CN102277342B - Mannose and mutants thereof - Google Patents
Mannose and mutants thereof Download PDFInfo
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- CN102277342B CN102277342B CN 201110234218 CN201110234218A CN102277342B CN 102277342 B CN102277342 B CN 102277342B CN 201110234218 CN201110234218 CN 201110234218 CN 201110234218 A CN201110234218 A CN 201110234218A CN 102277342 B CN102277342 B CN 102277342B
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Abstract
The invention belongs to the technical field of microbial engineering and provides high-enzymatic-activity mannose and mutants thereof. The invention provides six mannose genes obtained by cloning from different strains of bacillus licheniformis and mannose coded by the genes; and bioinformatics analysis on the difference and genetic relationship between the 6 mutants is performed. The mannose provided by the invention has high activity under a condition of a pH value of 7, the optimal temperature of the mannose is mild, and the mannose can be used as an enzyme preparation for feed purpose.
Description
Technical field
The present invention relates to mannase and mutant thereof, belong to the microbial engineering field.
Background technology
β-Isosorbide-5-Nitrae-D-mannase (EC 3.2.1.78) is called again mannase, and it can be hydrolyzed contains β-Isosorbide-5-Nitrae-D-MANNOSE glycosidic bond, generates mannooligo saccharide or mannocarolose, belongs to the hemicellulose enzyme.
At occurring in nature, mannosans is to be only second to cellulosic second largest reproducible hemicellulose carbohydrate, extensively is present in plant cell wall, and especially in seeds of leguminous plant, semi-lactosi-mannosans content is up to more than 20% of dry weight.Simultaneously, the structure of mannosans also has a variety of, as semi-lactosi-mannosans, glucose-mannosans and semi-lactosi-glucose-mannosans etc.The structure of semi-lactosi-mannosans be main chain by the mannosans that β-Isosorbide-5-Nitrae-the D-MANNOSE glycosidic bond is formed by connecting, side chain is α-1, the semi-lactosi that 6-semi-lactosi glycosidic bond connects.Glucose-mannosans main chain is to be formed by connecting by β-Isosorbide-5-Nitrae-D-MANNOSE glycosidic bond, but on its main chain, some seminoses are replaced by glucose.
Hemicellulase comprises mannonase zytase and dextranase etc., is widely used in the industries such as cellulose raw producing and ethanol, food, feed and papermaking.Fodder enzyme preparation will adapt to the digestive tube characteristics of animal such as pig and bird etc., need to have acid resistance strong, simultaneously in characteristics such as pH6.0 ~ 7.0 scope activity are the strongest.Soybean meal is the important source material of producing feed, if add appropriate mannase in feed, can improve the digestibility of feed, can reduce again feed cost (Yang Hongkun etc., 2007, feed research; Duan Lei etc., 2008, feed research; Fan Zhiheng, 2008, fodder industry).
A lot of microorganisms can produce mannase, comprise aspergillus, genus bacillus or even streptomycete etc.But the enzyme running water of the mannase that the screening by bacterial classification and the natural bacterial strain of mutagenic and breeding produce is flat≤and 100U/ml, often can not satisfy industrial needs (Yang Wenbo, 1995, microorganism circular).Therefore, clone gene carries out heterogenous expression and obtains the focus that efficient expression engineering is current exploitation mannase (Ding Hongbiao etc., 2006, herding and feed; Tan Xiuhua etc., 2005, the microorganism journal).
As fodder additives, seeking alive, the acidproof and optimum temperuture of high enzyme is focus and the difficult point of current research near the mannase of body temperature.And the protein engineering transformation is to obtain one of important means of ideal protein.Orthogenesis is the important means (Mao Shaoming etc., 2007, microorganism circular) that obtains desirable beta-mannanase for feeding.Orthogenesis is to utilize random mutation and screen the mass mutation body and obtain target protein, and its workload is large, also very large (Zhang Hongying etc., 1999, Science Bulletin of randomness; Xu Qinkun etc., 2005, the biotechnology communication).Although and design and rational workload less often is difficult to find suitable amino acid sites suddenly change transformation (Zhang Xiuyan etc., 2006, biotechnology magazine; Wang Fanye etc., 2006, use chemical industry).
The invention provides the mannase of pH partial neutral and the mutant of a plurality of enzymes thereof.These mutant are to clone to obtain from the different strains of Bacillus licheniformis, incomplete homology between them, and homology is 99%.Although the homology of these mutant is very high, there is very large difference in active (specific activity) to each other.This illustrates that these sites that suddenly change/replace are to affect its active critical sites, this for the orthogenesis of mannase and design and rational by alternative critical sites and direction are provided.
Summary of the invention
The purpose of this invention is to provide mannase and variant thereof that high enzyme is lived.
The invention provides new restructuring mannase and mutant thereof, the aminoacid sequence of wherein said restructuring mannase:
(a) comprise be selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,, SEQ ID NO:10 or SEQ ID NO:12 one of them; Perhaps
(b) be selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,, SEQ ID NO:10 or SEQ ID NO:12 replace, lack or add one or several amino-acid residues on one of them and obtain.
In one embodiment, the aminoacid sequence of described seminase is SEQ ID NO:2, and the nucleotides sequence of its encoding gene is classified SEQ ID NO:1 as.
In one embodiment, the aminoacid sequence of described seminase is SEQ ID NO:4, and the nucleotides sequence of its encoding gene is classified SEQ ID NO:3 as.
In one embodiment, the aminoacid sequence of described seminase is SEQ ID NO:6,, the nucleotides sequence of its encoding gene is classified SEQ ID NO:5 as.
In one embodiment, the aminoacid sequence of described seminase is SEQ ID NO:8, and the nucleotides sequence of its encoding gene is classified SEQ ID NO:7 as.
In one embodiment, the aminoacid sequence of described seminase is SEQ ID NO:10, and the nucleotides sequence of its encoding gene is classified SEQ ID NO:9 as.
In one embodiment, the aminoacid sequence of described seminase is SEQ ID NO:12, and the nucleotides sequence of its encoding gene is classified SEQ ID NO:11 as.
Enzyme experimental result alive shows: mannase provided by the invention has good enzyme and lives.Wherein amino acid is that the high specific activity mannase of SEQ ID NO:12 has good stability.Bathed 5 minutes at 70 ℃, 80 ℃, 85 ℃ and 90 ℃ of Water Unders, can also keep respectively 90%, 30%, 20% and 18% enzyme to live.The optimal pH analysis shows, this enzyme all has activity in the pH5.0-9.0 scope, and 2 optimal pH effect peaks, i.e. pH6.5 and pH8.5 are arranged.
