CN107083374B - The beta-mannase enzyme mutant and its encoding gene and application that enzymatic activity improves - Google Patents
The beta-mannase enzyme mutant and its encoding gene and application that enzymatic activity improves Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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Abstract
The beta-mannase enzyme mutant and its encoding gene that are improved the present invention provides enzymatic activity and application.The present invention is with can be in pichia pastoris yeastPichia pastorisBased on the middle 'beta '-mannase for carrying out high efficient expression, the enzyme gene is mutated using fallibility PCR method, direct mutation is picked out by high-throughput screening method again, by two-wheeled fallibility PCR, finally obtain the mutant that 4 enzyme activity significantly improve, respectively MAN-M01, MAN-M02, MAN-M03 and MAN-M04, its enzyme activity is compared with original 'beta '-mannase, enzyme activity has been respectively increased 40%, 58%, 80% and 64%, present invention obtains the 'beta '-mannases that enzymatic activity greatly improves, it is advantageously implemented its application in business, to reduce production cost.
Description
Technical field
The invention belongs to genetic engineering and enzyme engineering field, the 'beta '-mannase that particular content is related to enzymatic activity raising is prominent
Variant and its encoding gene and application.
Background technique
Mannosan is the second largest component of hemicellulose, is widely present in nature, it is various plants cell wall
Main constituents.Mannosan content in many vegetality feedstuffs is very high, it improves the viscosity of feed, influences
Absorption and utilization of the animal to nutriment, is a kind of anti-nutritional factors.'beta '-mannase is hydrolysis with β-Isosorbide-5-Nitrae-D- pyrrole
Mannose of muttering is the endo hydrolysis enzyme of main chain, and substrate specificity is manna oligosacchride and mannocarolose.'beta '-mannase is in feed work
A kind of feed addictive is used as in industry, the mannosan in the diet that can effectively degrade improves the digestibility of feed, improves animal
Growth performance eliminates the anti-oxidant action of mannosan.'beta '-mannase is widespread in nature, and microorganism is β-
The main source of mannase.Currently, the strain for being applied to industrial production 'beta '-mannase is also mostly bacillus, song
Mould, yeast etc..The beta-mannase gene of wild type is already available to higher enzyme activity after being overexpressed, and improves existing
The enzyme activity of some 'beta '-mannases and the performance of enzyme have the performance of the control effect of its production cost most important
Effect.It is to improve beta-mannase production of enzyme and performance using the technological transformations beta-mannase gene such as protein engineering
Important means.
Fallibility PCR (error-prone PCR) technology is to react item by changing when carrying out PCR amplification using Taq enzyme
Part (concentration of 4 kinds of dNTP in such as change system), improves the frequency of mutation of Taq enzyme, thus into target gene with certain frequency
Rate introduces mutation, then screens mutant as needed.And continuous error-prone PCR (sequential error prone PCR) plan
Summary may obtain better mutation effect, i.e., the direct mutation gene obtained last PCR amplification is as PCR amplification next time
Template, continuously repeatedly carry out random mutagenesis, accumulate the forward mutation assay expanded each time and generate important beneficial
Mutation.
Summary of the invention
The beta-mannase enzyme mutant and its encoding gene that are improved the object of the present invention is to provide enzymatic activity and application.
The present invention improves its enzymatic activity by random mutation beta-mannase enzyme amino acid sequence, it is a further object of the present invention to provide
The recombinant vector of the beta-mannase gene obtained comprising above-mentioned mutation.The beta-mannase gene that mutation obtains is inserted
Enter between the suitable restriction enzyme site of expression vector, makes its nucleotide sequence is operable to be connected with expression regulation sequence
It connects, obtains expression of recombinant yeast plasmid.It is described the present invention also provides the recombinant bacterial strain comprising above-mentioned beta-mannase gene
Bacterial strain is E.coli and yeast.
It is a further object of the present invention to provide above-mentioned beta-mannase enzyme mutants in preparing animal feed additive
Application.The animal is pig, chicken, duck and ox, and the feed is corn-soybean meal diet.
For achieving the above object, the present invention is achieved by the following scheme:
The present invention provides beta-mannase enzyme mutant MAN-M01, the beta-mannase enzyme mutant MAN-M01
Amino acid sequence as shown in SEQ ID NO:3, the mutant MAN-M01 is SEQ ID NO:1 by amino acid sequence
306th amino acids of 'beta '-mannase become proline from serine and obtain.
The present invention provides the encoding genes of the beta-mannase enzyme mutant MAN-M01.
The present invention provides beta-mannase enzyme mutant MAN-M02, the beta-mannase enzyme mutant MAN-M02
Amino acid sequence as shown in SEQ ID NO:4, the mutant MAN-M02 is SEQ ID NO:1 by amino acid sequence
191st amino acids of 'beta '-mannase, which become arginine, the 306th amino acids from cysteine, becomes dried meat ammonia from serine
Acid obtains.
The present invention provides the encoding genes of the beta-mannase enzyme mutant MAN-M02.
The present invention provides beta-mannase enzyme mutant MAN-M03, the beta-mannase enzyme mutant MAN-M03
Amino acid sequence as shown in SEQ ID NO:5, the mutant MAN-M03 is SEQ ID NO:1 by amino acid sequence
292nd amino acids of 'beta '-mannase, which become glutamine, the 306th amino acids from glutamic acid, becomes dried meat ammonia from serine
Acid obtains.
The present invention provides the encoding genes of the beta-mannase enzyme mutant MAN-M03.
