CN103667216B - A kind of mannase and application thereof - Google Patents
A kind of mannase and application thereof Download PDFInfo
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- CN103667216B CN103667216B CN201310606719.5A CN201310606719A CN103667216B CN 103667216 B CN103667216 B CN 103667216B CN 201310606719 A CN201310606719 A CN 201310606719A CN 103667216 B CN103667216 B CN 103667216B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2491—Beta-mannosidase (3.2.1.25), i.e. mannanase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01025—Beta-mannosidase (3.2.1.25), i.e. mannanase
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Abstract
The present invention relates to gene engineering technology field, specifically provide a kind of mannase gene and the application thereof that derive from subtilis.The present invention, by being imported in pichia spp by the mannase gene of subtilis, constructs the Pichia yeast engineering of this gene recombinant expressed.Described Pichia yeast engineering energy high expression mannase, shake-flask fermentation enzyme activity reaches 912U/mL.The suitableeest action pH of restructuring mannase is 4, and optimum temperature is 58 DEG C, can be widely used in feed additive field.
Description
Technical field
The invention belongs to microbial engineering field, be specifically related to a kind of mannase and application thereof.
Background technology
Mannase is a kind of hydrolysis of hemicellulose enzyme, and with internal-cutting way degraded β-Isosorbide-5-Nitrae glycosidic link, the non-reducing end of degraded product is seminose, and its substrate specificity comprises glucomannan, polygalactomannan and beta-mannase etc.It can not only reduce enteron aisle viscosity, promotes the absorption and digestion of nutritive substance, but also can eliminate the beta-mannase that is rich in beans to the interference of glucose absorption, greatly improves the energy digestibility of grouts especially dregs of beans; Meanwhile, after with the addition of mannase, the resistibility of animal and reguarity all increase.
Mannans material, as the second largest component of hemicellulose, is distributed widely in occurring in nature.It is the main moiety of all leguminous plants cell wallss, in other vegetality feedstuff, content is also very high, is respectively 22.7%, 11.9%, 19.6% and 33.7% as polygalactomannan in dregs of beans, wheat, rapeseed meal, wheat bran accounts for non-starch polysaccharide content.
The main daily ration of China pig, chicken is corn-soybean meal diet, although the digestibility of the monogastric animal such as pig, chicken to corn is higher, is only 50% ~ 60% to the capacity usage ratio of dregs of beans.The reason that monogastric animal is so low to the utilization ratio of dregs of beans energy may be contain in dregs of beans 22.7% hemicellulose be can not by the non-starch polysaccharide of simple stomach animal digestion.Add mannase in feed can to degrade mannosans, reduce alimentary canal content viscosity, destroy plant feed cell wall structure, nutrition mass-energy is fully contacted with digestive ferment, improve the activity of endogenous enzyme, improve the functions such as the integrity of intestinal microflora and raising intestinal mucosa.Therefore, be Hot Contents to the research of mannase at present.
Summary of the invention
The object of this invention is to provide a kind of novel mannase and application thereof.The present invention by will derive from subtilis (
bacillus subtilis) mannase gene be transformed in pichia spp, build and obtain the engineering strain of high efficiency recombinant expressed mannase, thus make up the deficiencies in the prior art.
One aspect of the present invention provides a kind of mannase deriving from subtilis, it is characterized in that:
A () its aminoacid sequence is the mannase of SEQ ID NO:1;
B () amino acid in (a) replaces, lacks or adds one or several amino acid obtains, there is the enzyme of Mannanase Activity.
For the gene of above-mentioned mannase of encoding, wherein a kind of coding nucleotide sequence is SEQ ID NO:2.
Another aspect of the present invention relates to the application of above-mentioned mannase in feed.
The present invention, by being imported in pichia spp by the mannase gene of subtilis, constructs the Pichia yeast engineering of this gene recombinant expressed.Described Pichia yeast engineering energy high expression mannase, shake-flask fermentation enzyme activity reaches 912U/mL.The suitableeest action pH of restructuring mannase is 4, and optimum temperature is 58 DEG C.Under the condition reducing dietary digestibility of energy value, by adding mannase of the present invention in daily ration, experimental group broiler chicken day weight gain, fatten index than positive control group respectively high by 2%, 2.6%, feedstuff-meat ratio reduces 5.6% than positive control group, illustrates that mannase of the present invention can significantly improve the utilization ratio of feed, improves the day weight gain of broiler chicken and fattens index, reduce feed-weight ratio, thus the usage quantity of feed in livestock and poultry cultivation can be reduced, resource of saving food and aquaculture cost, increase productivity effect.