On the other hand, the present invention also provides a kind of method for preparing the mannase of recombinating, and the method comprises:
(a) extract the genomic dna of Bacillus licheniformis;
(b) take the genomic dna that extracts as template, carry out pcr amplification;
(c) pcr amplification product is cloned into expression vector;
(d) recombinant expression vector that obtains with step (c) transforms expressive host;
(e) separate, identify and the purifying expression product.
In a preferred embodiment of the invention, the primer pair of described pcr amplification use is
P1:GCACACACCGTTTCTCCGGTG, and
P2：CACGACAGGCGTCAAAGAATCG。
In a preferred embodiment of the invention, described pcr amplification condition is: 95 ℃ of 4min; 94 ℃ of 30s; 55 ℃ of 40s, 30 circulations of 72 ℃ of 1min; 72 ℃ of 7min.
In a preferred embodiment of the invention, described expression vector is pET28.
In a preferred embodiment of the invention, described expressive host is e. coli bl21.
In specific embodiments of the present invention, the nucleotide sequence of described amplified production is respectively SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11.
In a preferred embodiment of the invention; the recombinant expression vector of SEQ ID NO:11 that utilized the pPIC9K plasmid construction; and transform the Pichia pastoris GS115 bacterial strain, obtained the Pichia yeast engineering of high efficient expression, for fermentation and the application of mass-producing are laid a good foundation.
The application analyzes with biosoftware DNAMAN6.0 and shows the sudden change in various degree that has different loci between described enzyme coding gene.For studying these sudden changes to the impact of the enzyme activity, carried out these mutator genes recombinant expressed respectively.By the Ni-agarose purification of recombinant proteins and analyze its zymologic property.The zymologic property analysis shows that there is obvious difference in the enzyme (specific activity) alive of 6 mutant, illustrates that these site amino-acid residues are the critical sites that affect the mannosans enzymic activity.The invention provides 6 mannase mutant genes of Bacillus licheniformis and corresponding aminoacid sequence thereof.Exist between 6 mannosans enzyme amino acid sequences and have sudden change on 20,27,31,48,64,78,81,89,102,105,122,123,179,187,195,234,248,254,255,292,302,312,313 and 325 sites.The larger difference that the sudden change of these site amino-acid residues causes enzyme to live, this design and rational and orthogenesis for mannase provides reference.
On the other hand, the present invention also provides a kind of mensuration mannosans enzyme testing method, and the method comprises:
(Sigma company Batch#125K0091) is substrate with 0.6% locust bean gum mannosans.Take 0.1M acetic acid-sodium-acetate (pH5.5) as damping fluid, with 37 ℃ of balance 20min of mannosans substrate; With 37 ℃ of balance 10min of enzyme liquid to be measured.Get 4 test tubes, add respectively enzyme liquid 2ml, get wherein 3 conducts and measure pipe, add respectively the 2ml substrate solution, another adds 5ml DNS solution as blank tube, 37 ℃ ± 0.5 ℃ water-bath 30 minutes, to measure pipe for three and add respectively 5ml DNS solution, blank tube adds the 2ml substrate solution, reaction is 5 minutes in boiling water bath, be settled to 25ml after cooling, with the blank tube zeroing, in spectrophotometer 540nn place's survey absorbancy.The enzyme definition of living is under the condition of 37 ℃ of pH5.5, and the amount that the per minute hydrolysis substrate produces the 1 required enzyme liquid of μ mol seminose is a mannosans activity unit.Determining the protein quantity is with reference to the Bradford method.
Description of drawings
Fig. 1 is illustrated is with DNAMAN compare of analysis protein sequence
Fig. 2 is illustrated is the phylogeny of Clustal pxrd analysis protein sequence
Fig. 3 is illustrated is that Man6 zymologic property optimum temperuture and pH analyze
Advantage of the present invention:
The mutant of a plurality of mannase genes of clone from different lichem bacillus strains, and analyze mannase mutant zymetology characteristic.These mannase amino acid sequence homologies are up to more than 99%, but the sudden change of different loci replaces the significant difference that has caused enzymic activity.This provides the selection site for the orthogenesis of protein and protein engineering transformation, lays the foundation for selecting highly active mannase to carry out the high efficient expression of allos
Embodiment
Following examples are to set forth content of the present invention for explanation better, and the relevant technician in this area can understand better and grasp the present invention by embodiment.But, the case that protection of the present invention and claim scope are not limited to provide.
The clone of embodiment 1 Bacillus licheniformis mannase gene
1.1 extract the total genomic dna of Bacillus licheniformis different strains
With the 6 bacillus licheniformis incubated overnight that preserve in this laboratory, respectively get 1.5ml, centrifugal 1 minute of 12000rpm is except supernatant; Add 200 μ l lysis buffers (60mM Tris-HCl, pH7.8,20mM Na-Ac, 1mM EDTA, 1.5% SDS), acutely blow and beat with pipettor; Add 66 μ l 5M sodium perchlorate solution mixings, centrifugal 10 minutes of 12000rpm gets supernatant; Add the extracting of equal-volume phenol once, centrifugal 2 minutes of 12000rpm gets supernatant; Added the equal-volume isopropanol precipitating 5 minutes, centrifugal 5 minutes of 12000rpm; 70% twice of washing with alcohol; DNA with drying is dissolved in ddH at last
2O。
Gene clone
The genome DNA of extracting in 1.1 utilizes respectively primer pair (GCACACACCGTTTCTCCGGTG and CACGACAGGCGTCAAAGAATCG) to carry out pcr amplification as template.The pcr amplification condition is 95 ℃ of 4min; 94 ℃ of 30S; 55 ℃ of 40S, 30 circulations of 72 ℃ of 1min; 72 ℃ of 7min.Utilize gel to reclaim test kit and reclaim pcr amplification product.
Sequencing analysis
The amplified production that reclaims in 1.2 is connected respectively to pMD18 T-carrier, and corresponding cloning vector is called after pMDT-Man1, pMDT-Man2, pMDT-Man3, pMDT-Man4, pMDT-Man5 and pMDT-Man6 respectively; At last positive colony is delivered to the Huada Gene Research Center, Beijing and carry out sequencing analysis.Sequencing result is: the nucleotide sequence of Man1, Man2, Man3, Man4, Man5, Man6 is respectively SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11.The aminoacid sequence of its proteins encoded is respectively SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12.
