CN102277342A - Mannose and mutants thereof - Google Patents
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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-1,4-D-mannase (EC 3.2.1.78) is called mannase again, and it can hydrolysis contain β-1, and 4-D-seminose 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 β-1, the mannosans that 4-D-seminose glycosidic bond is formed by connecting, side chain then are α-1, the semi-lactosi that 6-semi-lactosi glycosidic bond connects.Glucose-mannosans main chain is by β-1, and 4-D-seminose glycosidic bond is formed by connecting, but some seminoses are replaced by glucose on its main chain.
Hemicellulase comprises mannase, zytase and dextranase etc., is widely used in 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., and it is strong to have acid resistance, 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 an amount of mannase in feed, can improve the digestibility of feed, can reduce feed cost (Yang Hongkun etc., 2007, feed research again; 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 screening by bacterial classification and the mannase that natural bacterial strain produced of mutagenic and breeding 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 the 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 the ideal beta-mannanase for feeding.Orthogenesis is to utilize random mutation and screen the mass mutation body and obtain target protein, and its workload is big, randomness also very big (Zhang Hongying etc., 1999, Science Bulletin; Xu Qinkun etc., 2005, the biotechnology communication).Though and the design and rational workload is less relatively, often be difficult to find suitable amino acid sites suddenly change transformation (Zhang Xiuyan etc., 2006, biotechnology magazine; Wang Fan industry 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 big difference in active to each other (than living).This illustrates that these sudden changes/replacement site is to influence 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 reorganization mannase and mutant thereof, the aminoacid sequence of wherein said reorganization mannase:
(a) comprise be selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,, SEQ IDNO: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 IDNO: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 height ratio of the SEQ IDNO:12 mannase of living has good stability.Water-bath is 5 minutes under 70 ℃, 80 ℃, 85 ℃ and 90 ℃ of conditions, can also keep 90%, 30%, 20% and 18% enzyme to live respectively.The optimal pH analysis shows that 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 this method comprises:
(a) genomic dna of extraction Bacillus licheniformis;
(b) be template with the genomic dna that extracts, 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 of described pcr amplification use is to being
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 an e. coli bl21.
In specific embodiments of the present invention, the nucleotide sequence of described amplified production is respectively SEQ ID NO:1, SEQ IDNO: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 pichia spp GS115 bacterial strain, obtained the Pichia yeast engineering that efficiently expresses, for the 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 the described enzyme coding gene.For studying the influence of these sudden changes, carried out these mutator genes recombinant expressed respectively to the enzyme activity.By the Ni-agarose purification of recombinant proteins and analyze its zymologic property.The zymologic property analysis shows that there is evident difference in the enzyme of 6 mutant (than living) alive, illustrates that these site amino-acid residues are to influence the active critical sites of mannase.The invention provides 6 mannase mutant genes of Bacillus licheniformis and amino acid sequence corresponding 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 sudden change of these site amino-acid residues cause that enzyme lives than big-difference, 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 this method comprises:
(Sigma company Batch#125K0091) is substrate with 0.6% locust bean gum mannosans.With 0.1M acetic acid-sodium-acetate (pH5.5) is 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 enzyme liquid 2ml respectively, get wherein 3 conducts and measure pipe, add the 2ml substrate solution respectively, another adds 5ml DNS solution as blank pipe, 37 ℃ ± 0.5 ℃ water-bath 30 minutes, to measure pipe for three and add 5ml DNS solution respectively, blank pipe adds the 2ml substrate solution, reaction is 5 minutes in boiling water bath, be settled to 25ml after the cooling,, survey absorbancy at spectrophotometer 540nn place with the zeroing of blank pipe.Enzyme is lived and defined is under the condition of 37 ℃ of pH5.5, and the amount that the per minute hydrolysis substrate produces the required enzyme liquid of 1 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 to be with DNAMAN compare of analysis protein sequence
Fig. 2 is illustrated to be the phylogeny of Clustal pxrd analysis protein sequence
Fig. 3 is illustrated to be 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 proteinic orthogenesis and protein engineering transformation, efficiently expresses and lays the foundation for selecting highly active mannase to carry out 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 grasps the present invention by embodiment.But the case that provided is provided for protection of the present invention and claim scope.
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 removes supernatant; Add 200 μ l lysis buffers (60mM Tris-HCl, pH7.8,20mM Na-Ac, 1mM EDTA 1.5%SDS), acutely blows and beats 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; Twice of 70% washing with alcohol; At last exsiccant DNA is dissolved in ddH
2O.
1.2 gene clone
With the genome DNA extracted in 1.1 is template, utilizes primer that (GCACACACCGTTTCTCCGGTG and CACGACAGGCGTCAAAGAATCG) carried out pcr amplification respectively.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.
1.3 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, SEQID 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.The corresponding nucleic acids 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 result's 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, N31S, L48T, V64I, E78K, 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 proteic aminoacid sequence of biosoftware Clustal pxrd analysis, 6 mutant sibships are near especially, see Fig. 2.
