property
Text has very much, wherein focusing primarily upon through genetic recombination, complex mutation, improvement screening technique, optimizing to fermentation condition
The methods of come improve its enzyme activity and thermal stability (Q55Q785, TQ925, TQ929.2, TQ925TQ920.1, Q939.9,
TS261.11, TQ925, TS201.25TS201.3, Q814, Q814.1Q936), glucose tolerance (Q949.327.103,
Q556.2Q786) catalytic efficiency improve the characteristics of (TQ925), heat-resisting, the alkaline-resisting and ion of resistance to part metals (Q93-331,
S216.2, Q949.327.1Q943.2, TQ925.5, R282.71Q556.2) etc..And the β-improved with pepsin resistance
Glucuroide has not been reported yet.
Summary of the invention
Primary and foremost purpose of the invention is to provide the beta-glucosidase of a kind of pair of trypsase stomach function regulating protease resistant raising.
The present invention is to carry out rite-directed mutagenesis to the gene (referred to as BGL1 gene) of beta-glucosidase.By Trichoderma viride
The beta-glucosidase obtained in (Trichoderma viride), gene have been sequenced, and GENBANK accession number is
FJ882071.1.The amino acid sequence of the maturation protein of the enzyme is SEQ ID NO.1.
The present invention is based on the screenings of the method for enzymatic reaction transition and calculation biology, and obtaining one plant has trypsase
β-glucose glycosidase saltant of stomach function regulating protease resistant improvement.Trypsase half-life period is extended compared with wild type
(enzymatic hydrolysis condition is that PH is 7.6 to 30min, and concentration is that the trypsase of 0.175mg/ml digests 0.25mg/ml's under the conditions of 37 DEG C
BGL1), to pepsin half-life period, compared with wild type extension 6min, (enzymatic hydrolysis condition is that PH is 1.2, and concentration is the stomach egg of 0.2mg/ml
White enzyme digests the BGL1 of 0.25mg/ml under the conditions of 37 DEG C).It is named as mBGL1Q91C；R601H；R683W。
It is SEQ ID that the Fixedpoint mutation modified beta-glucosidase of one kind of the present invention, which is by amino acid sequence,
NO.1 multiple amino acid substitutions are manufactured in the beta-glucosidase of Trichoderma viride (Trichoderma viride) and
It generates, to the beta-glucosidase that trypsase stomach function regulating protease resistant improves, the amino acid substitution is the 91st, 601
With 683 substitutions.
The further feature of Fixedpoint mutation modified beta-glucosidase according to the present invention, it is described at the 91st
Amino acid substitution is to replace glutamine with cysteine；It is to replace arginine with group in 601 amino acid substitutions；?
683 amino acid substitutions are to replace arginine with tryptophan；The amino of the Fixedpoint mutation modified beta-glucosidase
Acid sequence is SEQ ID NO.2.
The mutant beta-glucosidase (mBGL1) of the present invention for acting on cellobiose and short chain cell-oligosaccharide,
Through artificial pancreatic juice (PH 7.6, concentration are that the trypsase of 0.175mg/ml digests the BGL1 of 0.25mg/ml under the conditions of 37 DEG C)
Handle 60min, the resistance half-life period of trypsase is extended 30 minutes than wild-type enzyme, through simulated gastric fluid (pH 1.2,
Concentration is the pepsin of 0.15mg/mL under the conditions of 37 DEG C) after digestion process 60min, to pepsin resistance half-life period compared with
Wild type extends 6min, other zymologic properties and wild-type enzyme are almost the same.
Further, the present invention provides a kind of DNA moleculars, encode of the present invention to trypsase and stomach cardia
The beta-glucosidase that enzyme resistance improves.
Preferably, the nucleotides sequence of DNA molecular of the present invention is classified as SEQ ID NO.3.
It is a further object to provide a kind of carriers, contain DNA molecular of the present invention.
A further object of the present invention is to provide a kind of host cell, contains DNA molecular of the present invention, Huo Zhehan
There is carrier of the present invention.
Above-mentioned carrier and host cell can be prepared by technological means well known in the art.
The present invention also provides the beta-glucosidases of the present invention improved to trypsase stomach function regulating protease resistant
Production method, comprising: cultivate host cell of the present invention under conditions of being suitable for beta-glucosidase expression of enzymes, and from training
It supports and separates the beta-glucosidase in base.
When DNA molecular of the present invention is inserted into the carrier with correct reading frame to be suitably orientated, or
It is transferred in the host cell, the DNA molecular can express in any eukaryon or prokaryotic expression system.Many places
Master-carrier system may serve to expression protein coding sequence.Host-vector system includes but is not limited to: using bacteriophage, matter
The bacterium of grain or clay conversion；Microorganism containing yeast vector, such as yeast；With the mammalian cell system of virus infection；
With the insect cell system of virus infection；With the plant cell system of bacterium infection.Currently preferred carrier includes that virus carries
Body, plasmid, clay or oligonucleotides.