The sequential analysis of embodiment 2 mannases
Sequencing result BLAST compare of analysis on NCBI is shown these sequences and mannase gene height homology.Utilize biosoftware DNAMAN6.0 analysis of nucleic acids sequence to find that 6 sequences are not complete homologies.Corresponding nucleotide sequence is translated as aminoacid sequence, and difference called after Man1, Man2, Man3, Man4, Man5 and Man6.To the amino acid sequence homologous compare of analysis, there is sudden change in the result demonstration with next site or several site, that is: 20,27,31,48,64,78,81,89,102,105,122,123,179,187,195,234,248,254,255,292,302,312,313 and 325 sites.The possible replacement of the amino-acid residue in these sites is Y20N, D27N, N31 S, L48 T, V64I, E78 K, V81D, S89R, R102Q, L105M, S122P, S123N, S179T, A187E, R195Q, H234Y, H248Y, D254E, Q255E, N292K, G302E, G312D, A313T, D325E, the sequence alignment analytical results is seen Fig. 1.The phylogeny of the aminoacid sequence of biosoftware Clustal pxrd analysis albumen, 6 mutant sibships are near especially, see Fig. 2.
Recombinant expressed and the purifying of embodiment 3 mannases in intestinal bacteria
Take plasmid pMDT-Man1, pMDT-Man2, pMDT-Man3, pMDT-Man4, pMDT-Man5 and pMDT-Man6 as template, utilize primer (AGA respectively
GCTAGC GCACACACCGTTTCTCCGGTG---
NheI and ACA
CTCGAG CACGACAGGCGTCAAAGAATCG---
XhoI) carry out pcr amplification, the pcr amplification condition is 95 ℃ of 4min; 94 ℃ of 30S; 55 ℃ of 40S, 30 circulations of 72 ℃ of 1min; 72 ℃ of 7min.The amplified production gel first carries out after reclaiming
NheThe I enzyme is cut, and then reclaims enzyme and cuts product and carry out
XhoThe I enzyme is cut.Equally, expression plasmid pET28a is also carried out respectively
NheThe I enzyme cut and
XhoThe I enzyme is cut.With the T4 ligase enzyme, double digestion being produced product is that clone gene is connected a ℃ connection and is spent the night with expression vector.At last, import e. coli bl21 connecting product.Corresponding positive colony expression plasmid is called after pET-Man1, pET-Man2, pET-Man3, pET-Man4, pET-Man5 and pET-Man6 respectively.
Positive colony colony inoculation 5ml LB substratum, 37 ℃ added 1mM IPTG abduction delivering 4-5 hour when being cultured to OD600=0.3.Then centrifugal collection express cell adds 1mL lysis buffer (50 mM NaH in-20 ℃ of frozen spending the night
2PO
4, 300 mM NaCl, 10 mM imidazole) and carry out ultrasonic disruption, frequently carry out microscopic examination, the method that provides according to the Ni-NTA SPIN test kit of QIAGEN company after cell is fully broken is carried out protein purification and is reclaimed.Electrophoresis result shows that recovery obtains corresponding target protein.
(Sigma company Batch#125K0091) is substrate with 0.6% locust bean gum mannosans.Be determined at the activity of restructuring mannase purified in embodiment 3, step is as follows: take 0.1M acetic acid-sodium-acetate (pH5.5) as damping fluid, with 37 ℃ of balance 20min of mannosans substrate; With 37 ℃ of balance 10min of enzyme liquid to be measured.Get 4 test tubes, add respectively enzyme liquid 2ml, get wherein 3 conducts and measure pipe, add respectively the 2ml substrate solution, another adds 5ml DNS solution as blank tube, 37 ℃ ± 0.5 ℃ water-bath 30 minutes, to measure pipe for three and add respectively 5ml DNS solution, blank tube adds the 2ml substrate solution, reaction is 5 minutes in boiling water bath, be settled to 25ml after cooling, with the blank tube zeroing, in spectrophotometer 540nn place's survey absorbancy.The enzyme definition of living is under the condition of 37 ℃ of pH5.5, and the amount that the per minute hydrolysis substrate produces the 1 required enzyme liquid of μ mol seminose is a mannosans activity unit.Determining the protein quantity is with reference to the Bradford method.
The work of purifying protein enzyme is that 5.5 ~ 5620IU/ml does not wait, and specific activity is determined as 12 ~ 7020IU/mg and does not wait.Wherein albumen Man4 and the Man5 activity of SEQ ID NO.4 and SEQ ID NO.5 coding are extremely low.
In addition, also measured Man6 the enzyme activity under different pH and temperature condition, wherein damping fluid is respectively 0.1M acetic acid-sodium-acetate buffer (pH4.0, pH 5.0, pH 6.0), 0.1M SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic damping fluid (pH6.5,7.0,7.5,8.0) and 0.1M glycine-sodium hydrate buffer solution (pH8.5,9.0,10.0).Result as shown in Figure 3, optimum temperuture is 50 ℃, is 2 peaks the suitableeest at pH6.5 and pH8.5, and the mannase enzyme is lived very stable between pH6.0-7.0.
The expression of embodiment 5 in pichia spp
5.1 the structure of expression plasmid pPIC-Man2
Design primer Man2-F and Man2-R(ATA
GAATTCGCACACACCGTTTCTCCGGTG [
EcoRI] and ATA
GCGGCCGCCACGACAGGCGTCAAAGAATCG[
NotI]) take plasmid pET-Man2 as template, carry out pcr amplification take Man2-F and Man2-R as primer.The PCR condition is: 95 ℃ of 4min; 94 ℃ of 40s, 54 ℃ of 40s, 72 ℃ of 1min, totally 30 circulations; 72 ℃ of 7min.Gel reclaims the PCR product, and with
EcoRI and
NotThe I double digestion.Equally, with
EcoRI and
NotThen I double digestion pPIC9K cuts product to two enzymes and connects the rear importing bacillus coli DH 5 alpha that spends the night, and obtains recombinant expression plasmid pPIC-Man6.
The structure of recombinant bacterial strain
Expression plasmid pPIC-Man2 uses
SalAfter the I restriction enzyme digestion and electrophoresis is identified, concentrated through the ethanol precipitation, measure DNA concentration, save backup with 3 μ g/ μ L concentration dilution plasmid fragments.Preparation Pichia pastoris GS115 Electroporation-competent cells, be resuspended at last in the electrophoretic buffer of 1 mL precooling (contain 1mM MgCl2,10mM HEPES, 250mM sucrose, pH 7.8).Add 5 μ L linearizing recombinant plasmids in 80 μ L competent cells; Electricity transforms (condition is 1500V, 200 Ω, 25 μ F); Coat at last MM flat board (MM nutrient media components: 1.34%YNB, 4 * 10
-5The % vitamin H, 0.5% methyl alcohol), select recombinant bacterial strain.