Recombinant expressed and the purifying of embodiment 3 mannases in intestinal bacteria
Be template with plasmid pMDT-Man1, pMDT-Man2, pMDT-Man3, pMDT-Man4, pMDT-Man5 and pMDT-Man6 respectively, utilize primer (AGAGCTAGCGCACACACCGTTTCTCCGGTG---Nhe I and ACACTCGAGCACGACAGGCGTCAAAGAATCG---XhoI) to 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.After the amplified production gel reclaims, carry out Nhe I enzyme earlier and cut, reclaim enzyme then and cut product and carry out Xho I enzyme and cut.Equally, expression plasmid pET28a also being carried out Nhe I enzyme respectively cuts with Xho I enzyme and cuts.With the T4 ligase enzyme double digestion being produced product is that clone gene is connected for 4 ℃ with expression vector and spends the night.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 when being cultured to OD600=0.3 4-5 hour.Centrifugal collection express cell adds 1mL lysis buffer (50mM NaH then in-20 ℃ of frozen spending the night
2P0
4, 300mM NaCl, 10mM imidazole) and carry out ultrasonic disruption, carry out microscopic examination frequently, treat that method that the fully broken back of cell is provided according to the Ni-NTA SPIN test kit of QIAGEN company carries out protein purification and reclaim.Electrophoresis result shows that recovery obtains corresponding target albumen.
(Sigma company Batch#125K0091) is substrate with 0.6% locust bean gum mannosans.Be determined at the activity of reorganization mannase purified among the embodiment 3, step is as follows: with 0.1M acetic acid-sodium-acetate (pH5.5) is 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 enzyme liquid 2ml respectively, get wherein 3 conducts and measure pipe, add the 2ml substrate solution respectively, another adds 5ml DNS solution as blank pipe, 37 ℃ ± 0.5 ℃ water-bath 30 minutes, to measure pipe for three and add 5ml DNS solution respectively, blank pipe adds the 2ml substrate solution, reaction is 5 minutes in boiling water bath, be settled to 25ml after the cooling,, survey absorbancy at spectrophotometer 540nn place with the zeroing of blank pipe.Enzyme is lived and defined is under the condition of 37 ℃ of pH5.5, and the amount that the per minute hydrolysis substrate produces the required enzyme liquid of 1 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 is determined as 12～7020IU/mg than living and does not wait.Wherein SEQ ID NO.4 and SEQ ID NO.5 encoded protein Man4 and Man5 activity 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).The 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
GCACACACCGTTTCTCCGGTG[EcoRI] and ATA
CACGACAGGCGTCAAAGAATCG[Not I]) be template with plasmid pET-Man2, be that primer carries out pcr amplification with Man2-F and Man2-R.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 Eco RI and Not I double digestion.Equally,, then two enzymes are cut product and connect the back importing bacillus coli DH 5 alpha that spends the night, obtain recombinant expression plasmid pPIC-Man6 with Eco RI and Not I double digestion pPIC9K.
5.2 the structure of recombinant bacterial strain
Expression plasmid pPIC-Man2 concentrates through ethanol sedimentation after identifying with Sal I restriction enzyme digestion and electrophoresis, measures DNA concentration, preserves standby with 3 μ g/ μ L concentration dilution plasmid fragments.Preparation pichia spp GS115 electricity transformed competence colibacillus cell, be resuspended at last in the electrophoretic buffer of 1mL precooling (contain 1mM MgCl2,10mM HEPES, 250mM sucrose, pH 7.8).In 80 μ L competent cells, add 5 μ L linearizing recombinant plasmids; Electricity transforms (condition is 1500V, 200 Ω, 25 μ F); Coat MM flat board (MM nutrient media components: 1.34%YNB, 4 * 10 at last
-5The % vitamin H, 0.5% methyl alcohol), select recombinant bacterial strain.
5.3 shake a bottle abduction delivering
Engineering bacteria be will select and 5ml BMGY (1% yeast extract, 2% peptone, 1.34%YNB, 4 * 10 will be inoculated in
-5The % vitamin H, 1% 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 per 12 hours.
5.4 enzyme activity determination
Different time sampling and measuring supernatant enzyme is lived, and the result shows that it is 600IU/mL that the highest enzyme of its supernatant liquor is lived.
Claims (10)
1. reorganization mannase and mutant thereof, the aminoacid sequence of wherein said reorganization mannase:
(a) comprise be selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,, SEQ IDNO:10 or SEQ IDNO: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 IDNO:10 or SEQ ID NO:12 replace, lack or add one or several amino-acid residues on one of them and obtain.
2. the described reorganization mannase of claim 1, its aminoacid sequence be SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8,, SEQ ID NO:10 or SEQ ID NO:12.
3. the dna molecular of coding claim 1 or 2 described mannases.
4. the described dna molecular of claim 3, its nucleotides sequence is classified SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11 as.
5. a recombinant expression plasmid wherein contains claim 3 or 4 described dna moleculars.
6. expressive host, it is transformed by the described recombinant expression plasmid of claim 5.
7. the described expressive host of claim 6, it is selected from intestinal bacteria (Escherichia coli), yeast or genus bacillus.
8. the described expressive host of claim 6, wherein said yeast is pichia pastoris phaff (Pichia pastoris).
9. the described expressive host of claim 6, wherein said genus bacillus is subtilis (Bacillus subtilis) or Bacillus licheniformis (Bacillus licheniformis).
10. method for preparing the mannase of recombinating, this method comprises:
(a) genomic dna of extraction Bacillus licheniformis;
(b) be template with the genomic dna that extracts, 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.
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| CN107083374A (en) * | 2017-06-28 | 2017-08-22 | 青岛红樱桃生物技术有限公司 | β mannosans enzyme mutant and its encoding gene and application that enzymatic activity is improved |
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| CN116004585A (en) * | 2022-12-30 | 2023-04-25 | 山东龙昌动物保健品有限公司 | Eucalyptus essential oil mutant enzyme preparation and application thereof in livestock cultivation |
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| TWI494431B (en) * | 2013-04-17 | 2015-08-01 | Genozyme Biotech Inc | Mannanase having improved enzymatic activity |
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