Currently preferred host is eukaryotic system such as Pichia pastoris；Currently preferred protein expression method is red to finish
Yeast secreted expression.
Detailed description of the invention
Fig. 1 is SDS-PAGE protein electrophoresis figure, is purpose protein band at black arrow meaning, and size is 78.4Kd left
It is right；Swimming lane 1 is wild type beta-glucosidase protein B GL1wt；Swimming lane 2 is the wild type Pichia pastoris without target gene
SMD1168 control sample；Swimming lane 3 is mutant beta-glucosidase zymoprotein mBGL1Q91C；R601H；R683W.Electrophoresis result shows prominent
Variant mBGL1Q91C；R601H；R683WBand meets expection.
Fig. 2 is wild type BGL1 of the present inventionwtWith mutant mBGL1Q91C；R601H；R683WEnzyme activity determination standard it is bent
Line.
Fig. 3 is wild type BGL1 of the present inventionwtWith mutant mBGL1Q91C；R601H；R683WAlbumen is at trypsase
Manage the preceding and residual protein scanning result figure after trypsin treatment.
Fig. 4 is wild type BGL1 of the present inventionwtWith mutant mBGL1Q91C；R601H；R683WAlbumen is at pepsin
Manage the preceding and residual protein scanning result figure after pepsin.
Fig. 5 is wild type BGL1wtWith mutant mBGL1Q91C；R601H；R683WAlbumen is before trypsin treatment and through pancreas egg
Specific activity of enzyme measurement result figure after white enzymatic treatment.
Fig. 6 is wild type BGL1wtWith mutant mBGL1Q91C；R601H；R683WAlbumen is before pepsin and through stomach egg
Specific activity of enzyme measurement result figure after white enzymatic treatment.
Specific embodiment
Term used by herein is unless otherwise indicated the normally understood meaning of those skilled in the art institute.With
It is lower that the definition for some specific terms being used in the present invention is provided.
“BGL1wt" indicating wild type beta-glucosidase, gene is with italic BGL1wtIt indicates.
“mBGL1Q91C；R601H；R683W" indicating mutant beta-glucosidase mBGL1, gene is with mBGL1Q91C；R601H；R683W
It indicates.
Embodiment 1: the synthesis of beta-glucosidase gene
The present invention use the wild type beta-glucosidase in Trichoderma viride source gene (GenBank number of registration for
FJ882071.1), (other commercial companies with full genome synthesis can equally complete) is synthesized by Jin Weizhi company.
Embodiment 2: beta-glucosidase (bgl1) and cloning vector TaoxThe connection of+PgHT+PB
1. mbgl1 target gene and cloning vector Taox+PgHT+PB are used restriction enzyme EcoRI and SpeI/ respectively
For XbaI in 37 DEG C of digestion 10min, digestion condition is as follows:
2. two target fragments are separately recovered in digestion products after 1% agarose gel electrophoresis, connected with T4DNA ligase
It connects, linked system is as follows:
| Bgl1 digestion products | 0.6pmol |
| Cloning vector digestion products | 0.08pmol |
| T4DNA enzyme | 1μl |
| 10 × buffer | 1μl |
| Total amount | 10μl |
With 16 DEG C of connection 12h of ligase, connection product uses plasmid extraction kit after converting the amplification of DH5a competent cell
Plasmid is extracted, has two band of 7.0kb and 3.8kb as the result is shown with electrophoresis is run after EcoRI and PstI double digestion, shows to connect into
Function is determined as beta-glucosidase gene by DNA sequencing.
Embodiment 3: genetic fragment Paox+SS1 is connect with cloning vector M+Taox+PgHT+PB
1. recalling in the cloning vector Paox+SS1+PB that genetic fragment Paox+SS1 is saved by this research institute, EcoRI is used
With SpeI enzymes double zyme cutting and purification and recovery obtains；
2. cloning vector M+Taox+PgHT+PB is obtained by embodiment 2, genetic fragment Paox+SS1 and cloning vector M+
The connection method of Taox+PgHT+PB is the same as embodiment 2.
Embodiment 4: rite-directed mutagenesis
By using the principle being mutually distinguishable between enzymatic reaction transition state theory and protein molecular, and calculate chemistry
Method carries out on 4.5 software platform of Discovery Studio, and inventor determines to the 91st, the 601st, the 683rd ammonia
Base acid carries out rite-directed mutagenesis, the mutant mBGL1 after mutationS84C；Q643P；G696VGene is by the Suzhou gold only limited public affairs of intelligence biotechnology
Department's synthesis.Gene chemical synthesis can also be completed by other commercial companies synthesized with full genome.