The shaking flask abduction delivering
Engineering bacteria be will select and 5ml BMGY (1% yeast extract, 2% peptone, 1. 34 % YNB, 4 * 10 will be inoculated in
-5% vitamin H, l% glycerine), 30 ℃ of overnight incubation, centrifugal collection thalline adds 50ml BMMY inducing culture (1% yeast extract, 2% peptone, 1. 34 % YNB, 4 * 10 to thalline
-5% vitamin H, 0.5% methyl alcohol), added 50 μ L methyl alcohol in every 12 hours.
Enzyme activity determination
Different time sampling and measuring supernatant enzyme is lived, and result shows that the highest enzyme work of its supernatant liquor is 600 IU/mL.
Sequence table
＜110〉Qingdao Continent Biotech Co., Ltd.
＜120〉mannase and mutant thereof
<130>
<160> 12
<170> PatentIn version 3.4
<210> 1
<211> 1008
<212> DNA
＜213〉recombination sequence
<220>
＜221〉encoding gene
<222> (1)..(1008)
<400> 1
gcacacaccg tttctccggt gaatccgaat gcccagccga cgacgaaagc ggtgatgaac 60
tggcttgccc acctgcccaa tcggacggaa aaccgagtga tgtccggggc attcggagga 120
tacagccttg acacattttc gctggctgaa gccgaccgga tcaaacaagc aacaggacag 180
ctgccagcca tatacggctg cgattatgca aggggatggc tggagccgga agagatcgcc 240
gatacgattg actacagctg caacagcgat ttgatcgcat actggaaaag cggaggcatt 300
ccgcaaatca gcctgcacct cgcaaacccc gcgtttactt cgggtcatta taaaactcag 360
atttcaaaca gccagtatga gagaatttta gattcttcca cgcccgaagg aaaacggctt 420
gaggcgatgc tgagcaaaat cgccgatggc cttcaggagc ttgaaaatga aggcgtgccc 480
gttctattca gaccccttca tgaaatgaac ggcgaatggt tctggtgggg gctgacgcaa 540
tataatcaaa aagacagcgc gagaatctcc ttatacaaac ggctctatgt gaaaatctat 600
gactatatga caaagacaag aggcttggat catctgttgt gggtgtatgc gcctgacgcc 660
aacagagact ttaaaacaga cttttatccg ggcgcatcat atgttgacat cgtcgggctt 720
gacgcttatt ttgatgaccc gcacgccatt gatggctacg atcagctcac atctctgaac 780
aagccgtttg cctttacaga ggtcgggcca cagacgacaa acggcgggct ggattacgcg 840
cggtttatcc atgcaatcaa agagaaatac ccgaatacga cgtacttcct ggcgtggaac 900
gatgggtgga gccctgctgt gaataaggga gcgggcgccc tctatcttca tccatggacg 960
ctgaataagg gagacatctg ggacggcgat tctttgacgc ctgtcgtg 1008
<210> 2
<211> 336
<212> PRT
＜213〉recombination sequence
<220>
＜221〉mannase
<222> (1)..(336)
<400> 2
Ala His Thr Val Ser Pro Val Asn Pro Asn Ala Gln Pro Thr Thr Lys
1 5 10 15
Ala Val Met Asn Trp Leu Ala His Leu Pro Asn Arg Thr Glu Asn Arg
20 25 30
Val Met Ser Gly Ala Phe Gly Gly Tyr Ser Leu Asp Thr Phe Ser Leu
35 40 45
Ala Glu Ala Asp Arg Ile Lys Gln Ala Thr Gly Gln Leu Pro Ala Ile
50 55 60
Tyr Gly Cys Asp Tyr Ala Arg Gly Trp Leu Glu Pro Glu Glu Ile Ala
65 70 75 80
Asp Thr Ile Asp Tyr Ser Cys Asn Ser Asp Leu Ile Ala Tyr Trp Lys
85 90 95
Ser Gly Gly Ile Pro Gln Ile Ser Leu His Leu Ala Asn Pro Ala Phe
100 105 110
Thr Ser Gly His Tyr Lys Thr Gln Ile Ser Asn Ser Gln Tyr Glu Arg
115 120 125
Ile Leu Asp Ser Ser Thr Pro Glu Gly Lys Arg Leu Glu Ala Met Leu
130 135 140
Ser Lys Ile Ala Asp Gly Leu Gln Glu Leu Glu Asn Glu Gly Val Pro
145 150 155 160
Val Leu Phe Arg Pro Leu His Glu Met Asn Gly Glu Trp Phe Trp Trp
165 170 175
Gly Leu Thr Gln Tyr Asn Gln Lys Asp Ser Ala Arg Ile Ser Leu Tyr
180 185 190
Lys Arg Leu Tyr Val Lys Ile Tyr Asp Tyr Met Thr Lys Thr Arg Gly
195 200 205
Leu Asp His Leu Leu Trp Val Tyr Ala Pro Asp Ala Asn Arg Asp Phe
210 215 220
Lys Thr Asp Phe Tyr Pro Gly Ala Ser Tyr Val Asp Ile Val Gly Leu
225 230 235 240
Asp Ala Tyr Phe Asp Asp Pro His Ala Ile Asp Gly Tyr Asp Gln Leu
245 250 255
Thr Ser Leu Asn Lys Pro Phe Ala Phe Thr Glu Val Gly Pro Gln Thr
260 265 270
Thr Asn Gly Gly Leu Asp Tyr Ala Arg Phe Ile His Ala Ile Lys Glu
275 280 285
Lys Tyr Pro Asn Thr Thr Tyr Phe Leu Ala Trp Asn Asp Gly Trp Ser
290 295 300
Pro Ala Val Asn Lys Gly Ala Gly Ala Leu Tyr Leu His Pro Trp Thr
305 310 315 320
Leu Asn Lys Gly Asp Ile Trp Asp Gly Asp Ser Leu Thr Pro Val Val
325 330 335
<210> 3
<211> 1008
<212> DNA
＜213〉recombination sequence
<220>
＜221〉encoding gene
<222> (1)..(1008)
<400> 3
gcacacaccg tttctccggt gaacccgaat gcccagccga cgacgaaagc ggtgatgtac 60
tggctcgccc acctgcccga tcggacggaa agccgggtga tgtccggggc gttcggagga 120
tacagcctcg acacattttc aacggctgaa gccgaccgga tcaaacaggc aacaggacag 180
ctgccggcca tatacggctg cgattatgca agagggtggc tggagccgga aaagatcgcc 240
gatacgattg actacagctg caaccgtgat ttgatcgcat actggaaaag cggaggcatt 300
