CN104711241B - A kind of acidic beta dextranase NGlu and its gene and application - Google Patents
A kind of acidic beta dextranase NGlu and its gene and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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Abstract
The present invention relates to genetic engineering and field of fermentation engineering, in particular it relates to a kind of beta glucan enzyme NGlu and its gene and application.The invention provides a kind of new beta glucan enzyme NGlu, it has the amino acid sequence as shown in SEQ ID NO.1, and present invention also offers the gene GNGlu for encoding above-mentioned new beta glucan enzyme, its nucleotide sequence is as shown in SEQ ID NO.2, and present invention also offers the recombinant vector comprising said gene, recombinant bacterial strain and its application.The new beta glucan enzyme of the present invention can largely reduce production cost in the industrial production by that can reach higher fermentation level after high efficient expression, it is had a good application prospect in the industry such as feed, brewing.
Description
Technical field
The present invention relates to genetic engineering and field of fermentation engineering, in particular it relates to a kind of 1,4 beta-glucanase NGlu
And its gene and application.
Background technology
Beta glucan is widely present in plant cell wall, is to connect what is formed by the glycosidic bond of β -1,3 and β-Isosorbide-5-Nitrae mixing
Dextran polymer, is a kind of structural SNSP (Non-starch polysaccharides, NSP), especially in standing grain
Content is higher in the aleurone and Formation of Endosperm Cell Walls of cereal (such as barley, oat, rye and wheat).Beta glucan is divided into water-soluble
Property and two kinds of water-insoluble, the large percentage shared by water miscible beta glucan can be with water formation sticky mass, and β-Portugal
Glycan concentration is higher, and viscosity is bigger.
1,4 beta-glucanase (β-Glucanase, E.C 3.2.1.73) is a kind of hydrolysis of hemicellulose enzyme, is mainly derived from micro-
Biology, including bacterium (bacillus), fungi (aspergillus, Mucor, trichoderma etc.) and rumen microorganism etc..1,4 beta-glucanase can be hydrolyzed
β-glycosidic bond forms the small-molecule substance of solubility, the industries such as feed, brewing is can be widely applied to, with good market
Application prospect.
1st, application of the 1,4 beta-glucanase in terms of feed
Developed rapidly with the feed industry of China, the shortage problem of feedstuff is increasingly serious, some are unconventional to raise
Material raw material such as barley, wheat, palm kernel meal etc. be also widely applied, but in these raw materials the ANFs such as beta glucan content compared with
Height, on the one hand because nonruminant can not secrete endogenous 1,4 beta-glucanase, it is impossible to digestibility and utilization beta glucan, another aspect β-
The presence of glucan can make the chyme in animal alimentary canal produce higher vicidity, influence the digestion of other nutriments to inhale
Receive, therefore beta glucan turns into a kind of main ANFs in feed, seriously reduces the digestibility and utilization of these raw materials.
1,4 beta-glucanase is as a kind of fodder enzyme, and the anti-nutrition that can efficiently solve beta glucan in feedstuff is made
With it, which is acted on, mainly includes the following aspects：
(1) 1,4 beta-glucanase can effectively degrade the beta glucan in feedstuff, improve the digestive utilization ratio of feed.
(2) 1,4 beta-glucanase can improve the ecological environment and function of animal alimentary canal.Beta glucan enzyme hydrolysis beta glucan
Afterwards, the viscosity of chyme is substantially reduced, and intestine evacuation velocity is improved, and is reduced in enteron aisle and is fermented, and what especially anaerobe was fermented can
Energy property, so as to reduce the generation of animal intestinal tract disease.
(3) promote growth of animal, improve body's immunity.Research shows, in the high level SNSP basal diets such as wheat class
Add after NSP enzymes based on 1,4 beta-glucanase, it is outer except digesting and assimilating for nutritional ingredient can be strengthened, moreover it is possible to influence animal refreshing
Through endocrine metabolic diseases, so as to influence metabolism, promote growth.
2nd, application of the 1,4 beta-glucanase in terms of brewing
Beer is as a kind of popular low alcoholic beverage, and consumption figure is in ascendant trend year by year, according to statistics, in 2010
State's beer production reaches 43,000,000 tons, occupies the first in the world within continuous 9 years.