The present invention provides beta-mannase enzyme mutant MAN-M04, the beta-mannase enzyme mutant MAN-M04
Amino acid sequence as shown in SEQ ID NO:6, the mutant MAN-M04 is SEQ ID NO:1 by amino acid sequence
337th amino acids of 'beta '-mannase, which become asparagine, the 306th amino acids from aspartic acid, becomes dried meat from serine
Propylhomoserin obtains.
The present invention provides the encoding genes of the beta-mannase enzyme mutant MAN-M04.
The present invention provides the encoding gene containing the MAN-M01 or the coding bases containing the MAN-M02
The expression vector of the encoding gene of the encoding gene or MAN-M04 containing described in of cause or the MAN-M03 containing described in.
The present invention provides beta-mannase enzyme mutant MAN-M01, MAN-M02, MAN-M03 or MAN-M04 to be used for
Prepare the application in animal feed additive.
Compared with prior art, advantages of the present invention and have the technical effect that the present invention passes through to from aspergillus niger
The 'beta '-mannase of (Aspergillus niger) using fallibility PCR method to 'beta '-mannase carbohydrase MAN-1 gene into
Row transformation, reuses high-throughput screening method and picks out direct mutation, by two-wheeled fallibility PCR, finally obtains 4 enzyme activity and significantly mentions
High mutant, respectively MAN-M01, MAN-M02, MAN-M03 and MAN-M04, enzyme activity and original 'beta '-mannase phase
Than enzyme activity has been respectively increased 40%, 58%, 80% and 64%, the 'beta '-mannase carbohydrase mutant that the present invention obtains and open country
Raw type is compared, and the enzymatic activity of mutant is significantly improved, and is advantageously implemented its application in business, is produced into reduce
This.
Detailed description of the invention
Fig. 1 is beta-mannase enzyme mutant MAN-M01 and wild-type amino acid sequence comparison result；
Fig. 2 is beta-mannase enzyme mutant MAN-M02 and wild-type amino acid sequence comparison result；
Fig. 3 is beta-mannase enzyme mutant MAN-M03 and wild-type amino acid sequence comparison result；
Fig. 4 is beta-mannase enzyme mutant MAN-M04 and wild-type amino acid sequence comparison result；
Fig. 5 is the enzyme activity of beta-mannase enzyme mutant MAN-M01, MAN-M02, MAN-M03 and MAN-M04 and wild type
Comparative measurements result.
Specific embodiment
Technical solution of the present invention is further described in detail in the following with reference to the drawings and specific embodiments.Below in conjunction with
The present invention will be further described for embodiment, it should be pointed out that the present embodiment for explaining only the invention, rather than to this hair
The limitation of bright range.
Embodiment 1: fallibility PCR (error-prone PCR) method constructs 'beta '-mannase MAN-1 mutated library
'beta '-mannase MAN-1 from aspergillus niger is made of 362 amino acid (see SEQ ID NO:1).According to
Reported MAN-1 gene sequence information, full genome synthetic method have synthesized 'beta '-mannase MAN-1 complete genome sequence (such as
Shown in Genbank ID KM213619.1).The gene both ends of synthesis also have EcoR I and Not I restriction enzyme site, in order to
Expression vector is attached.Use MAN-1 (nucleotide sequence is shown in SEQ ID NO:2) for template amplification 'beta '-mannase carbohydrase
MAN-1 gene is randomly incorporated into mutation using GeneMorph II random mutation PCR kit (Stratagene).
The primer are as follows:
5′-GCGCGAATTCCTGCCGAAAGCCTCCCCTGCACCG-3 ' (SEQ ID NO:7)；
5′-TAAAGCGGCCGCTTAGGCGCTATCAATAGCAGCAAC-3 ' (SEQ ID NO:8)；Distinguish at underscore
For EcoR I and Not I restriction enzyme site.
Reaction condition are as follows: 94 DEG C of initial denaturations 10min, 94 DEG C of denaturation 60s, 60s and 72 DEG C of extension 1min20s of 58 DEG C of annealing,
Totally 30 circulations.After 1% agarose electrophoresis, target gene fragment is recycled using plastic recovery kit.
By target fragment with EcoR I and Not I it is double digested after, pET 21a (+) carrier of digestion identical as process
(ampicillin resistance) is attached reaction with Ligase.The segment connected is converted to e. coli bl21-DE3, coating
LB plate containing ampicillin, 37 DEG C of inversion cultures, after there is transformant on plate, picking monoclonal to 96 orifice plates,
Contain 150uL LB culture medium (containing 1mM IPTG, 50ng/mL ampicillin) in every hole, while the inoculation of each orifice plate is wild
Orifice plate is placed in -20 DEG C as control, 30 DEG C of 220rpm shake culture 12h by type gene expression bacterial strain, and multigelation broken wall obtains
Obtain the crude enzyme liquid containing 'beta '-mannase.It takes 20uL crude enzyme liquid to be added to 96 new orifice plates respectively with the volley of rifle fire, 20uL β-is added
After mannosan zymolyte (carob) reaction, enzyme activity is measured with DNS method.
The bacterial strain for taking enzyme activity higher than wild type MAN-1 carries out repeating screening verification into 96 new well culture plates.Finally
1 mutant is screened, is MAN-M01, enzyme activity is higher than wild type control, and picking monoclonal send gene sequencing company to be sequenced.
Sequencing result is shown, as shown in Figure 1, epicycle fallibility PCR obtains the mutant an of simple point mutation containing S306P
MAN-M01 (its amino acid sequence is SEQ ID NO:3).