Embodiment
Below in conjunction with example, method of the present invention is described further, the experimental technique of unreceipted actual conditions in embodiment, usually can condition routinely, condition as described in " Molecular Cloning: A Laboratory guide " that J. Pehanorm Brooker (Sambrook) etc. is write, or run according to the condition that manufacturer advises.Those skill in the art related can understand better by embodiment and grasp the present invention.But protection of the present invention and right are not limited to provided case.
the clone of embodiment 1 mannase gene
The fresh subtilis CGMCC1.897(of picking is purchased from China General Microbiological culture presevation administrative center) single bacterium colony is in 5mL LB liquid nutrient medium, 37 DEG C of shaking table 200rpm shaking culture are spent the night, and karyomit(e) is extracted in the explanation of extracting test kit (Tian Gen biochemical technology company limited) according to Tiangen bacterial genomes.
According to mannase homologous sequence design primer on NCBI, with subtilis CGMCC1.897 karyomit(e) for template, upstream and downstream primer is utilized to increase, amplification condition is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 40s, 60 DEG C of annealing 40s, 72 DEG C extend 60s, after 30 circulations, 72 DEG C extend 10min.Gel reclaims test kit and reclaims pcr amplification product, by this product called after
man-1.
Will
man-1be cloned into pMD18-T carrier, build and obtain plasmid pT-Man-1, deliver to Hua Da gene sequencing center, Beijing and check order, record
man-1gene order is SEQ ID NO:1, and its encoding amino acid sequence is SEQ ID NO:2.This sequence is carried out the comparison of NCBI homologous sequence, determine
man-1gene is a new mannase gene, is up to 88% with existing open sequence similarity.
the structure of embodiment 2 expression vector
With plasmid pT-Man-1 for template, utilize primer to carry out pcr amplification, pcr amplification condition is 95 DEG C of 4min; 94 DEG C of 30S; 55 DEG C of 40S, 72 DEG C of 1.2min 30 circulations; 72 DEG C of 7min.Gel reclaims amplified production;
ecorI and Not I double digestion.Expression plasmid pPIC9K is also carried out
ecorI and Not I double digestion; With T4 ligase enzyme above-mentioned double digestion product 4 DEG C connected and spend the night.Finally, connection product is imported e. coli bl21.Corresponding positive colony expression plasmid called after pPIC-Man-1.
the structure of embodiment 3 Pichia yeast engineering
Expression plasmid pPIC-Man-1 is used
salafter the qualification of I restriction enzyme digestion and electrophoresis, concentrate through alcohol settling, measure DNA concentration, save backup with 3 μ g/ μ L concentration dilution plasmid fragments.Prepare Pichia pastoris GS115 Electroporation-competent cells, be finally resuspended in the electrophoretic buffer of 1 mL precooling (containing 1mM MgCl
2, 10mM HEPES, 250mM sucrose, pH 7.8).5 μ L linearizing recombinant plasmids are added in 80 μ L competent cells; Electricity transforms (condition is 1500V, 200 Ω, 25 μ F); Finally coat MM flat board (MM nutrient media components: 1.34%YNB, 4 × 10
-5% vitamin H, 0.5% methyl alcohol), select a recombinant bacterial strain, called after pichia spp Man-1(
pichia pastorisman-1).
embodiment 4 is fermented and zymologic property measures
Pichia yeast engineering Man-1 is inoculated in 5ml BMGY (1% yeast extract, 2% peptone, 1. 34 % YNB, 4 × 10
-5% vitamin H, l% glycerine), 30 DEG C of overnight incubation, collected by centrifugation thalline, adds 50ml BMMY inducing culture (1% yeast extract, 2% peptone, 1. 34 % YNB, 4 × 10 thalline
-5% vitamin H, 0.5% methyl alcohol), within every 12 hours, add 50 μ L methyl alcohol, inducing culture 5 days, 200rpm, centrifugal 5 minutes, get fermented liquid supernatant liquid, carry out zymologic property and tolerance analysis.
mannase enzyme activity determination method is as described below:
With 0.6% locust bean gum mannosans (Sigma company, Batch#125K0091) for substrate, with 0.1M Acetic acid-sodium acetate (pH5.5) for damping fluid, by mannan substrate 37 DEG C balance 20min, by enzyme liquid to be measured 37 DEG C balance 10min.Get 4 test tubes, add enzyme liquid 2ml respectively, wherein manage as measuring for 3, add 2ml substrate solution respectively, another was as blank tube, adds 5ml DNS solution, 37 DEG C ± 0.5 DEG C water-bath 30 minutes.Then three mensuration pipes add 5ml DNS solution respectively, and blank tube adds 2ml substrate solution, reacts 5 minutes, be settled to 25ml after cooling in boiling water bath.With blank tube zeroing, survey absorbancy at spectrophotometer 540nn place.
Mannase enzyme is lived definition: 37 DEG C, under the condition of pH5.5, the amount that per minute hydrolysis substrate produces enzyme liquid needed for 1 μm of ol seminose is a mannosans activity unit.Determining the protein quantity is with reference to Bradford method.