Embodiment 5: wild type BGL1 and mutant mBGL1Q91C；R601H；R683WGene integrates Pichia pastoris genome respectively
And the secreting, expressing of recombinant protein
In order to improve the integration efficiency for singly examining shellfish expression cassette on Pichia chromosome, with restriction enzyme XbaI and
SpeI is by expression cassette Paox+SS1+M+Taox+ PgHT is from Paox+SS1+M+TaoxIt is cut on+PgHT+PB and is used together kits
Recycling.The recipient bacterium of this experiment is Pichia pastoris SMD1168, uses MD plate to carry out preliminary screening after electrotransformation, then picking
Monoclonal on MD plate cultivates 1 day measurement colony diameter d1 and darkened features in aesculin-ironic citrate screening and culturing medium
The diameter d2 of circle.The value of R=d1/d2 is calculated, the lesser bacterial strain of R value is screened.
Embodiment 6: wild type BGL1wtAnd mutant mBGL1Q91C；R601H；R683WThe SDS-PAGE electrophoresis detection of recombinant protein
(1) separation gel for configuring 10mL 10%, uses micropipettor encapsulating into glass plate, directly until from short after mixing
2~3 centimeters stop at the top of glass plate, then seal glue surface with distilled water, and can gently lift gel maker one end and then put down makes glue
Face is smooth, polymerize after 40min to distilled water is abandoned, sucks excessive moisture with filter paper；
(2) the concentration glue for configuring 4mL 5%, is uniformly filled on separation gel, is inserted into the pillar of dimension while answering
It avoids generating bubble, polyase 13 0min waits being gelled solid；
(3) electrophoresis tank is installed, electrophoresis liquid will be filled in slot, volume is preferably greater than the half of electrophoresis sump volume, and the glue made is moved
Enter in electrophoresis tank, carefully takes out sample comb；
(4) successively point sample, point sample amount should not be excessive, and the every hole 15uL is suitable；
(5) 120V is first set when electrophoresis starts and runs glue, voltage is changed to 180V and continues electrophoresis by indicator to concentration glue part,
Purpose band can stop electrophoresis when going to middle position (purpose band corresponds to the respective strap of Maker, would know that in advance)；
(6) gel is carefully peeled, after coomassie brilliant blue R_250 dyes 30min, destainer decolourizes to the shallower albumen one of background
Band is clear；
(7) gel imaging and result is observed.SDS-PAGE protein electrophoresis result is as shown in Figure 1.
Embodiment 7: wild type BGL1wtAnd mutant mBGL1Q91C；R601H；R683WThe trypsase and stomach cardia of recombinant protein
Enzyme Resistance detecting
By wild BGL1wtAnd mBGL1Q91C；R601H；R683WMutation trypsase and pepsin disappear under the conditions of 37 DEG C
Change.15ul is taken out in 0,10,20,30,40,50,60min and 0,20,40,60,80,100,120min respectively and 5ul egg is added
White electrophoretic buffer, which terminates, to be digested and boils 5min immediately, and it is remaining wild then to carry out the detection of SDS-PAGE electrophoresis detection
BGL1wtAnd mBGL1Q91C；R601H；R683WThe protein content of mutant, calculating half-life period evaluate them to trypsase and pepsin
The effect of resistance rationality.As a result as shown in Figure 3 and Figure 4.
Embodiment 8: detection activity rating wild type BGL1wtAnd mutant mQ91C；R601H/R683WThe trypsase of recombinant protein
Resistance
1. by wild BGL1wtAnd mBGL1Q91C；R601H；R683WMutant uses trypsase and pepsin in 37 DEG C of items respectively
The BGL1 of 0.25mg/mL is digested under partwtOr mBGL1Q91C；R601H；R683W60min is handled, boils 5min immediately.Then enzyme activity is carried out
Measurement.Wilder BGL1wtAnd mBGL1Q91C；R601H；R683WMutant respectively through trypsase and pepsin before and after
Remain enzyme activity.
2. enzyme activity determination:
Enzyme activity definition: using 5mmol/L pNPG solution as substrate, under the conditions of pH is 4.5,50 DEG C, it is catalyzed bottom per minute
Enzyme amount needed for object generates the pNP of 1umol is 1 enzyme activity unit (U/ml).
Specific activity of enzyme definition: by the units of the enzyme in every milligram of protein, it is defined as Rate activity (U/mg).
(1) the 0.2M Na for being 4.5 with pH2HPO4- 0.1M citrate buffer solution configures 5mmol/L pNPG solution.
(2) the 0.2M Na of 40ul is added into 96 orifice plates2HPO4- 0.1M citrate buffer solution, adds 75ul's
5mmol/L pNPG solution, 50 DEG C of preheating 5min of metal bath.