(the 306th amino acids become proline, corresponding DNA from serine to MAN-M01:Mutation:306S → P
Sequence becomes CCA from TCC).
Embodiment 2: the second takes turns the building of the construction and screening mutated library of fallibility PCR mutated library
The mutant MAN-M01 that first round fallibility PCR method is screened extracts the mould that plasmid makees the second wheel fallibility PCR
Plate, the building process of mutated library, the primer used, PCR reaction condition, with embodiment 1.It is same to obtain by above procedure
A large amount of mutant gene segment.The mutant that building obtains is transferred to E. coli expression strains BL21-DE3, screening is high
With MAN-M01 to compare when active direct mutation, remaining operation is same as Example 2, the bacterium for taking activity higher than saltant type MAN-M01
Strain carries out repeating screening into 96 new well culture plates.Screen 3 enzymatic activitys raising mutant, be named as MAN-M02,
MAN-M03 and MAN-M04.Picking monoclonal send gene sequencing company to be sequenced.
Sequencing result is shown, as shown in Fig. 2, epicycle fallibility PCR obtains the mutant of two point mutation of S306P and C191R
MAN-M02, amino acid sequence are SEQ ID NO:4.
(the 191st amino acids become arginine from cysteine to MAN-M02:Mutation:191C → R, corresponding
DNA sequence dna becomes AGA from TGT)；
(the 306th amino acids become proline from serine to Mutation:306S → P, and corresponding DNA sequence dna is by TCC
Become CCA).
As shown in figure 3, epicycle fallibility PCR obtains the mutant MAN-M03 of two point mutation of S306P and E292Q, amino
Acid sequence is SEQ ID NO:5.
(the 292nd amino acids become glutamine from glutamic acid to MAN-M03:Mutation:292E → Q, corresponding
DNA sequence dna becomes CAA from GAA)；
(the 306th amino acids become proline from serine to Mutation:306S → P, and corresponding DNA sequence dna is by TCC
Become CCA).
As shown in figure 4, epicycle fallibility PCR obtains the mutant MAN-M04 of two point mutation of S306P and D337N, amino
Acid sequence is SEQ ID NO:6.
(the 337th amino acids become asparagine from aspartic acid to MAN-M04:Mutation:337D → N, corresponding
DNA sequence dna becomes GGT from GAT)；
(the 306th amino acids become proline from serine to Mutation:306S → P, and corresponding DNA sequence dna is by TCC
Become CCA).
Embodiment 3: the building of pichia pastoris engineered strain
Using primer described in embodiment 1, the mutant obtained using embodiment 1 and embodiment 2 carries out PCR expansion as template
Increase, has obtained the beta-mannase enzyme mutant gene that 4 both ends have EcoR I and Not I restriction enzyme site.PCR reaction condition
Are as follows: 94 DEG C of denaturation 5min；94 DEG C of denaturation 30s, 56 DEG C of renaturation 30s, 72 DEG C of extension 1min20s, 30 circulations, 72 DEG C extend
10min。
The beta-mannase enzyme mutant gene segment that above-mentioned clone is obtained and original beta-mannase gene MAN-1
Genetic fragment is connected through restriction enzyme EcoR I and Not I double digestion with the pPIC9k carrier for also passing through double digestion,
Construction of expression vector pPIC9K-MAN-M01, pPIC9K-MAN-M02, pPIC9K-MAN-M03, pPIC9K-MAN-M04 and
pPIC9K-MAN-1。
The above expression vector is linearized with Sac I, linearized fragment fragment purification kit (TaKaRa
MiniBEST DNA Fragment Purifibation Kit) after purified pool, passes through electrotransformation method and convert Pichia pastoris
GS115 is coated with MD plate.The bacterium colony grown on MD plate is applied to concentration and successively gradually rises (1mg/mL, 2mg/
ML, 4mg/mL, 8mg/mL) Geneticin YPD plate on screen multicopy positive transformant, obtain Pichia pastoris recombination
Bacterial strain.
By the transformant of 5 genes be respectively designated as Pichia pastoris MAN-M01 (Pichia pastoris MAN-M01),
Pichia pastoris MAN-M02 (Pichia pastoris MAN-M02), Pichia pastoris MAN-M03 (Pichia pastoris MAN-
M03), Pichia pastoris MAN-M04 (Pichia pastoris MAN-M04) and Pichia pastoris MAN-1 (Pichia
PastorisMAN-1), the transformant for picking them separately each gene is transferred in BMGY culture medium, and 30 DEG C, 220rpm shaken cultivation
After 18h, centrifugation obtains thallus, and appropriate thallus is transferred in BMMY culture medium, and cell concentration is made to reach OD600=1, and 30 DEG C,
220rpm continues shaken cultivation, per the methanol for adding volume of culture 1% for 24 hours.After inducing expression 4d, medium centrifugal is obtained
Clearly, supernatant is subjected to beta-mannase enzyme activity determination.
'beta '-mannase enzyme activity detection method:
The active measurement of 'beta '-mannase: drawing 2mL by appropriate diluted enzyme solution, be added in scale test tube, then
The mannan solution that 2mL concentration is 0.6% (w/v) is added, shakes 3s, incubates 30min at 50 DEG C.5mL DNS reagent is added,
Uniformly, boiling water bath heats 5min, is cooled to room temperature for concussion, adds water constant volume to 25mL, absorbance is measured at 540nm.