Need to use barley in Beer Brewage, wherein beta glucan content is up to 4%-8%, β present in barley-
Glucan can increase wheat juice viscosity in saccharification, and the polymer substance such as easy adsorbed proteins causes wheat poor " hardened ", so as to lead
Cause wheat juice and beer filtration difficult, reduce wheat juice yield；In addition, barley beta-glucan can not be utilized or decomposed, Yi Yin by yeast
Beer haze and gel precipitation are played, the stability of beer is influenceed, the quality of beer is reduced, the residual of beta glucan can cause beer
Wine body is muddy, durable lather power is reduced and it is not strong to hang cup power.
Due to the influence of beta glucan in barley, the economic loss caused every year to beer industry is estimated on 2,000,000,000 yuan of left sides
The right side, a problem as puzzlement beer industry development.Nearest research and applicable cases shows that 1,4 beta-glucanase can be effectively
The beta glucan in beer saccharification process barley is removed, this problem can be fundamentally solved.
1,4 beta-glucanase is applied in Beer Brewage, can decompose the beta glucan gel in barley and malt, reduces beer
The turbidity of wine, improves beer product quality, so as to achieve good economic benefit.At present, the U.S., Japan, Germany, plus take
It is big to wait country using main enzyme preparation of the 1,4 beta-glucanase as brewing industry.
1,4 beta-glucanase is as a kind of brewing enzyme, and it, which is acted on, mainly includes the following aspects：
(1) effectively degraded barley endosperm cell in beta glucan, increase yeast can tunning, improve Beer Brewage obtain
Rate.1,4 beta-glucanase can decompose the very high beta glucan of viscosity in barley in specific manner, can loose barley endosperm cell membrane, promote
Enter the excessive of cellular content, utilization rate of the yeast to raw material is improved, so as to improve beer production.
(2) wort viscosity is reduced, improves strainability, the rate of filtration is improved, the limpid degree of the wheat juice after filtering is improved.
(3) colloidal stability of beer is improved, the cold concrete caused by beta glucan is removed.
In addition to use above, 1,4 beta-glucanase can be additionally used in terms of sugaring, environmental protection and resource processing, application prospect
It is boundless.
The invention provides a kind of new 1,4 beta-glucanase, and can in high efficient expression in Pichia pastoris this eukaryotic system,
With important industrial application value.
The content of the invention
It is an object of the invention to provide a kind of new 1,4 beta-glucanase NGlu.
It is a further object of the present invention to provide the gene GNGlu for encoding above-mentioned new 1,4 beta-glucanase.
It is a further object of the present invention to provide the recombinant vector for including above-mentioned new 1,4 beta-glucanase NGlu.
It is a further object of the present invention to provide the recombinant bacterial strain for including above-mentioned new beta glucan enzyme gene GNGlu.
It is a further object of the present invention to provide the method for preparing above-mentioned new 1,4 beta-glucanase NGlu.
It is a further object of the present invention to provide above-mentioned new 1,4 beta-glucanase NGlu application.
The present invention obtains new 1,4 beta-glucanase NGlu, its amino acid sequence from aspergillus niger (Aspergillus niger)
Row are as shown in SEQ ID NO.1：
The zymoprotein is made up of 354 amino acid, is analyzed through SIGNAIP, and signal peptide sequence is not present in the amino acid sequence
Row, its theoretical pI/Mw：4.98/37165.33.
Present invention also offers the gene GNGlu for encoding above-mentioned new 1,4 beta-glucanase, the sequence of the gene is 1065bp,
Specifically as shown in SEQ ID NO.2：
After testing, enzyme enzyme activity under conditions of pH 2.0~6.5 is stable, belongs to acidic dextranase, through proteasome
Outer hydrolysising experiment, show the 1,4 beta-glucanase that obtains of the present invention have good resistance to pepsin and Trypsin Induced ability with
And preferable resistance to elevated temperatures.Under the conditions of simulation brewing, the enzyme also has good water to the beta glucan in malt
Solve effect.
Present invention also offers the recombinant vector for including above-mentioned new beta glucan enzyme gene GNGlu, preferably pPICz α
A-GNGlu.It is preferably to insert the new beta glucan enzyme gene of the present invention as the most preferred embodiment of the present invention
Enter between EcoR I and the NotI restriction enzyme sites on plasmid pPICz α A, make the nucleotide sequence positioned at AOX1 start
The downstream of son is simultaneously regulated and controled by it, obtains expression of recombinant yeast plasmid pPICz α A-GNGlu.