ccgcaaatca gcatgcacct cgcaaacccc gcgtttactt cgggtcatta taaaactcag 360
atttcaaaca gccagtatga gagaatttta gattcttcca ctcctgaagg aaagcggctt 420
gaggcgatgc tgagcaaaat cgcggacggc ctccaggagc ttgaaaatga aggcgtgccc 480
gttctattca gaccccttca cgaaatgaac ggcgaatggt tctggtgggg gctgacgcaa 540
tataatcaaa aagacagcga aagaatctcc ttgtacaaac agctctatgt gaaaatctat 600
gactatatga caaaaacaag aggcctggat catctcttgt gggtgtatgc gccggacgcc 660
aacagagact ttaaaacgga cttttatccg ggcgcatcat atgtggacat tgtcgggctt 720
gacgcttatt ttgatgaccc gtacgccatt gatggctacg aagagctcac atcgctgaac 780
aagccgtttg cctttacaga agtcggaccg cagacgacaa acggcgggct ggattacgcg 840
cggtttatcc atgccatcaa agaaaaatac ccgaaaacga cgtacttcct ggcgtggaac 900
gatgagtgga gcccggctgt gaataagggt gcggacaccc tctatcttca tccatggacg 960
ctgaataaag gagagatatg ggacggcgat tctttgacgc ctgtcgtg 1008
<210> 4
<211> 336
<212> PRT
＜213〉recombination sequence
<220>
＜221〉mannase
<222> (1)..(336)
<400> 4
Ala His Thr Val Ser Pro Val Asn Pro Asn Ala Gln Pro Thr Thr Lys
1 5 10 15
Ala Val Met Tyr Trp Leu Ala His Leu Pro Asp Arg Thr Glu Ser Arg
20 25 30
Val Met Ser Gly Ala Phe Gly Gly Tyr Ser Leu Asp Thr Phe Ser Thr
35 40 45
Ala Glu Ala Asp Arg Ile Lys Gln Ala Thr Gly Gln Leu Pro Ala Ile
50 55 60
Tyr Gly Cys Asp Tyr Ala Arg Gly Trp Leu Glu Pro Glu Lys Ile Ala
65 70 75 80
Asp Thr Ile Asp Tyr Ser Cys Asn Arg Asp Leu Ile Ala Tyr Trp Lys
85 90 95
Ser Gly Gly Ile Pro Gln Ile Ser Met His Leu Ala Asn Pro Ala Phe
100 105 110
Thr Ser Gly His Tyr Lys Thr Gln Ile Ser Asn Ser Gln Tyr Glu Arg
115 120 125
Ile Leu Asp Ser Ser Thr Pro Glu Gly Lys Arg Leu Glu Ala Met Leu
130 135 140
Ser Lys Ile Ala Asp Gly Leu Gln Glu Leu Glu Asn Glu Gly Val Pro
145 150 155 160
Val Leu Phe Arg Pro Leu His Glu Met Asn Gly Glu Trp Phe Trp Trp
165 170 175
Gly Leu Thr Gln Tyr Asn Gln Lys Asp Ser Glu Arg Ile Ser Leu Tyr
180 185 190
Lys Gln Leu Tyr Val Lys Ile Tyr Asp Tyr Met Thr Lys Thr Arg Gly
195 200 205
Leu Asp His Leu Leu Trp Val Tyr Ala Pro Asp Ala Asn Arg Asp Phe
210 215 220
Lys Thr Asp Phe Tyr Pro Gly Ala Ser Tyr Val Asp Ile Val Gly Leu
225 230 235 240
Asp Ala Tyr Phe Asp Asp Pro Tyr Ala Ile Asp Gly Tyr Glu Glu Leu
245 250 255
Thr Ser Leu Asn Lys Pro Phe Ala Phe Thr Glu Val Gly Pro Gln Thr
260 265 270
Thr Asn Gly Gly Leu Asp Tyr Ala Arg Phe Ile His Ala Ile Lys Glu
275 280 285
Lys Tyr Pro Lys Thr Thr Tyr Phe Leu Ala Trp Asn Asp Glu Trp Ser
290 295 300
Pro Ala Val Asn Lys Gly Ala Asp Thr Leu Tyr Leu His Pro Trp Thr
305 310 315 320
Leu Asn Lys Gly Glu Ile Trp Asp Gly Asp Ser Leu Thr Pro Val Val
325 330 335
<210> 5
<211> 1008
<212> DNA
＜213〉recombination sequence
<220>
＜221〉encoding gene
<222> (1)..(1008)
<400> 5
gcacacaccg tttctccggt gaacccgaat gcccagccga cgacgaaagc ggtgatgaac 60
tggcttgccc acctgcccaa tcggacggaa aatcgggtaa tgtccggggc attcggagga 120
tacagccttg acacgttctc gctggctgaa gccgaccgga tcaaacaggc aacaggacag 180
ctgccagccg tatacggctg cgattatgca agaggatggc tggagccgga ggagatcgcc 240
gttacgattg actacagctg caacagcgat ttgatcgcat actggaaaag cggaggcata 300
ccgcaaatca gcctgcacct cgcaaacccc gcgtttactt cgggtcatta taaaactcag 360
atttcgaaca gccagtatga gagaatttta gattcttcca cacccgaagg aaaacggctt 420
gaggcgatgc tgagcaaaat cgccgatggc cttcaggagc ttgaaaatga aggtgtgccc 480
gttctgttca gaccccttca cgaaatgaac ggcgaatggt tctggtgggg actgtcgcaa 540
tataatcaaa aagacagcgc gagaatctcc ttgtacaaac ggctctatgt gaaaatctat 600
gactatatga caaagacaag aggcttggat catctgttgt gggtgtatgc gcctgatgcc 660
aacagagact ttaaaacaga cttttatccg ggcgcatcac atgttgacat cgtcgggctt 720
gacgcttatt ttgatgaccc gcacgccatt gatggctacg atcagctcac atctctgaac 780
aagccgtttg cctttacaga ggtcgggcca cagacgacaa acggcgggct ggattacgcg 840
cggtttatcc atgcaatcaa agagaaatat ccgaatacga cgtacttcct ggcgtggaac 900
gatgggtgga gccctgctgt gaataaggga gcgggcgccc tctatcttca tccatggacg 960
ctgaataaag gagacatctg ggacggcgat tctttgacgc ctgtcgtg 1008
<210> 6
<211> 336
<212> PRT
＜213〉recombination sequence
<220>
＜221〉mannase
<222> (1)..(336)
<400> 6
Ala His Thr Val Ser Pro Val Asn Pro Asn Ala Gln Pro Thr Thr Lys
1 5 10 15
Ala Val Met Asn Trp Leu Ala His Leu Pro Asn Arg Thr Glu Asn Arg
20 25 30
Val Met Ser Gly Ala Phe Gly Gly Tyr Ser Leu Asp Thr Phe Ser Leu
35 40 45
Ala Glu Ala Asp Arg Ile Lys Gln Ala Thr Gly Gln Leu Pro Ala Val
50 55 60