DNS preparation of reagents method: weighing 3,5- dinitrosalicylic acid 3.15g, adds water 500mL, after stirring water-bath to 45 DEG C,
It is slowly added to the sodium hydroxide solution of 100mL 0.2g/mL, while being stirred continuously and (being noted: being added until solution is as clear as crystal
During sodium hydroxide, solution temperature does not exceed 48 DEG C).It is gradually added Rochelle salt 91.0g, phenol 2.50g again
With anhydrous sodium sulfite 2.50g.Water supplement 300mL simultaneously, is stirred continuously, until the substance of addition is completely dissolved.It is cooled to room
Wen Hou is settled to 1000ml with deionized water.Solution answers as clear as crystal (as muddiness should be prepared again) at this time, is stored in brown bottle
In and be uniformly mixed, be kept in dark place.Standard curve is demarcated after storing 7 days at room temperature, validity period is 6 months, is marked again within every 2 months
Determine curve.
Enzyme-activity unit and definition: poly- from the sweet dew that concentration is 3mg/ml per minute under conditions of 37 DEG C, pH value are 5.5
Enzyme amount required for degradation release 1umol reduced sugar is an enzyme activity unit U in sugar juice.
Enzyme activity determination result: as shown in Figure 5.Measurement obtains Pichia pastoris MAN-1 fermented supernatant fluid according to the method described above
Enzyme activity is 198U/mL, and Pichia pastoris MAN-M01, Pichia pastoris MAN-M02, Pichia pastoris MAN-M03 and Pichia pastoris MAN-
The enzyme activity of M04 fermented supernatant fluid is respectively 278U/mL, 312U/mL, 356U/mL and 325U/mL, and enzyme activity level is respectively increased
40%, 58%, 80% and 64%.
Embodiment 4: the cultivation application experiment of 'beta '-mannase
4.1 experimental design
Experiment uses single factor test completely randomized design, selection weight is close from 150 AA meat cocks 36, and random point
It is handled at 6, each processing sets 6 repetitions, 1 chicken of each repetition.Positive control group, negative control group and 4 experimental groups are set respectively.
Experimental design is shown in Table 1, and each experimental group adds 'beta '-mannase provided by the present invention, the daily ration composition of each experimental group and nutrition
It is horizontal that see Table 2 for details.Animal is freely eaten and drinking-water；Routinely immune programme is immunized, and records Broiler chicks morbidity and death
Situation.
1. experimental design of table
| Grouping | Daily ration |
| Positive control group | Corn bean pulp type basal diet |
| Negative control group | 50Kcal/Kg energy is reduced on the basis of corn bean pulp type basal diet. |
| Test 1 group | Negative control group daily ration+'beta '-mannase 250U/Kg |
| Test 2 groups | Negative control group daily ration+'beta '-mannase 500U/Kg |
| Test 3 groups | Negative control group daily ration+'beta '-mannase 1000U/Kg |
| Test 4 groups | Negative control group daily ration+'beta '-mannase 1500U/Kg |
The composition and trophic level of each experimental group basal diet of table 2.
4.2 sample treatments and testing index
Excrement method is received using complete, daily defecation sample is placed on freezen protective in -20 DEG C of refrigerator-freezer, will be received after experiment periods
After all 4 days excrement samples of collection mix, takes the 2/5 of its total fresh excrement sample to be placed on 70 DEG C and be made air-dry sample, dry matter to be measured, energy
And crude protein.
The measurement of energy uses kjeldahl apparatus using oxygen bomb calorimeter measurement, the measurement of crude protein.
4.3 results and analysis
3. 'beta '-mannase of table to Broiler chicks be averaged feed intake, dry matter, energy and apparent digestibility of crude protein shadow
It rings
Addition various dose 'beta '-mannase apparently disappears to be averaged feed intake, dry matter, energy and crude protein of Broiler chicks
The influence of rate is shown in Table 3.From table 3 it can be seen that reducing 50Kcal/Kg metabolizable energy value corn-soybean meal compared with negative control group
The 'beta '-mannase (1 to 4 group of experiment) of 250U/Kg, 500U/Kg, 1000U/Kg, 1500U/Kg are added in type daily ration respectively
Diet dry matter, energy apparent digestibility can be improved in various degree, wherein the 'beta '-mannase group of addition 500U/Kg is (real
Test 2 groups) diet dry matter, energy apparent digestibility compared with negative control group (reduce 50Kcal/Kg energy bins) improve 2.23% (P <
0.05), 2.18% (P < 0.05), calibration control group improve 1.77% and 1.15%.Add various dose 'beta '-mannase pair
Broiler chicks crude protein metabolic rate and average feed intake do not influence (P > 0.05).
The 'beta '-mannase of various dose is added in this experiment in Broiler chicks corn-soybean meal diet, studies β-sweet dew
Dextranase is averaged feed intake to Broiler chicks, and the influence of dry matter, energy and apparent digestibility of crude protein inquires into it in Broiler chicks day
Function and effect in grain.The experimental results showed that adding various dose in the corn-soybean meal diet for reducing 50Kcal/kg energy
'beta '-mannase can improve the dry matter of feed, energy apparent digestibility in various degree, to improve the utilization of feed
Rate reduces feeding cost.
The present embodiment 4 is for the ease of embodying the application of 'beta '-mannase of the present invention, however it is not limited to which broiler chicken is answered
With can be used for the feeding of other fowl poultry kinds because the 'beta '-mannase can be added in basal diet.Usually may be used
To be added in its mixed feed in the breeding process such as pig, rabbit.