Present invention also offers the recombinant bacterial strain for including above-mentioned new beta glucan enzyme gene GNGlu, preferably finish red ferment
Female recombinant bacterial strain X33.
Present invention also offers a kind of method for preparing new 1,4 beta-glucanase NGlu, it is characterised in that it is main include with
Lower step：
1) host cell X33 is converted with above-mentioned recombinant vector pPICz α A-GNGlu, screening obtains recombinant bacterial strain；
2) recombinant bacterial strain, induction production 1,4 beta-glucanase are cultivated；And
3) reclaim and purify expressed new 1,4 beta-glucanase NGlu.
Present invention also offers above-mentioned new 1,4 beta-glucanase NGlu application, preferably it is used as feed, brewing, system
The industrial circles such as sugar are used.The new 1,4 beta-glucanase of the present invention is a kind of acid 1,4 beta-glucanase, stability and heat resistance
It is excellent, and with good antipepsin and Trypsin Induced ability, comply fully with preferred various industrial circles
Using.The new 1,4 beta-glucanase of the present invention is existing with the market by that can reach higher fermentation level after fermentation expression
Various 1,4 beta-glucanases compare, with obvious market competition advantage.
Brief description of the drawings
Liquid fermentation conditional curve of the new 1,4 beta-glucanases of Fig. 1 in 25L tanks.
The optimal reactive temperature of the new 1,4 beta-glucanases of Fig. 2 determines curve.
The optimal reaction pH value of the new 1,4 beta-glucanases of Fig. 3 determines curve.
The heat resistance curve of the new 1,4 beta-glucanases of Fig. 4.
The bin stability energy test curve of the new 1,4 beta-glucanases of Fig. 5.
The new 1,4 beta-glucanase antipepsins of Fig. 6, the performance test of trypsase.
Influence of each metal ion species of Fig. 7 to new 1,4 beta-glucanase.
Embodiment
Do not make the experimental methods of molecular biology illustrated, equal reference in following examples《Molecular Cloning:A Laboratory guide》
Listed specific method is carried out in the book of (third edition) J. Pehanorm Brookers one, or is carried out according to kit and product description；
The reagent and biomaterial, unless otherwise specified, are commercially obtained.
Experiment material and reagent：
1st, bacterial strain and carrier
Coli strain Topl0, Pichia pastoris X33, carrier pPICzaA are purchased from Invitrogen companies.
2nd, enzyme and kit
Reverse transcriptase SuperScriptTMIII, RNA extracts kit are purchased from Invitrogen companies.Extraction of plasmid DNA is tried
Agent box, glue reclaim kit, PCR purification kits, ligase, restriction enzyme are purchased from Shanghai Sheng Gong companies.
3rd, culture medium
Basic salt culture medium (BSM culture mediums)：Ammonium dihydrogen phosphate 5%, potassium dihydrogen phosphate 0.5%, epsom salt
1.5%th, potassium sulfate 1.95%, calcium sulfate 0.1%, potassium hydroxide 0.1%, defoamer 0.03%.0.1MPa, 121 DEG C of sterilizings
30min, every liter plus 4.4 milliliters of inorganic salt solutions (PTM1) after cooling.
PTM1 (Trace salts solution)：Copper sulphate 0.6%, KI 0.018%, manganese sulfate monohydrate 0.3%, Sodium Molybdate Dihydrate
0.02%th, boric acid 0.002%, CoCL2 6H2O 0.05%, zinc chloride 2%, green-vitriol 6.5%, the concentrated sulfuric acid 0.5%, life
Add after thing element 0.02%, wherein biotin, the PTM1 autoclavings such as need are simultaneously cooled to addition after room temperature.
Embodiment 1, the fermented and cultured for producing 1,4 beta-glucanase Aspergillus niger strain and zymologic property test
Aspergillus niger strain AN0803 employed in the present invention before use, pass through multiple ultraviolet, nitrosoguanidine, sulphur
The single and multiple mutateds such as diethyl phthalate (DES), high energy ion beam, can produce alpha-galactosidase, 1,4 beta-glucanase, β-
The enzymes such as mannase.
Fermented and cultured is carried out to mutant strain AN0803, its fermentation medium composition is as follows：
Peptone 0.5%, bean cake powder 2%, wheat flour 2%, glucose 0.5%, potassium dihydrogen phosphate 0.25%, magnesium sulfate
0.10%, pH are natural.