(3) the suitably diluted enzyme solution of 10ul, 50 DEG C of reaction 30min are added to 96 orifice plates.Using the enzyme solution of inactivation as control
Group, every group of three repetitions.
(4) the 1M Na of 75ul is added2CO3Solution (enhancing develops the color and terminates reaction).It is stored at room temperature 5min.
(5) light absorption value is measured at OD405.
(6) concentration that pNP is calculated according to the standard curve made calculates enzyme activity further according to vis viva formula.
(7) enzyme activity calculation formula:
Enzyme activityWherein, C is to substitute into the bent pNP concentration (μm ol/mL) of mark；V1 is reaction volume
(mL)；N is extension rate；V2 is enzyme amount used (mL)；T is the enzyme reaction time.
(8) specific activity of enzyme calculation formula:
Specific activity of enzymeWherein, U is enzyme total activity (U/mL)；M is protein by weight (mg) in sample.
Enzyme activity determination standard curve is as shown in Fig. 2, 1M sodium carbonate liquor and 0.1M citric acid solution are mixed with the ratio of 1:2
It is even, as mixed liquor.The pNP solution of configured good 0.01M is diluted 10 times, obtains the pNP titer of 0.001M.According to the form below
Reagent is successively measured, measures OD after mixing405Light absorption value.
The standard curve of table 1:pNP concentration
Measurement result is as shown in Figure 5 and Figure 6.
Enzyme activity determination is the results show that mutant mBGL1Q91C；R601H；R683WThe trypsin-resistant of albumen is wild-type protein
2 times, pepsin resistance is 1.5 times of wild type.Wild type BGL1wtBefore albumen trypsase and pepsin
Enzyme ratio living is 362.3 ± 2.5U/mg, and residual enzyme is 108.7 ± 2.0U/mg than living after trypsase (artificial pancreatic juice) processing,
Residual enzyme is than living for 31.9 ± 2.0U/mg after pepsin (simulated gastric fluid) processing；Mutant mBGL1Q91C；R601H；R683WWithout
It is 385.7 ± 2.5U/mg that albumen trypsase and pepsin preferment ratio, which are lived, after trypsase (artificial pancreatic juice) processing
Residual enzyme is 308.5 ± 2.2U/mg than living, and it is 119.2 ± 2.5 that residual enzyme ratio is living after pepsin (simulated gastric fluid) processing；
Under the conditions of trypsase (artificial pancreatic juice) is existing, mutant mBGL1Q91C；R601H；R683WThe residual enzyme of albumen is than living than wild
Type BGL1 improves 2.8 ± 0.2 times；Under the conditions of pepsin (simulated gastric fluid) is existing, mutant mBGL1Q91C ；R601H；R683WThe residual enzyme of albumen is than living than wild type BGL1wtImprove 3.7 ± 0.3 times.
SEQUENCE LISTING
<110>Ji'nan University
<120>beta-glucosidase that a kind of pair of trypsase stomach function regulating protease resistant improves
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 744
<212> PRT
<213>Trichoderma viride (Trichoderma viride)
<400> 1
Met Arg Tyr Arg Thr Ala Ala Ala Leu Ala Leu Ala Thr Gly Pro Phe
1 5 10 15
Ala Arg Ala Asp Ser His Ser Thr Ser Gly Ala Ser Ala Glu Ala Val
20 25 30
Val Pro Pro Ala Gly Thr Pro Trp Gly Thr Ala Tyr Asp Lys Ala Lys
35 40 45
Ala Ala Leu Ala Lys Leu Asn Leu Gln Asp Lys Val Gly Ile Val Ser
50 55 60
Gly Val Gly Trp Asn Gly Gly Pro Cys Val Gly Asn Thr Ser Pro Ala
65 70 75 80