For piglet, adding mannase in its basal diet can effectively improve the production performance of piglet,
It shows as improving the daily gain of piglet, reduce feed-weight ratio, from the point of view of biochemical indicator, mannase can improve the immune of piglet
Function.For growing and fattening pigs, adding mannase in its basal diet can also be improved its production performance.In recent years, as
One kind of non-starch polysaccharide enzyme, mannase also starts to be applied to aquaculture, studies have shown that adding in aquatic feeds compound
The digestive utilization ratio of feed can be improved in mannase, enhances nonspecific immunity sexuality, and then promote fish and shrimps etc.
The growth of aquatic livestock.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although referring to aforementioned reality
Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these are modified or replace
It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.
SEQUENCE LISTING
<110>Qingdao red cherry Bioisystech Co., Ltd
<120>the beta-mannase enzyme mutant and its encoding gene and application that enzymatic activity improves
<130>
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 362
<212> PRT
<213>artificial sequence
<400> 1
Leu Pro Lys Ala Ser Pro Ala Pro Ser Thr Ser Ser Ser Ala Ala Ser
1 5 10 15
Thr Ser Phe Ala Ser Thr Ser Gly Leu Gln Phe Thr Ile Asp Gly Glu
20 25 30
Thr Gly Tyr Phe Ala Gly Thr Asn Ser Tyr Trp Ile Gly Phe Leu Thr
35 40 45
Asp Asn Ala Asp Val Asp Leu Val Met Gly His Leu Lys Ser Ser Gly
50 55 60
Leu Lys Ile Leu Arg Val Trp Gly Phe Asn Asp Val Thr Ser Gln Pro
65 70 75 80
Ser Ser Gly Thr Val Trp Tyr Gln Leu His Gln Asp Gly Lys Ser Thr
85 90 95
Ile Asn Thr Gly Ala Asp Gly Leu Gln Arg Leu Asp Tyr Val Val Ser
100 105 110
Ser Ala Glu Gln His Asp Ile Lys Leu Ile Ile Asn Phe Val Asn Tyr
115 120 125
Trp Thr Asp Tyr Gly Gly Met Ser Ala Tyr Val Ser Ala Tyr Gly Gly
130 135 140
Ser Gly Glu Thr Asp Phe Tyr Thr Ser Asp Thr Met Gln Ser Ala Tyr
145 150 155 160
Gln Thr Tyr Ile Lys Thr Val Val Glu Arg Tyr Ser Asn Ser Ser Ala
165 170 175
Val Phe Ala Trp Glu Leu Ala Asn Glu Pro Arg Cys Pro Ser Cys Asp
180 185 190
Thr Ser Val Leu Tyr Asn Trp Ile Glu Lys Thr Ser Lys Phe Ile Lys
195 200 205
Gly Leu Asp Ala Asp Arg Met Val Cys Ile Gly Asp Glu Gly Phe Gly
210 215 220
Leu Asn Ile Asp Ser Asp Gly Ser Tyr Pro Tyr Gln Phe Ser Glu Gly
225 230 235 240
Leu Asn Phe Thr Met Asn Leu Gly Ile Asp Thr Ile Asp Phe Gly Thr
245 250 255
Leu His Leu Tyr Pro Asp Ser Trp Gly Thr Ser Asp Asp Trp Gly Asn
260 265 270
Gly Trp Ile Thr Ala His Gly Ala Ala Cys Lys Ala Ala Gly Lys Pro
275 280 285
Cys Leu Leu Glu Glu Tyr Gly Val Thr Ser Asn His Cys Ser Val Glu
290 295 300
Gly Ser Trp Gln Lys Thr Ala Leu Ser Thr Thr Gly Val Gly Ala Asp
305 310 315 320
Leu Phe Trp Gln Tyr Gly Asp Asp Leu Ser Thr Gly Lys Ser Pro Asp
325 330 335
Asp Gly Asn Thr Ile Tyr Tyr Gly Thr Ser Asp Tyr Gln Cys Leu Val
340 345 350
Thr Asp His Val Ala Ala Ile Asp Ser Ala
355 360
<210> 2
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agctactgga tcggtttcct cactgacaac gcggacgtcg acctcgtcat gggccacctg 180
aagtcgtccg gcctcaagat cctccgcgtg tggggcttca acgatgtcac ctcgcagccc 240
tcctccggca cagtctggta ccaactgcac caggacggca aatcgacaat caacacgggt 300
gccgacggtc tccagcgcct cgactacgtc gtctcgtctg ccgaacagca cgacatcaaa 360
ctcatcatca acttcgtcaa ctactggacc gattacggtg gtatgtctgc gtacgtgagc 420
gcgtatggcg gatccggcga gacggatttc tataccagtg ataccatgca gagtgcctat 480
cagacatata tcaagacggt cgtggagcgg tacagtaact cctcggcggt gtttgcgtgg 540
gagttggcga atgagccgag atgtccgagt tgcgatactt ctgtgttgta taactggatt 600