Above liquid fermentation medium is prepared in inoculated aspergillus niger after the 30min that sterilized under 121 DEG C, 0.1MPa, cooling
AN0803 strains, are cultivated 4 days under the conditions of 30 DEG C, 200rpm.
After fermentation ends, appropriate enzyme liquid is taken to carry out zymologic property test, testing result shows, the action pH of 1,4 beta-glucanase
For 2.0~6.5, stability and heat resistance are good, can be widely applied to apply in the fields such as feed, brewing, sugaring, tool
There is good market application foreground.
The clone of embodiment 2, aspergillus niger (Aspergillus niger) beta glucan enzyme gene
With RNA extracts kits, the total serum IgE of Aspergillus Niger Mutant (AN0803) is extracted, according to reverse transcriptase
SuperScriptTMIII Reverse Transcriptase operating instructions synthesize the first chain cDNA, then using cDNA as template,
Design primer enters performing PCR amplification.
Expand the primer as follows：
F-NGlu(EcoRI)：
5’CAGTAGAATTCATGGACGCCGATGGTGGTC3’
R-NGlu(NotI)：
5’CAGTACGCGGCCGCTTAGGAGCTAGATTGGCACT3’
After PCR terminates, PCR primer is purified and EcoRI and NotI double digestions are carried out, then with passing through same double digestion
Carrier pPICzaA is connected, connection product conversion Escherichia coli Topl0 competent cells, is coated with added with antibiotic Zeocin's
YPD flat boards are screened, and obtain positive colony.The plasmid of positive colony is extracted, sample presentation to Shanghai Sheng Gong companies is sequenced.Sequencing knot
Fruit shows that the full length gene 1065bp of the coding 1,4 beta-glucanase encodes 354 amino acid, the recombinant expression carrier screened
It is named as GNGlu-pPICzaA.
Embodiment 3, the structure of the Pichia yeast engineering comprising new beta glucan enzyme gene GNGlu and screening
The recombinant expression carrier GNGlu-pPICzaA obtained is linearized using SacI, the restructuring after linearisation
The electroporated Pichia pastoris X33 of carrier, is coated on YPD (Zeo+) flat board after electricity conversion and is screened, obtain Pichia pastoris weight
Group bacterial strain, in quiescent culture 3-4 days at 30 DEG C.
Competent yeast cells electricity conversion condition：U=1500V, C=25 μ F, R=200 Ω.
Select the single bacterium colony grown on YPD (Zeo+) flat board and carry out shake flask fermentation culture, induce producing enzyme, filter out β-Portugal and gather
Carbohydrase level of production highest bacterial strain, numbering is GNGlu-pPICzaA-X33-36.
The high efficient expression of embodiment 4, novel acid 1,4 beta-glucanase recombinant bacterial strain
The novel acid 1,4 beta-glucanase recombinant bacterial strain GNGlu-pPICzaA-X33-36 progress high density obtained will be screened
Fermented and cultured.
10L basal salt medias (BSM) are prepared, after the sterilizing of 25L liquid fermentation tanks high temperature, normal temperature are cooled to.Use ammonia
The pH value of water regulation zymotic fluid is 4.60, accesses 1L liquid strains, and fermentation temperature is set as 30 DEG C, adjusts rotating speed and air mass flow control
Make dissolved oxygen in whole fermentation process and be more than more than 20%.
3 stages of whole fermentation process point：
First stage is the glycerine or glucose of thalline cultivation stage, stream plus sterilized 2L 50%, and culture 20-24 is small
When, stream glycerol adding or glucose it is complete after terminate；
Second stage is the hungry stage, glycerine or glucose feeding it is complete after, rise when dissolved oxygen rises to more than 80%, pH
Show that the stage terminates, schedule to last about 30min；
Phase III is induced expression stage, stream plus inducing culture, and keeps dissolved oxygen more than 20%, whole fermentation
Incubation time about 190 hours or so.
Timing sampling during the fermentation determines enzyme activity, and fermentation 192h zymotic fluid enzyme activity is 16250U/mL.Whole hair
The expression of novel acid 1,4 beta-glucanase is as shown in Figure 1 during ferment.
Embodiment 5, the activity analysis of novel acid 1,4 beta-glucanase and property are determined
Detected using DNS methods.
It is per minute to discharge 1 μ from concentration to be decomposed in 4mg/mL beta glucan solution under conditions of 37 DEG C and pH 5.5
Enzyme amount required for mol reduced sugars is an enzyme-activity unit (1U=1 μm of ol/min).