Ser Lys Ile Ser Tyr Pro Ser Leu Cys Leu Gln Asp Gly Pro Leu Gly
85 90 95
Val Arg Tyr Ser Thr Gly Ser Thr Ala Phe Thr Pro Gly Val Gln Ala
100 105 110
Ala Ser Thr Trp Asp Val Asn Leu Ile Arg Glu Arg Gly Gln Phe Ile
115 120 125
Gly Glu Glu Val Lys Ala Ser Gly Ile His Val Ile Leu Gly Pro Val
130 135 140
Ala Gly Pro Leu Gly Lys Thr Pro Gln Gly Gly Arg Asn Trp Glu Gly
145 150 155 160
Phe Gly Val Asp Pro Tyr Leu Thr Gly Ile Ala Met Gly Gln Thr Ile
165 170 175
Asn Gly Ile Gln Ser Val Gly Val Gln Ala Thr Ala Lys His Tyr Ile
180 185 190
Leu Asn Glu Gln Glu Leu Asn Arg Glu Thr Ile Ser Ser Asn Pro Asp
195 200 205
Asp Arg Thr Leu His Glu Leu Tyr Thr Trp Pro Phe Ala Asp Ala Val
210 215 220
Gln Ala Asn Val Ala Ser Val Met Cys Ser Tyr Asn Lys Val Asn Thr
225 230 235 240
Thr Trp Ala Cys Glu Asp Gln Tyr Thr Leu Gln Thr Val Leu Lys Asp
245 250 255
Gln Leu Gly Phe Pro Gly Tyr Val Met Thr Asp Trp Asn Ala Gln His
260 265 270
Thr Thr Val Gln Ser Ala Asn Ser Gly Leu Asp Met Ser Met Pro Gly
275 280 285
Thr Asp Phe Asn Gly Asn Asn Arg Leu Trp Gly Pro Ala Leu Thr Asn
290 295 300
Ala Val Asn Ser Asn Gln Val Pro Thr Ser Arg Val Asp Asp Met Val
305 310 315 320
Thr Arg Ile Leu Ala Ala Trp Tyr Leu Thr Gly Gln Asp Gln Ala Gly
325 330 335
Tyr Pro Ser Phe Asn Ile Ser Arg Asn Val Gln Gly Asn His Lys Thr
340 345 350
Asn Val Arg Ala Ile Ala Arg Asp Gly Ile Val Leu Leu Lys Asn Asp
355 360 365
Ala Asn Ile Leu Pro Leu Lys Lys Pro Ala Ser Ile Ala Val Val Gly
370 375 380
Ser Ala Ala Ile Ile Gly Asn His Ala Arg Asn Ser Pro Ser Cys Asn
385 390 395 400
Asp Lys Gly Cys Asp Asp Gly Ala Leu Gly Met Gly Trp Gly Ser Gly
405 410 415
Ala Val Asn Tyr Pro Tyr Phe Val Ala Pro Tyr Asp Ala Ile Asn Thr
420 425 430
Arg Ala Ser Ser Gln Gly Thr Gln Val Thr Leu Ser Asn Thr Asp Asn
435 440 445
Thr Ser Ser Gly Ala Ser Ala Ala Arg Gly Lys Asp Val Ala Ile Val
450 455 460
Phe Ile Thr Ala Asp Ser Gly Glu Gly Tyr Ile Thr Val Glu Gly Asn
465 470 475 480
Ala Gly Asp Arg Asn Asn Leu Asp Pro Trp His Asn Gly Asn Ala Leu
485 490 495
Val Gln Ala Val Ala Gly Ala Asn Ser Asn Val Ile Val Val Val His
500 505 510
Ser Val Gly Ala Ile Ile Leu Glu Gln Ile Leu Ala Leu Pro Gln Val
515 520 525
Lys Ala Val Val Trp Ala Gly Leu Pro Ser Gln Glu Ser Gly Asn Ala
530 535 540
Leu Val Asp Val Leu Trp Gly Asp Val Ser Pro Ser Gly Lys Leu Val
545 550 555 560
Tyr Thr Ile Ala Lys Ser Pro Asn Asp Tyr Asn Thr Arg Ile Val Ser
565 570 575
Gly Gly Ser Asp Ser Phe Ser Glu Gly Leu Phe Ile Asp Tyr Lys His
580 585 590
Phe Asp Asp Ala Asn Ile Thr Pro Arg Tyr Glu Phe Gly Tyr Gly Leu
595 600 605
Ser Tyr Thr Lys Phe Asn Tyr Ser Arg Leu Ser Val Leu Ser Thr Ala
610 615 620
Lys Ser Gly Pro Ala Thr Gly Ala Val Val Pro Gly Gly Pro Ser Asp
625 630 635 640
Leu Phe Gln Asn Val Ala Thr Val Thr Val Asp Ile Ala Asn Ser Gly
645 650 655
Gln Val Thr Gly Ala Glu Val Ala Gln Leu Tyr Ile Thr Tyr Pro Ser
660 665 670
Ser Ala Pro Arg Thr Pro Pro Lys Gln Leu Arg Gly Phe Ala Lys Leu
675 680 685
Asn Leu Thr Pro Gly Gln Ser Gly Thr Ala Thr Phe Asn Ile Arg Arg
690 695 700
Arg Asp Leu Ser Tyr Trp Asp Thr Ala Ser Gln Lys Trp Val Val Pro
705 710 715 720
Ser Gly Ser Phe Gly Ile Ser Val Gly Ala Ser Ser Arg Asp Ile Arg
725 730 735
Leu Thr Ser Thr Leu Ser Val Ala
740
<210> 2
<211> 744
<212> PRT
<213>improved amino acid sequence
<400> 2
Met Arg Tyr Arg Thr Ala Ala Ala Leu Ala Leu Ala Thr Gly Pro Phe
1 5 10 15