The enzyme measuring fermented liquid supernatant according to said determination method is lived as 912U/mL, illustrates that the Pichia yeast engineering that the present invention builds can high efficiency recombinant expressed mannase.
1, optimal pH analysis
The damping fluid being respectively 2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0 by pH value carries out dilution metering, under temperature 55 DEG C of conditions, measure enzyme live, live as 100% with the highest enzyme, calculate relative enzyme and live, do the relative enzyme of pH-curve alive.Result shows: the optimum pH of restructuring mannase of the present invention is 4.0.
2, optimum temperuture analysis
Respectively at 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, measure enzyme under the condition of pH5.5 and live, live as 100% with the highest enzyme, calculate relative enzyme and live, do temperature-enzyme curve alive relatively.Result shows: the optimum temperuture of restructuring mannase of the present invention is 58 DEG C.
the application of embodiment 5 mannase of the present invention in livestock and poultry cultivation
This experiment reduces the metabolizable energy of 150kcal/kg on broiler chicken Dog ration basis, in daily ration, then add restructuring mannase of the present invention.By the evaluation to meat chicken production performance, inquire into mannase to the improvement amplitude of low energy dietary digestibility of energy utilization ratio.
Purchase the white plumage meat cock of 1 age in days Roche 308, be divided into positive and negative control group and experimental group, each treatment group 16 repetition, each repetition 8 plumage chicken, the production test phase is 35 days; Positive control group: Normal Goods daily ration treatment group; Negative control group: reduce 150kcal/kg metabolizable energy on contrast daily ration basis; Test group: add 100-200 gram of/ton restructuring mannase (50000U/g) of the present invention on negative contrast daily ration basis, test-results is as shown in the table:
| Group | Day weight gain (g) | Feedstuff-meat ratio | Fatten index |
| Positive control group | 70.11±4.76 | 1.59±0.18 b | 340.12±40.69 |
| Negative control group | 67.12±2.06 | 1.63±0.23 b | 331.42±30.33 |
| Experimental group | 71.50±4.80 | 1.50±0.11 a | 349.07±39.85 |
Experimental result shows, under the condition reducing dietary digestibility of energy value, by adding mannase in daily ration, experimental group broiler chicken day weight gain, fatten index than positive control group respectively high by 2%, 2.6%, feedstuff-meat ratio reduces 5.6% than positive control group, illustrate that mannase of the present invention can significantly improve the utilization ratio of feed, improve the day weight gain of broiler chicken and fatten index, reducing feed-weight ratio, thus the usage quantity of feed in livestock and poultry cultivation can be reduced, resource of saving food and aquaculture cost, increase productivity effect.
SEQUENCE LISTING
<110> Qingdao Weilan Biology Group Co., Ltd.
<120> mannase and application thereof
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 360
<212> PRT
<213> mannase enzyme sequence
<400> 1
Met Leu Lys Leu Ile Ala Val Cys Leu Ser Ile Val Leu Leu Arg Leu
1 5 10 15
Gly Ala Ala Ser Ser Ile Glu Ala His Thr Val Tyr Pro Val Asn Pro
20 25 30
Asn Ala Gln Gln Thr Thr Lys Asp Ile Met Asn Trp Leu Ala His Leu
35 40 45
Leu Asn Arg Ser Asp Thr Arg Val Leu Ser Gly Val Phe Gly Gly Tyr
50 55 60
Ser Asp Val Thr Phe Ser Met Thr Glu Glu Asn Arg Leu Lys Asn Ala
65 70 75 80
Thr Gly Glu Cys Leu Ala Ile Tyr Gly Cys Asp Tyr Gly Arg Gly Trp
85 90 95
Leu Glu Thr Ala Asp Ile Thr Asp Thr Ile Asp Tyr Ser Cys Asn Ser
100 105 110