gagaagacga gtaagtttat taaggggttg gatgcggatc gtatggtttg tattggtgat 660
gagggcttcg gtctcaacat cgactcggac ggcagctacc cttatcaatt ctccgagggc 720
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cctgatagct ggggcacctc cgacgactgg ggcaacggct ggatcaccgc ccacggcgca 840
gcctgcaaag cggccggcaa gccatgtctc ctggaggaat acggagtcac ctcgaaccac 900
tgcagtgtgg agggctcgtg gcagaagaca gcgctcagca caacgggcgt cggcgcggat 960
ctgttctggc agtatggtga tgatttgagt accgggaagt cgccggatga tgggaatact 1020
atctactatg ggactagtga ttatcagtgc ctggtgacgg atcatgttgc tgctattgat 1080
agcgcctaa 1089
<210> 3
<211> 362
<212> PRT
<213>artificial sequence
<400> 3
Leu Pro Lys Ala Ser Pro Ala Pro Ser Thr Ser Ser Ser Ala Ala Ser
1 5 10 15
Thr Ser Phe Ala Ser Thr Ser Gly Leu Gln Phe Thr Ile Asp Gly Glu
20 25 30
Thr Gly Tyr Phe Ala Gly Thr Asn Ser Tyr Trp Ile Gly Phe Leu Thr
35 40 45
Asp Asn Ala Asp Val Asp Leu Val Met Gly His Leu Lys Ser Ser Gly
50 55 60
Leu Lys Ile Leu Arg Val Trp Gly Phe Asn Asp Val Thr Ser Gln Pro
65 70 75 80
Ser Ser Gly Thr Val Trp Tyr Gln Leu His Gln Asp Gly Lys Ser Thr
85 90 95
Ile Asn Thr Gly Ala Asp Gly Leu Gln Arg Leu Asp Tyr Val Val Ser
100 105 110
Ser Ala Glu Gln His Asp Ile Lys Leu Ile Ile Asn Phe Val Asn Tyr
115 120 125
Trp Thr Asp Tyr Gly Gly Met Ser Ala Tyr Val Ser Ala Tyr Gly Gly
130 135 140
Ser Gly Glu Thr Asp Phe Tyr Thr Ser Asp Thr Met Gln Ser Ala Tyr
145 150 155 160
Gln Thr Tyr Ile Lys Thr Val Val Glu Arg Tyr Ser Asn Ser Ser Ala
165 170 175
Val Phe Ala Trp Glu Leu Ala Asn Glu Pro Arg Cys Pro Ser Cys Asp
180 185 190
Thr Ser Val Leu Tyr Asn Trp Ile Glu Lys Thr Ser Lys Phe Ile Lys
195 200 205
Gly Leu Asp Ala Asp Arg Met Val Cys Ile Gly Asp Glu Gly Phe Gly
210 215 220
Leu Asn Ile Asp Ser Asp Gly Ser Tyr Pro Tyr Gln Phe Ser Glu Gly
225 230 235 240
Leu Asn Phe Thr Met Asn Leu Gly Ile Asp Thr Ile Asp Phe Gly Thr
245 250 255
Leu His Leu Tyr Pro Asp Ser Trp Gly Thr Ser Asp Asp Trp Gly Asn
260 265 270
Gly Trp Ile Thr Ala His Gly Ala Ala Cys Lys Ala Ala Gly Lys Pro
275 280 285
Cys Leu Leu Glu Glu Tyr Gly Val Thr Ser Asn His Cys Ser Val Glu
290 295 300
Gly Pro Trp Gln Lys Thr Ala Leu Ser Thr Thr Gly Val Gly Ala Asp
305 310 315 320
Leu Phe Trp Gln Tyr Gly Asp Asp Leu Ser Thr Gly Lys Ser Pro Asp
325 330 335
Asp Gly Asn Thr Ile Tyr Tyr Gly Thr Ser Asp Tyr Gln Cys Leu Val
340 345 350
Thr Asp His Val Ala Ala Ile Asp Ser Ala
355 360
<210> 4
<211> 362
<212> PRT
<213>artificial sequence
<400> 4
Leu Pro Lys Ala Ser Pro Ala Pro Ser Thr Ser Ser Ser Ala Ala Ser
1 5 10 15
Thr Ser Phe Ala Ser Thr Ser Gly Leu Gln Phe Thr Ile Asp Gly Glu
20 25 30
Thr Gly Tyr Phe Ala Gly Thr Asn Ser Tyr Trp Ile Gly Phe Leu Thr
35 40 45
Asp Asn Ala Asp Val Asp Leu Val Met Gly His Leu Lys Ser Ser Gly
50 55 60
Leu Lys Ile Leu Arg Val Trp Gly Phe Asn Asp Val Thr Ser Gln Pro
65 70 75 80
Ser Ser Gly Thr Val Trp Tyr Gln Leu His Gln Asp Gly Lys Ser Thr
85 90 95
Ile Asn Thr Gly Ala Asp Gly Leu Gln Arg Leu Asp Tyr Val Val Ser
100 105 110
Ser Ala Glu Gln His Asp Ile Lys Leu Ile Ile Asn Phe Val Asn Tyr
115 120 125
Trp Thr Asp Tyr Gly Gly Met Ser Ala Tyr Val Ser Ala Tyr Gly Gly
130 135 140
Ser Gly Glu Thr Asp Phe Tyr Thr Ser Asp Thr Met Gln Ser Ala Tyr
145 150 155 160
Gln Thr Tyr Ile Lys Thr Val Val Glu Arg Tyr Ser Asn Ser Ser Ala
165 170 175
Val Phe Ala Trp Glu Leu Ala Asn Glu Pro Arg Cys Pro Ser Arg Asp
180 185 190
Thr Ser Val Leu Tyr Asn Trp Ile Glu Lys Thr Ser Lys Phe Ile Lys
195 200 205
Gly Leu Asp Ala Asp Arg Met Val Cys Ile Gly Asp Glu Gly Phe Gly
210 215 220
Leu Asn Ile Asp Ser Asp Gly Ser Tyr Pro Tyr Gln Phe Ser Glu Gly
225 230 235 240
Leu Asn Phe Thr Met Asn Leu Gly Ile Asp Thr Ile Asp Phe Gly Thr
245 250 255