Tyr Gly Cys Asp Tyr Ala Arg Gly Trp Leu Glu Pro Glu Glu Ile Ala
65 70 75 80
Val Thr Ile Asp Tyr Ser Cys Asn Ser Asp Leu Ile Ala Tyr Trp Lys
85 90 95
Ser Gly Gly Ile Pro Gln Ile Ser Leu His Leu Ala Asn Pro Ala Phe
100 105 110
Thr Ser Gly His Tyr Lys Thr Gln Ile Ser Asn Ser Gln Tyr Glu Arg
115 120 125
Ile Leu Asp Ser Ser Thr Pro Glu Gly Lys Arg Leu Glu Ala Met Leu
130 135 140
Ser Lys Ile Ala Asp Gly Leu Gln Glu Leu Glu Asn Glu Gly Val Pro
145 150 155 160
Val Leu Phe Arg Pro Leu His Glu Met Asn Gly Glu Trp Phe Trp Trp
165 170 175
Gly Leu Ser Gln Tyr Asn Gln Lys Asp Ser Ala Arg Ile Ser Leu Tyr
180 185 190
Lys Arg Leu Tyr Val Lys Ile Tyr Asp Tyr Met Thr Lys Thr Arg Gly
195 200 205
Leu Asp His Leu Leu Trp Val Tyr Ala Pro Asp Ala Asn Arg Asp Phe
210 215 220
Lys Thr Asp Phe Tyr Pro Gly Ala Ser His Val Asp Ile Val Gly Leu
225 230 235 240
Asp Ala Tyr Phe Asp Asp Pro His Ala Ile Asp Gly Tyr Asp Gln Leu
245 250 255
Thr Ser Leu Asn Lys Pro Phe Ala Phe Thr Glu Val Gly Pro Gln Thr
260 265 270
Thr Asn Gly Gly Leu Asp Tyr Ala Arg Phe Ile His Ala Ile Lys Glu
275 280 285
Lys Tyr Pro Asn Thr Thr Tyr Phe Leu Ala Trp Asn Asp Gly Trp Ser
290 295 300
Pro Ala Val Asn Lys Gly Ala Gly Ala Leu Tyr Leu His Pro Trp Thr
305 310 315 320
Leu Asn Lys Gly Asp Ile Trp Asp Gly Asp Ser Leu Thr Pro Val Val
325 330 335
<210> 7
<211> 1008
<212> DNA
＜213〉recombination sequence
<220>
＜221〉encoding gene
<222> (1)..(1008)
<400> 7
gcacacaccg tttctccggt gaacccgaat gcccagccga cgacgaaagc ggtgatgaac 60
tggcttgccc acctgcccaa tcggacggaa aatcgggtaa tgtccggggc attcggagga 120
tacagccttg acacattctc gctggctgaa gccgaccgga tcaaacaggc aacaggacag 180
ctgccagccg tatacggctg cgattatgca agaggatggc tggagccgga ggagatcgcc 240
gatacgattg actacagctg caacagcgat ttgatcgcat actggaaaag cggaggcata 300
ccgcgaatca gcctgcacct cgcaaacccc gcgtttactt cgggtcatta taaaactcag 360
atttcgaaca gccagtatga gagaatttta gattcttcca cacccgaagg aaaacggctt 420
gaggcgatgc tgagcaaaat cgccgatggc cttcaggagc ttgaaaatga aggtgtgccc 480
gttctgttca gaccccttca cgaaatgaac ggcgaatggt tctggtgggg actgacgcaa 540
tataatcaaa aagacagcgc gagaatctcc ttgtacaaac ggctctatgt gaaaatctat 600
gactatatga caaagacaag aggcttggat catctgttgt gggtgtatgc gcctgatgcc 660
aacagagact ttaaaacaga cttttatccg ggcgcatcat atgttgacat cgtcgggctt 720
gacgcttatt ttgatgaccc gcacgccatt gatggctacg atcagctcac atctctgaac 780
aagccgtttg cctttacaga ggtcgggcca cagacgacaa acggcgggct ggattacgcg 840
cggtttatcc atgcaatcaa agagaaatat ccgaatacga cgtacttcct ggcgtggaac 900
gatgggtgga gccctgctgt gaataaggga gcgggcgccc tctatcttca tccatggacg 960
ctgaataaag gagacatctg ggacggcgat tctttgacgc ctgtcgtg 1008
<210> 8
<211> 336
<212> PRT
＜213〉recombination sequence
<220>
＜221〉mannase
<222> (1)..(336)
<400> 8
Ala His Thr Val Ser Pro Val Asn Pro Asn Ala Gln Pro Thr Thr Lys
1 5 10 15
Ala Val Met Asn Trp Leu Ala His Leu Pro Asn Arg Thr Glu Asn Arg
20 25 30
Val Met Ser Gly Ala Phe Gly Gly Tyr Ser Leu Asp Thr Phe Ser Leu
35 40 45
Ala Glu Ala Asp Arg Ile Lys Gln Ala Thr Gly Gln Leu Pro Ala Val
50 55 60
Tyr Gly Cys Asp Tyr Ala Arg Gly Trp Leu Glu Pro Glu Glu Ile Ala
65 70 75 80
Asp Thr Ile Asp Tyr Ser Cys Asn Ser Asp Leu Ile Ala Tyr Trp Lys
85 90 95
Ser Gly Gly Ile Pro Arg Ile Ser Leu His Leu Ala Asn Pro Ala Phe
100 105 110
Thr Ser Gly His Tyr Lys Thr Gln Ile Ser Asn Ser Gln Tyr Glu Arg
115 120 125
Ile Leu Asp Ser Ser Thr Pro Glu Gly Lys Arg Leu Glu Ala Met Leu
130 135 140
Ser Lys Ile Ala Asp Gly Leu Gln Glu Leu Glu Asn Glu Gly Val Pro
145 150 155 160
Val Leu Phe Arg Pro Leu His Glu Met Asn Gly Glu Trp Phe Trp Trp
165 170 175
Gly Leu Thr Gln Tyr Asn Gln Lys Asp Ser Ala Arg Ile Ser Leu Tyr
180 185 190
Lys Arg Leu Tyr Val Lys Ile Tyr Asp Tyr Met Thr Lys Thr Arg Gly
195 200 205
Leu Asp His Leu Leu Trp Val Tyr Ala Pro Asp Ala Asn Arg Asp Phe
210 215 220
Lys Thr Asp Phe Tyr Pro Gly Ala Ser Tyr Val Asp Ile Val Gly Leu
225 230 235 240
Asp Ala Tyr Phe Asp Asp Pro His Ala Ile Asp Gly Tyr Asp Gln Leu
245 250 255
Thr Ser Leu Asn Lys Pro Phe Ala Phe Thr Glu Val Gly Pro Gln Thr
260 265 270
Thr Asn Gly Gly Leu Asp Tyr Ala Arg Phe Ile His Ala Ile Lys Glu
275 280 285
Lys Tyr Pro Asn Thr Thr Tyr Phe Leu Ala Trp Asn Asp Gly Trp Ser
290 295 300
Pro Ala Val Asn Lys Gly Ala Gly Ala Leu Tyr Leu His Pro Trp Thr
305 310 315 320