Ala Arg Ala Asp Ser His Ser Thr Ser Gly Ala Ser Ala Glu Ala Val
20 25 30
Val Pro Pro Ala Gly Thr Pro Trp Gly Thr Ala Tyr Asp Lys Ala Lys
35 40 45
Ala Ala Leu Ala Lys Leu Asn Leu Gln Asp Lys Val Gly Ile Val Ser
50 55 60
Gly Val Gly Trp Asn Gly Gly Pro Cys Val Gly Asn Thr Ser Pro Ala
65 70 75 80
Ser Lys Ile Ser Tyr Pro Ser Leu Cys Leu Cys Asp Gly Pro Leu Gly
85 90 95
Val Arg Tyr Ser Thr Gly Ser Thr Ala Phe Thr Pro Gly Val Gln Ala
100 105 110
Ala Ser Thr Trp Asp Val Asn Leu Ile Arg Glu Arg Gly Gln Phe Ile
115 120 125
Gly Glu Glu Val Lys Ala Ser Gly Ile His Val Ile Leu Gly Pro Val
130 135 140
Ala Gly Pro Leu Gly Lys Thr Pro Gln Gly Gly Arg Asn Trp Glu Gly
145 150 155 160
Phe Gly Val Asp Pro Tyr Leu Thr Gly Ile Ala Met Gly Gln Thr Ile
165 170 175
Asn Gly Ile Gln Ser Val Gly Val Gln Ala Thr Ala Lys His Tyr Ile
180 185 190
Leu Asn Glu Gln Glu Leu Asn Arg Glu Thr Ile Ser Ser Asn Pro Asp
195 200 205
Asp Arg Thr Leu His Glu Leu Tyr Thr Trp Pro Phe Ala Asp Ala Val
210 215 220
Gln Ala Asn Val Ala Ser Val Met Cys Ser Tyr Asn Lys Val Asn Thr
225 230 235 240
Thr Trp Ala Cys Glu Asp Gln Tyr Thr Leu Gln Thr Val Leu Lys Asp
245 250 255
Gln Leu Gly Phe Pro Gly Tyr Val Met Thr Asp Trp Asn Ala Gln His
260 265 270
Thr Thr Val Gln Ser Ala Asn Ser Gly Leu Asp Met Ser Met Pro Gly
275 280 285
Thr Asp Phe Asn Gly Asn Asn Arg Leu Trp Gly Pro Ala Leu Thr Asn
290 295 300
Ala Val Asn Ser Asn Gln Val Pro Thr Ser Arg Val Asp Asp Met Val
305 310 315 320
Thr Arg Ile Leu Ala Ala Trp Tyr Leu Thr Gly Gln Asp Gln Ala Gly
325 330 335
Tyr Pro Ser Phe Asn Ile Ser Arg Asn Val Gln Gly Asn His Lys Thr
340 345 350
Asn Val Arg Ala Ile Ala Arg Asp Gly Ile Val Leu Leu Lys Asn Asp
355 360 365
Ala Asn Ile Leu Pro Leu Lys Lys Pro Ala Ser Ile Ala Val Val Gly
370 375 380
Ser Ala Ala Ile Ile Gly Asn His Ala Arg Asn Ser Pro Ser Cys Asn
385 390 395 400
Asp Lys Gly Cys Asp Asp Gly Ala Leu Gly Met Gly Trp Gly Ser Gly
405 410 415
Ala Val Asn Tyr Pro Tyr Phe Val Ala Pro Tyr Asp Ala Ile Asn Thr
420 425 430
Arg Ala Ser Ser Gln Gly Thr Gln Val Thr Leu Ser Asn Thr Asp Asn
435 440 445
Thr Ser Ser Gly Ala Ser Ala Ala Arg Gly Lys Asp Val Ala Ile Val
450 455 460
Phe Ile Thr Ala Asp Ser Gly Glu Gly Tyr Ile Thr Val Glu Gly Asn
465 470 475 480
Ala Gly Asp Arg Asn Asn Leu Asp Pro Trp His Asn Gly Asn Ala Leu
485 490 495
Val Gln Ala Val Ala Gly Ala Asn Ser Asn Val Ile Val Val Val His
500 505 510
Ser Val Gly Ala Ile Ile Leu Glu Gln Ile Leu Ala Leu Pro Gln Val
515 520 525
Lys Ala Val Val Trp Ala Gly Leu Pro Ser Gln Glu Ser Gly Asn Ala
530 535 540
Leu Val Asp Val Leu Trp Gly Asp Val Ser Pro Ser Gly Lys Leu Val
545 550 555 560
Tyr Thr Ile Ala Lys Ser Pro Asn Asp Tyr Asn Thr Arg Ile Val Ser
565 570 575
Gly Gly Ser Asp Ser Phe Ser Glu Gly Leu Phe Ile Asp Tyr Lys His
580 585 590
Phe Asp Asp Ala Asn Ile Thr Pro His Tyr Glu Phe Gly Tyr Gly Leu
595 600 605
Ser Tyr Thr Lys Phe Asn Tyr Ser Arg Leu Ser Val Leu Ser Thr Ala
610 615 620
Lys Ser Gly Pro Ala Thr Gly Ala Val Val Pro Gly Gly Pro Ser Asp
625 630 635 640
Leu Phe Gln Asn Val Ala Thr Val Thr Val Asp Ile Ala Asn Ser Gly
645 650 655
Gln Val Thr Gly Ala Glu Val Ala Gln Leu Tyr Ile Thr Tyr Pro Ser
660 665 670
Ser Ala Pro Arg Thr Pro Pro Lys Gln Leu Trp Gly Phe Ala Lys Leu
675 680 685
Asn Leu Thr Pro Gly Gln Ser Gly Thr Ala Thr Phe Asn Ile Arg Arg
690 695 700
Arg Asp Leu Ser Tyr Trp Asp Thr Ala Ser Gln Lys Trp Val Val Pro