Leu His Leu Tyr Pro Asp Ser Trp Gly Thr Ser Asp Asp Trp Gly Asn
260 265 270
Gly Trp Ile Thr Ala His Gly Ala Ala Cys Lys Ala Ala Gly Lys Pro
275 280 285
Cys Leu Leu Glu Glu Tyr Gly Val Thr Ser Asn His Cys Ser Val Glu
290 295 300
Gly Pro Trp Gln Lys Thr Ala Leu Ser Thr Thr Gly Val Gly Ala Asp
305 310 315 320
Leu Phe Trp Gln Tyr Gly Asp Asp Leu Ser Thr Gly Lys Ser Pro Asp
325 330 335
Asp Gly Asn Thr Ile Tyr Tyr Gly Thr Ser Asp Tyr Gln Cys Leu Val
340 345 350
Thr Asp His Val Ala Ala Ile Asp Ser Ala
355 360
<210> 5
<211> 362
<212> PRT
<213>artificial sequence
<400> 5
Leu Pro Lys Ala Ser Pro Ala Pro Ser Thr Ser Ser Ser Ala Ala Ser
1 5 10 15
Thr Ser Phe Ala Ser Thr Ser Gly Leu Gln Phe Thr Ile Asp Gly Glu
20 25 30
Thr Gly Tyr Phe Ala Gly Thr Asn Ser Tyr Trp Ile Gly Phe Leu Thr
35 40 45
Asp Asn Ala Asp Val Asp Leu Val Met Gly His Leu Lys Ser Ser Gly
50 55 60
Leu Lys Ile Leu Arg Val Trp Gly Phe Asn Asp Val Thr Ser Gln Pro
65 70 75 80
Ser Ser Gly Thr Val Trp Tyr Gln Leu His Gln Asp Gly Lys Ser Thr
85 90 95
Ile Asn Thr Gly Ala Asp Gly Leu Gln Arg Leu Asp Tyr Val Val Ser
100 105 110
Ser Ala Glu Gln His Asp Ile Lys Leu Ile Ile Asn Phe Val Asn Tyr
115 120 125
Trp Thr Asp Tyr Gly Gly Met Ser Ala Tyr Val Ser Ala Tyr Gly Gly
130 135 140
Ser Gly Glu Thr Asp Phe Tyr Thr Ser Asp Thr Met Gln Ser Ala Tyr
145 150 155 160
Gln Thr Tyr Ile Lys Thr Val Val Glu Arg Tyr Ser Asn Ser Ser Ala
165 170 175
Val Phe Ala Trp Glu Leu Ala Asn Glu Pro Arg Cys Pro Ser Cys Asp
180 185 190
Thr Ser Val Leu Tyr Asn Trp Ile Glu Lys Thr Ser Lys Phe Ile Lys
195 200 205
Gly Leu Asp Ala Asp Arg Met Val Cys Ile Gly Asp Glu Gly Phe Gly
210 215 220
Leu Asn Ile Asp Ser Asp Gly Ser Tyr Pro Tyr Gln Phe Ser Glu Gly
225 230 235 240
Leu Asn Phe Thr Met Asn Leu Gly Ile Asp Thr Ile Asp Phe Gly Thr
245 250 255
Leu His Leu Tyr Pro Asp Ser Trp Gly Thr Ser Asp Asp Trp Gly Asn
260 265 270
Gly Trp Ile Thr Ala His Gly Ala Ala Cys Lys Ala Ala Gly Lys Pro
275 280 285
Cys Leu Leu Gln Glu Tyr Gly Val Thr Ser Asn His Cys Ser Val Glu
290 295 300
Gly Pro Trp Gln Lys Thr Ala Leu Ser Thr Thr Gly Val Gly Ala Asp
305 310 315 320
Leu Phe Trp Gln Tyr Gly Asp Asp Leu Ser Thr Gly Lys Ser Pro Asp
325 330 335
Asp Gly Asn Thr Ile Tyr Tyr Gly Thr Ser Asp Tyr Gln Cys Leu Val
340 345 350
Thr Asp His Val Ala Ala Ile Asp Ser Ala
355 360
<210> 6
<211> 362
<212> PRT
<213>artificial sequence
<400> 6
Leu Pro Lys Ala Ser Pro Ala Pro Ser Thr Ser Ser Ser Ala Ala Ser
1 5 10 15
Thr Ser Phe Ala Ser Thr Ser Gly Leu Gln Phe Thr Ile Asp Gly Glu
20 25 30
Thr Gly Tyr Phe Ala Gly Thr Asn Ser Tyr Trp Ile Gly Phe Leu Thr
35 40 45
Asp Asn Ala Asp Val Asp Leu Val Met Gly His Leu Lys Ser Ser Gly
50 55 60
Leu Lys Ile Leu Arg Val Trp Gly Phe Asn Asp Val Thr Ser Gln Pro
65 70 75 80
Ser Ser Gly Thr Val Trp Tyr Gln Leu His Gln Asp Gly Lys Ser Thr
85 90 95
Ile Asn Thr Gly Ala Asp Gly Leu Gln Arg Leu Asp Tyr Val Val Ser
100 105 110
Ser Ala Glu Gln His Asp Ile Lys Leu Ile Ile Asn Phe Val Asn Tyr
115 120 125
Trp Thr Asp Tyr Gly Gly Met Ser Ala Tyr Val Ser Ala Tyr Gly Gly
130 135 140
Ser Gly Glu Thr Asp Phe Tyr Thr Ser Asp Thr Met Gln Ser Ala Tyr
145 150 155 160
Gln Thr Tyr Ile Lys Thr Val Val Glu Arg Tyr Ser Asn Ser Ser Ala
165 170 175
Val Phe Ala Trp Glu Leu Ala Asn Glu Pro Arg Cys Pro Ser Cys Asp
180 185 190
Thr Ser Val Leu Tyr Asn Trp Ile Glu Lys Thr Ser Lys Phe Ile Lys
195 200 205
Gly Leu Asp Ala Asp Arg Met Val Cys Ile Gly Asp Glu Gly Phe Gly
210 215 220
Leu Asn Ile Asp Ser Asp Gly Ser Tyr Pro Tyr Gln Phe Ser Glu Gly
225 230 235 240
Leu Asn Phe Thr Met Asn Leu Gly Ile Asp Thr Ile Asp Phe Gly Thr
245 250 255
Leu His Leu Tyr Pro Asp Ser Trp Gly Thr Ser Asp Asp Trp Gly Asn
260 265 270