Leu Asn Lys Gly Asp Ile Trp Asp Gly Asp Ser Leu Thr Pro Val Val
325 330 335
<210> 9
<211> 1008
<212> DNA
＜213〉recombination sequence
<220>
＜221〉encoding gene
<222> (1)..(1008)
<400> 9
gcacacaccg tttctccggt gaacccgaat gcccagccga cgacgaaagc ggtgatgaac 60
tggctcgccc acctgcccaa tcggacggaa agccgggtga tgtccggggc gttcggagga 120
tacagcctcg acacattttc aacggctgaa gccgaccgga tcaaacaggc aacaggacag 180
ctgccggcca tatacggctg cgattatgca agaggatggc tggagccgga aaagatcgcc 240
gatacgattg actacagctg caaccgtgat ctgatcgcat actggaaaag cggaggcatt 300
ccgcaaatca gcatgcacct cgcaaacccc gcgtttactt cgggtcatta taaaactcag 360
attccaaaca gccagtatga gagaatttta gattcttcca ctcctgaagg aaagcggctt 420
gaggcgatgc tgagcaaaat cgcggacggc ctccaggagc ttgaaaatga aggcgtgccc 480
gttctattca gaccccttca cgaaatgaac ggcgaatggt tctggtgggg gctgacgcaa 540
tataatcaaa aagacagcga aagaatctcc ttgtacaaac agctctatgt gaaaatctat 600
gactatatga caaaaacaag aggcctggat catctcttgt gggtgtatgc gccggacgcc 660
aacagagact ttaaaacgga cttttatccg ggcgcatcat atgtggacat tgtcgggctt 720
gacgcttatt ttgatgaccc gtacgccatt gatggctacg aggagctcac atcgctgaac 780
aagccgtttg cctttacaga agtcggaccg cagacgacaa acggcgggct ggattacgcg 840
cggtttatcc atgccatcaa agaaaaatac ccgaaaacga cgtacttcct ggcgtggaac 900
gatgagtgga gcccggctgt gaataaggga gcggacaccc tctatcttca tccatggacg 960
ctgaataaag gagagatatg ggacggcgat tctttgacgc ctgtcgtg 1008
<210> 10
<211> 336
<212> PRT
＜213〉recombination sequence
<220>
＜221〉mannase
<222> (1)..(336)
<400> 10
Ala His Thr Val Ser Pro Val Asn Pro Asn Ala Gln Pro Thr Thr Lys
1 5 10 15
Ala Val Met Asn Trp Leu Ala His Leu Pro Asn Arg Thr Glu Ser Arg
20 25 30
Val Met Ser Gly Ala Phe Gly Gly Tyr Ser Leu Asp Thr Phe Ser Thr
35 40 45
Ala Glu Ala Asp Arg Ile Lys Gln Ala Thr Gly Gln Leu Pro Ala Ile
50 55 60
Tyr Gly Cys Asp Tyr Ala Arg Gly Trp Leu Glu Pro Glu Lys Ile Ala
65 70 75 80
Asp Thr Ile Asp Tyr Ser Cys Asn Arg Asp Leu Ile Ala Tyr Trp Lys
85 90 95
Ser Gly Gly Ile Pro Gln Ile Ser Met His Leu Ala Asn Pro Ala Phe
100 105 110
Thr Ser Gly His Tyr Lys Thr Gln Ile Pro Asn Ser Gln Tyr Glu Arg
115 120 125
Ile Leu Asp Ser Ser Thr Pro Glu Gly Lys Arg Leu Glu Ala Met Leu
130 135 140
Ser Lys Ile Ala Asp Gly Leu Gln Glu Leu Glu Asn Glu Gly Val Pro
145 150 155 160
Val Leu Phe Arg Pro Leu His Glu Met Asn Gly Glu Trp Phe Trp Trp
165 170 175
Gly Leu Thr Gln Tyr Asn Gln Lys Asp Ser Glu Arg Ile Ser Leu Tyr
180 185 190
Lys Gln Leu Tyr Val Lys Ile Tyr Asp Tyr Met Thr Lys Thr Arg Gly
195 200 205
Leu Asp His Leu Leu Trp Val Tyr Ala Pro Asp Ala Asn Arg Asp Phe
210 215 220
Lys Thr Asp Phe Tyr Pro Gly Ala Ser Tyr Val Asp Ile Val Gly Leu
225 230 235 240
Asp Ala Tyr Phe Asp Asp Pro Tyr Ala Ile Asp Gly Tyr Glu Glu Leu
245 250 255
Thr Ser Leu Asn Lys Pro Phe Ala Phe Thr Glu Val Gly Pro Gln Thr
260 265 270
Thr Asn Gly Gly Leu Asp Tyr Ala Arg Phe Ile His Ala Ile Lys Glu
275 280 285
Lys Tyr Pro Lys Thr Thr Tyr Phe Leu Ala Trp Asn Asp Glu Trp Ser
290 295 300
Pro Ala Val Asn Lys Gly Ala Asp Thr Leu Tyr Leu His Pro Trp Thr
305 310 315 320
Leu Asn Lys Gly Glu Ile Trp Asp Gly Asp Ser Leu Thr Pro Val Val
325 330 335
<210> 11
<211> 1007
<212> DNA
＜213〉recombination sequence
<220>
＜221〉encoding gene
<222> (1)..(1007)
<400> 11
gcacacaccg tttctccggt gaacccgaat gcccagccga cgacgaaagc ggtgatgaac 60
tggctcgccc acctgcccaa tcggacggaa agccgggtga tgtccggggc gttcggagga 120
tacagcctcg acacattttc aacggctgaa gccgaccgga tcaaacaggc aacaggacag 180
ctgccggcca tatacggctg cgattatgca agaggatggc tggagccgga aaagatcgcc 240
gatacgattg actacagctg caaccgtgat ttgatcgcat actggaaaag cggaggcatt 300
ccgcaaatca gcatgcacct cgcaaacccc gcgtttactt cgggtcatta taaaactcag 360
attccaagca gccagtatga gagaatttta gattcttcca ctcctgaagg aaagcggctt 420
gaggcgatgc tgagcaaaat cgcggacggc ctccaggagc ttgaaaatga aggcgtgccc 480
gttctattca gaccccttca cgaaatgaac ggcgaatggt tctggtgggg gctgacgcaa 540
tataatcaaa aagacagcga aagaatctcc ttgtacaaac agctctatgt gaaaatctat 600
gactatatga caaaaacaag aggcctggat catctcttgt gggtgtatgc gccggacgcc 660
aacagagact ttaaaacgga cttttatccg ggcgcatcat atgtggacat tgtcgggctt 720
gacgcttatt ttgatgaccc gtacgccatt gatggctacg aagagctcac atcgctgaac 780
aagccgtttg cctttacaga agtcggaccg cagacgacaa acggcgggct ggattacgcg 840
cggtttatcc atgccatcaa agaaaaatac ccgaaaacga cgtacttcct ggcgtggaac 900