705 710 715 720
Ser Gly Ser Phe Gly Ile Ser Val Gly Ala Ser Ser Arg Asp Ile Arg
725 730 735
Leu Thr Ser Thr Leu Ser Val Ala
740
<210> 3
<211> 2235
<212> DNA
<213>improved nucleic acid sequence
<400> 3
atgcgttacc gaacagcagc tgcgctggca cttgccactg ggccctttgc tagggcagac 60
agtcactcaa catcgggggc ctcggctgag gcagttgtac ctcctgcagg gactccatgg 120
ggaaccgcgt acgacaaggc gaaggccgca ttggcaaagc tcaatctcca agataaggtc 180
ggcatcgtga gcggtgtcgg ctggaacggc ggtccttgcg ttggaaacac atctccggcc 240
tccaagatca gctatccatc gctatgcctt caagacggac ccctcggtgt tcaatactcg 300
acaggcagca cagcctttac gccgggcgtt caagcggcct cgacgtggga tgtcaatttg 360
atccgcgaac gtggacagtt catcggtgag gaggtgaagg cctcggggat tcatgtcata 420
cttggtcctg tggctgggcc gctgggaaag actccgcagg gcggtcgcaa ctgggagggc 480
ttcggtgtcg atccatatct cacgggcatt gccatgggtc aaaccatcaa cggcatccag 540
tcggtaggcg tgcaggcgac agcgaagcac tatatcctca acgagcagga gctcaatcga 600
gaaaccattt cgagcaaccc agatgaccga actctccatg agctgtatac ttggccattt 660
gccgacgcgg ttcaggccaa tgtcgcttct gtcatgtgct cgtacaacaa ggtcaatacc 720
acctgggcct gcgaggatca gtacacgctg cagactgtgc tgaaagacca gctggggttc 780
ccaggctatg tcatgacgga ctggaacgca cagcacacga ctgtccaaag cgcgaattct 840
gggcttgaca tgtcaatgcc tggcacagac ttcaacggta acaatcggct ctggggtcca 900
gctctcacca atgcggtaaa tagcaatcag gtccccacga gcagagtcga cgatatggtg 960
actcgtatcc tcgccgcatg gtacttgaca ggccaggacc aggcaggcta tccgtcgttc 1020
aacatcagca gaaatgttca aggaaaccac aagaccaatg tcagggcaat tgccagggac 1080
ggcatcgttc tgctcaagaa tgacgccaac atcctgccgc tcaagaagcc cgctagcatt 1140
gccgtcgttg gatctgccgc aatcattggt aaccacgcca gaaactcgcc ctcgtgcaac 1200
gacaaaggct gcgacgacgg ggccttgggc atgggttggg gttccggcgc cgtcaactat 1260
ccgtacttcg tcgcgcccta cgatgccatc aataccagag cgtcttcgca gggcacccag 1320
gttaccttga gcaacaccga caacacgtcc tcaggcgcat ctgcagcaag aggaaaggac 1380
gtcgccatcg tcttcatcac cgccgactcg ggtgaaggct acatcaccgt ggagggcaac 1440
gcgggcgatc gcaacaacct ggatccgtgg cacaacggca atgccctggt ccaggcggtg 1500
gccggtgcca acagcaacgt cattgttgtt gtccactccg ttggcgccat cattctggag 1560
cagattcttg ctcttccgca ggtcaaggcc gttgtctggg cgggtcttcc ttctcaggag 1620
agcggcaatg cgctcgtcga cgtgctgtgg ggagatgtca gcccttctgg caagctggtg 1680
tacaccattg cgaagagccc caatgactat aacactcgca tcgtttccgg cggcagtgac 1740
agcttcagcg agggatcgtt catcgactat aagcacttcg acgacgccaa tatcacgccg 1800
cggtacgagt tcggctatgg actgtcttac accaagttca actactcacg cctctccgtc 1860
ttgtcgaccg ccaagtctgg tcctgcgact ggggccgttg tgccgggagg cccgagtgat 1920
ctgttccaga atgtcgcgac agtcaccgtt gacatcgcaa actctggcca agtgactggt 1980
gccgaggtag cccagctgta catcacctac ccatcttcag cacccccgac ccctccgaag 2040
cagctgcgag gctttgccaa gctgaacctc acgcctggtc agagcggaac agcaacgttc 2100
aacatccgac gacgagatct cagctactgg gacacggctt cgcagaaatg ggtggtgccg 2160
tcggggtcgt ttggcatcag cgtgggagcg agcagccggg atatcaggct gacgagcact 2220
ctgtcggtag cgtag 2235
Claims (7)
1. the beta-glucosidase that a kind of pair of trypsase stomach function regulating protease resistant improves, it is characterised in that: it is by amino acid
Sequence is that manufacturing in the beta-glucosidase of Trichoderma viride (Trichoderma viride) for SEQ ID NO.1 is multiple
Amino acid substitution and generate, to the stronger enzyme of trypsase stomach function regulating protease resistant, the amino acid substitution is the 91st,
601 and 683 substitutions.