Gly Trp Ile Thr Ala His Gly Ala Ala Cys Lys Ala Ala Gly Lys Pro
275 280 285
Cys Leu Leu Glu Glu Tyr Gly Val Thr Ser Asn His Cys Ser Val Glu
290 295 300
Gly Pro Trp Gln Lys Thr Ala Leu Ser Thr Thr Gly Val Gly Ala Asp
305 310 315 320
Leu Phe Trp Gln Tyr Gly Asp Asp Leu Ser Thr Gly Lys Ser Pro Asp
325 330 335
Asn Gly Asn Thr Ile Tyr Tyr Gly Thr Ser Asp Tyr Gln Cys Leu Val
340 345 350
Thr Asp His Val Ala Ala Ile Asp Ser Ala
355 360
<210> 7
<211> 34
<212> DNA
<213>artificial sequence
<400> 7
gcgcgaattc ctgccgaaag cctcccctgc accg 34
<210> 8
<211> 36
<212> DNA
<213>artificial sequence
<400> 8
taaagcggcc gcttaggcgc tatcaatagc agcaac 36
Claims (10)
1. beta-mannase enzyme mutant MAN-M01, which is characterized in that the beta-mannase enzyme mutant MAN-M01's
For amino acid sequence as shown in SEQ ID NO:3, the mutant MAN-M01 is the β-of SEQ ID NO:1 by amino acid sequence
306th amino acids of mannase become proline from serine and obtain.
2. the encoding gene of beta-mannase enzyme mutant MAN-M01 described in claim 1.
3. beta-mannase enzyme mutant MAN-M02, which is characterized in that the beta-mannase enzyme mutant MAN-M02's
For amino acid sequence as shown in SEQ ID NO:4, the mutant MAN-M02 is the β-of SEQ ID NO:1 by amino acid sequence
191st amino acids of mannase, which become arginine, the 306th amino acids from cysteine, becomes proline from serine
It obtains.
4. the encoding gene of beta-mannase enzyme mutant MAN-M02 as claimed in claim 3.
5. beta-mannase enzyme mutant MAN-M03, which is characterized in that the beta-mannase enzyme mutant MAN-M03's
For amino acid sequence as shown in SEQ ID NO:5, the mutant MAN-M03 is the β-of SEQ ID NO:1 by amino acid sequence
292nd amino acids of mannase, which become glutamine, the 306th amino acids from glutamic acid, becomes proline from serine
It obtains.
6. the encoding gene of beta-mannase enzyme mutant MAN-M03 described in claim 5.
7. beta-mannase enzyme mutant MAN-M04, which is characterized in that the beta-mannase enzyme mutant MAN-M04's
For amino acid sequence as shown in SEQ ID NO:6, the mutant MAN-M04 is the β-of SEQ ID NO:1 by amino acid sequence
337th amino acids of mannase, which become asparagine, the 306th amino acids from aspartic acid, becomes dried meat ammonia from serine
Acid obtains.
8. the encoding gene of beta-mannase enzyme mutant MAN-M04 as claimed in claim 7.
9. the encoding gene containing MAN-M01 as claimed in claim 2 or the coding containing MAN-M02 as claimed in claim 4
Gene or the encoding gene containing MAN-M03 as claimed in claim 6 or the volume containing MAN-M04 according to any one of claims 8
The expression vector of code gene.
10. claim 1,3,5 and 7 described in any item beta-mannase enzyme mutant MAN-M01, MAN-M02, MAN-M03
Or MAN-M04 is in the application being used to prepare in animal feed additive.
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| CN107760700B (en) * | 2017-09-30 | 2020-11-06 | 武汉轻工大学 | Beta-mannase gene, recombinant expression vector, strain, beta-mannase and preparation method and application thereof |
| CN111321173B (en) * | 2018-12-14 | 2022-05-31 | 南京百斯杰生物工程有限公司 | Application of mannase in alcohol fermentation |
| CN112410322B (en) * | 2020-11-30 | 2022-05-13 | 河北科技大学 | Bacillus licheniformis beta-mannase mutant and application thereof |
| CN118048345A (en) * | 2022-11-16 | 2024-05-17 | 青岛蔚蓝生物集团有限公司 | High specific activity acidic mannanase mutant |
| CN116004585B (en) * | 2022-12-30 | 2024-05-31 | 山东龙昌动物保健品股份有限公司 | Eucalyptus essential oil mutant enzyme preparation and application thereof in livestock cultivation |
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