Ser Leu Ile Ser Tyr Cys Arg Arg Gly Gly Leu Pro Gln Val Arg Leu
115 120 125
His Leu Ala Asn Pro Ala Phe Gln Phe Gly Asn Tyr Lys Thr Ala Ile
130 135 140
Lys Lys Thr Leu Tyr Lys Asn Ile Leu Asp Pro Ser Thr Val Glu Gly
145 150 155 160
Lys Arg Leu Glu Ala Leu Leu Ser Lys Ile Ala Asp Gly Leu Thr Gln
165 170 175
Leu Arg Phe Gln Gly Val Thr Val Leu Phe Arg Pro Val Leu Glu Met
180 185 190
Asn Gly Glu Trp Phe Trp Trp Gly Leu Thr Gly Tyr Asn Gln Lys Asp
195 200 205
Thr Glu Arg Ile Ser Leu Tyr Lys Glu Leu Tyr Lys Lys Ile Tyr Arg
210 215 220
Tyr Met Thr Glu Thr Arg Gly Leu Asp Asn Leu Phe Arg Val Tyr Leu
225 230 235 240
Pro Asp Ala Asn Arg Asp Phe Lys Thr Asp Phe Tyr Pro Gly Ser Ser
245 250 255
Tyr Val Asp Ile Thr Gly Leu Asp Ala Tyr Ser Thr Asp Pro Tyr Ala
260 265 270
Ile Ser Gly Tyr Asp Ala Met Met Ser Leu Lys Arg Leu Ser Ala Phe
275 280 285
Val Glu Thr Gly Pro Ser Gly Asn Ile Gly Asn Phe Asp Tyr Ala Ala
290 295 300
Ser Ile Lys Ala Ile Arg Gln Lys Tyr Pro Glu Thr Thr Tyr Phe Leu
305 310 315 320
Thr Trp Asp Glu Gln Leu Arg Pro Val Ser Asn Gln Gly Ala Gln Ser
325 330 335
Leu Tyr Gln Asn Ser Trp Thr Leu Asn Lys Gly Glu Ile Leu Glu Leu
340 345 350
Arg Ser Leu Lys Pro Ile Val Asp
355 360
<210> 2
<211> 1083
<212> DNA
<213> mannase gene sequence
<400> 2
atgcttaaat tgatagccgt ctgcctgtct attgttttat tgcgcttagg agccgccagt 60
tcgattgaag cacatacagt ttatcctgtt aatccaaatg cccagcagac gacaaaagat 120
atcatgaact ggctggcgca cctgctcaac cgttccgata ccagggtctt gtccggtgtg 180
ttcggcgggt acagcgatgt cactttttca atgacagagg aaaaccgctt gaaaaacgcg 240
acgggagagt gtctcgcaat ctacggctgt gactatggga gaggatggct ggaaacagcg 300
gatatcaccg atactatcga ttacagctgc aacagcagct tgatctcata ctgtagaagg 360
ggcggtctcc ctcaagtcag gctgcatctt gcaaatccgg cttttcaatt cggaaactat 420
aaaacggcca tcaaaaagac cctgtacaaa aacatcctgg acccttcaac tgttgaagga 480
aaacggcttg aggcgctgct cagcaaaatc gccgacggcc ttactcagct gagatttcaa 540
ggcgtcaccg ttctgttcag gcctgttctc gaaatgaacg gcgagtggtt ctggtggggg 600
ctgacaggct acaaccaaaa agacacggaa agaatctcgc tgtacaaaga gctttacaag 660
aagatatacc gctatatgac agagacaaga ggattggata acctgttcag ggtgtatttg 720
cctgatgcca acagagactt taaaacagac ttctacccag gctcatctta tgtggatatt 780
accggtctgg acgcttactc cacggatccg tatgcgattt caggttatga tgcaatgatg 840
tctctgaaaa gactgtctgc ctttgtcgaa accggtccgt ccggcaatat cggaaacttt 900
gattatgctg cgtctattaa ggcgatcagg caaaagtatc ccgagaccac gtactttttg 960
acatgggatg aacaattgag gcctgtgtcc aatcaaggcg cgcaaagcct ttatcaaaac 1020
agctggacat taaataaagg cgaaattttg gaattgaggt ccttgaagcc gatcgtggac 1080
taa 1083
Claims (4)
1. a mannase, is characterized in that, the aminoacid sequence of described mannase is SEQ ID NO:1.
2. for the gene of mannase described in claim 1 of encoding.
3. gene as claimed in claim 2, its nucleotides sequence is classified as SEQ ID NO:2.
4. the application of mannase described in claim 1 in feed.
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| CN103667216B true CN103667216B (en) | 2015-10-28 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102943068A (en) * | 2012-09-05 | 2013-02-27 | 周海燕 | Mannase Man23 and gene modification thereof |
-
2013
- 2013-11-27 CN CN201310606719.5A patent/CN103667216B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102943068A (en) * | 2012-09-05 | 2013-02-27 | 周海燕 | Mannase Man23 and gene modification thereof |
Non-Patent Citations (3)
| Title |
|---|
| β-mannanase ameliorates viscosity-associated depression of growth in broiler chickens fed guar germ and hull fractions;LEE J T 等;《Poultry Science》;20031231;第82卷(第12期);1925-1931 * |
| β-甘露聚糖酶在家禽饲料中应用的研究进展;辛总秀;《畜牧与饲料科学》;20111231;第32卷(第5期);28-30 * |
| 异甘露聚糖酶基因的克隆表达及酶学性质分析;汪立平 等;《食品工业科技》;20121231;第33卷(第16期);第213页标题、摘要、左栏第1段、右栏第1段，第216页第3节结论 * |
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