gatgagtgga gcccggctgt gaataaggga gcggacaccc tctatcttca tccatggacg 960
ctgaataaag gagagatatg ggacggcgat tctttgacgc ctgtgtg 1007
<210> 12
<211> 335
<212> PRT
＜213〉recombination sequence
<220>
＜221〉mannase
<222> (1)..(335)
<400> 12
Ala His Thr Val Ser Pro Val Asn Pro Asn Ala Gln Pro Thr Thr Lys
1 5 10 15
Ala Val Met Asn Trp Leu Ala His Leu Pro Asn Arg Thr Glu Ser Arg
20 25 30
Val Met Ser Gly Ala Phe Gly Gly Tyr Ser Leu Asp Thr Phe Ser Thr
35 40 45
Ala Glu Ala Asp Arg Ile Lys Gln Ala Thr Gly Gln Leu Pro Ala Ile
50 55 60
Tyr Gly Cys Asp Tyr Ala Arg Gly Trp Leu Glu Pro Glu Lys Ile Ala
65 70 75 80
Asp Thr Ile Asp Tyr Ser Cys Asn Arg Asp Leu Ile Ala Tyr Trp Lys
85 90 95
Ser Gly Gly Ile Pro Gln Ile Ser Met His Leu Ala Asn Pro Ala Phe
100 105 110
Thr Ser Gly His Tyr Lys Thr Gln Ile Pro Ser Ser Gln Tyr Glu Arg
115 120 125
Ile Leu Asp Ser Ser Thr Pro Glu Gly Lys Arg Leu Glu Ala Met Leu
130 135 140
Ser Lys Ile Ala Asp Gly Leu Gln Glu Leu Glu Asn Glu Gly Val Pro
145 150 155 160
Val Leu Phe Arg Pro Leu His Glu Met Asn Gly Glu Trp Phe Trp Trp
165 170 175
Gly Leu Thr Gln Tyr Asn Gln Lys Asp Ser Glu Arg Ile Ser Leu Tyr
180 185 190
Lys Gln Leu Tyr Val Lys Ile Tyr Asp Tyr Met Thr Lys Thr Arg Gly
195 200 205
Leu Asp His Leu Leu Trp Val Tyr Ala Pro Asp Ala Asn Arg Asp Phe
210 215 220
Lys Thr Asp Phe Tyr Pro Gly Ala Ser Tyr Val Asp Ile Val Gly Leu
225 230 235 240
Asp Ala Tyr Phe Asp Asp Pro Tyr Ala Ile Asp Gly Tyr Glu Glu Leu
245 250 255
Thr Ser Leu Asn Lys Pro Phe Ala Phe Thr Glu Val Gly Pro Gln Thr
260 265 270
Thr Asn Gly Gly Leu Asp Tyr Ala Arg Phe Ile His Ala Ile Lys Glu
275 280 285
Lys Tyr Pro Lys Thr Thr Tyr Phe Leu Ala Trp Asn Asp Glu Trp Ser
290 295 300
Pro Ala Val Asn Lys Gly Ala Asp Thr Leu Tyr Leu His Pro Trp Thr
305 310 315 320
Leu Asn Lys Gly Glu Ile Trp Asp Gly Asp Ser Leu Thr Pro Val
325 330 335
Claims (8)
1. restructuring mannase, the aminoacid sequence of wherein said restructuring mannase is SEQ ID NO:4.
2. the DNA molecular of coding claim 1 described mannase.
3. DNA molecular claimed in claim 2, its nucleotides sequence is classified SEQ ID NO:3 as.
4. a recombinant expression plasmid, wherein contain the described DNA molecular of claim 2 or 3.
5. expressive host, it is transformed by recombinant expression plasmid claimed in claim 4.
6. expressive host claimed in claim 5, its be selected from intestinal bacteria (
Escherichia coli), yeast or genus bacillus.
7. expressive host claimed in claim 6, wherein said yeast be pichia pastoris phaff (
Pichia pastoris).
8. expressive host claimed in claim 6, wherein said genus bacillus be subtilis (
Bacillus subtilis) or Bacillus licheniformis (
Bacillus licheniformis).
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| TWI494431B (en) * | 2013-04-17 | 2015-08-01 | Genozyme Biotech Inc | Mannanase having improved enzymatic activity |
| CN103740682B (en) * | 2014-01-17 | 2016-01-06 | 北京科为博生物科技有限公司 | A kind of high temperature resistant acidic 'beta '-mannase Man-L30 and gene thereof and application |
| CN107083374B (en) * | 2017-06-28 | 2019-11-26 | 青岛红樱桃生物技术有限公司 | The beta-mannase enzyme mutant and its encoding gene and application that enzymatic activity improves |
| CN111363735B (en) * | 2020-04-09 | 2021-07-20 | 中国海洋大学 | Beta-mannase heat-resistant mutant, recombinant bacterium and application thereof |
| CN116218817A (en) * | 2022-12-30 | 2023-06-06 | 山东龙昌动物保健品有限公司 | Beta-mannase mutant beta-ManM, additive prepared from beta-mannase mutant beta-ManM and bile acid and application of additive |
| CN116042581B (en) * | 2022-12-30 | 2024-05-10 | 山东龙昌动物保健品股份有限公司 | Co-expression mutant enzyme recombinant bacterium, preparation thereof and application thereof in preparation of feed additive for aquatic animals |
| CN117467648B (en) * | 2023-12-27 | 2024-04-23 | 潍坊新希望六和饲料科技有限公司 | Application of yeast zymolyte in preparation of feed additive |
| CN118048346A (en) * | 2024-04-15 | 2024-05-17 | 北京科为博生物科技有限公司 | Enzyme mutant with improved thermal stability, gene and application thereof |
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| Publication number | Publication date |
|---|---|
| CN102277342A (en) | 2011-12-14 |
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