2. the beta-glucosidase according to claim 1 improved to trypsase stomach function regulating protease resistant, feature exist
In: the amino acid substitution at the 91st is to replace glutamine with cysteine；It is to use group in 601 amino acid substitutions
Replace arginine；It is to replace arginine with tryptophan in the 683rd amino acid substitution；Fixedpoint mutation modified β-the Portugal
The amino acid sequence of polyglycoside enzyme is SEQ ID NO.2.
3. a kind of DNA molecular, it is characterised in that: it encodes and as claimed in claim 2 mentions to trypsase stomach function regulating protease resistant
High beta-glucosidase.
4. DNA molecular according to claim 3, it is characterised in that: its nucleotides sequence is classified as SEQ ID NO.3.
5. a kind of carrier, it is characterised in that: it contains DNA molecular described in claim 3 or 4.
6. a kind of host cell, it is characterised in that: it contains DNA molecular described in claim 3 or 4, or wants containing having the right
Carrier described in asking 5.
7. a kind of production of the beta-glucosidase according to claim 1 improved to trypsase stomach function regulating protease resistant
Method, which is characterized in that the described method includes: under conditions of being suitable for beta-glucosidase expression of enzymes described in culture claim 6
Host cell, and separate from culture medium the beta-glucosidase.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111876399A (en) * | 2020-07-13 | 2020-11-03 | 中国水产科学研究院黄海水产研究所 | Arctic-pole-derived beta-glucosidase gene, and encoded protein and application thereof |
| CN114774385A (en) * | 2022-03-11 | 2022-07-22 | 暨南大学 | Zearalenone hydrolase with improved resistance to trypsin and pepsin |
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| CN1816631A (en) * | 2003-05-02 | 2006-08-09 | 诺维信股份有限公司 | Variants of beta-glucosidases |
| CN102712916A (en) * | 2009-09-18 | 2012-10-03 | 诺维信股份有限公司 | Polypeptides having beta-glucosidase activity and polynucleotides encoding same |
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2018
- 2018-10-26 CN CN201811258430.8A patent/CN109355274B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1816631A (en) * | 2003-05-02 | 2006-08-09 | 诺维信股份有限公司 | Variants of beta-glucosidases |
| CN102712916A (en) * | 2009-09-18 | 2012-10-03 | 诺维信股份有限公司 | Polypeptides having beta-glucosidase activity and polynucleotides encoding same |
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| LIU,Z.ET AL.: "ACCESSION NO：FJ882071，Trichoderma viride strain AS 3.3711 beta-D-glucoside glucohydrolase I (bgl1) mRNA, complete cds", 《GENBANK》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111876399A (en) * | 2020-07-13 | 2020-11-03 | 中国水产科学研究院黄海水产研究所 | Arctic-pole-derived beta-glucosidase gene, and encoded protein and application thereof |
| CN111876399B (en) * | 2020-07-13 | 2022-03-01 | 中国水产科学研究院黄海水产研究所 | Arctic-pole-derived beta-glucosidase gene, and encoded protein and application thereof |
| CN114774385A (en) * | 2022-03-11 | 2022-07-22 | 暨南大学 | Zearalenone hydrolase with improved resistance to trypsin and pepsin |
| CN114774385B (en) * | 2022-03-11 | 2024-02-02 | 暨南大学 | Trypsin-like enzyme and pepsin resistance improved zearalenone hydrolase |
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| Publication number | Publication date |
|---|---|
| CN109355274B (en) | 2021-07-27 |
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