1st, novel acid 1,4 beta-glucanase is determined than living
The zymotic fluid of 1,4 beta-glucanase is passed through into ion exchange column (Q SepharoseTMFast Flow Ion
Exchanger) purified, protein concentration is determined using modified form Bradford kits (Shanghai Sheng Gong companies), this is drawn
The 1,4 beta-glucanase of resistance to novel acid is than living for 1815U/mg.
2nd, the optimal reactive temperature of novel acid 1,4 beta-glucanase and pH are determined
(1) optimal reactive temperature is determined：Enzyme liquid is diluted into suitable multiple, reacted under 20~80 DEG C of different temperatures, is determined
The enzyme activity of the novel acid 1,4 beta-glucanase.Using highest enzyme activity as 100%, the enzyme activity measured at other temperature by comparison,
Obtain relative enzyme activity at this temperature.The optimal reactive temperature curve of the enzyme is shown in Fig. 2.
(2) optimal reaction pH is determined：Different pH buffer solution is prepared, the enzyme liquid after dilution is entered under pH 2.0~7.0
Row detection, obtains the enzyme activity of the 1,4 beta-glucanase under the conditions of each pH, and uses highest enzyme activity for 100%, other pH conditions
Under detect result by comparison, that is, obtain the relative enzyme activity under different pH testing conditions.The optimal reaction pH curves of the enzyme are shown in
Fig. 3.
Testing result shows that the optimal reactive temperature of the recombined new 1,4 beta-glucanase is 55 DEG C, and optimal reaction pH is
4.0, it is a kind of acid 1,4 beta-glucanase.
3rd, the heat resistance of novel acid 1,4 beta-glucanase is determined
Enzyme liquid is diluted into suitable multiple, at different temperatures (60-100 DEG C) processing 10min, with untreated sample enzyme activity
Power is control, determines the relative enzyme activity of the acid 1,4 beta-glucanase, heat resistance testing result is shown in Fig. 4.
Testing result shows that the acid 1,4 beta-glucanase handles 10min at 85 DEG C, and it is left that remaining enzyme activity still can reach 80%
The right side, heat resistance is good.
4th, novel acid 1,4 beta-glucanase bin stability is determined
The novel acid 1,4 beta-glucanase solid sample is placed in and stored at room temperature, monthly timing sampling, it is poly- according to β-Portugal
The detection method of carbohydrase, detects the sample enzyme activity of different holding times, to determine the stability of the enzyme.Novel acid β-Portugal
The bin stability measurement result of dextranase is shown in Fig. 5.
As a result show that the stability of the novel acid 1,4 beta-glucanase is good, preserve 1 year at ambient temperature, it is remaining
Enzyme activity still can reach 88% or so.
5th, novel acid 1,4 beta-glucanase antipepsin, the digestic property of trypsase are determined
The 0.1mL 1,4 beta-glucanases are taken, 0.5mL 0.1mg/mL pepsins (the concentration 100U/mL of pH 2.0) are separately added into
With 0.5mL 0.1mg/mL trypsase (the concentration 200U/mL of pH 7.0), enzyme activity is surveyed after handling different time in 37 DEG C.Detection knot
Fruit is as shown in Figure 6.
After wherein pepsin 90min, enzymatic activity is still up to 85%, after trypsin treatment 90min, remaining
82%.This illustrates that this 1,4 beta-glucanase has excellent antipepsin and Trypsin Induced ability.
6th, influence of the metal ion to enzyme activity
1mM K will be contained+、Mg2+、Ca2+、Fe2+、Fe3+、Zn2+、Mn2+、Co2+、Cu2+Acetic acid-sodium acetate buffer solution
(pH5.5) mixed with diluting the enzyme liquid of suitable multiple, room temperature places 1h, using untreated enzyme liquid as control, as a result such as Fig. 7 institutes
Show.
As a result show, Ca2+、Mg2+、Co2+There are certain activation, K to enzyme activity+、Fe2+、Zn2+、Mn2+Enzyme activity is influenceed
Less, Fe3+、Cu2+Then there is a certain degree of inhibitory action to enzyme activity.
Claims (9)
1. a kind of 1,4 beta-glucanase NGlu, it is characterised in that its amino acid sequence is as shown in SEQ ID NO. 1.
2. a kind of beta glucan enzyme gene GNGlu, it is characterised in that the 1,4 beta-glucanase described in coding claim 1.
3. beta glucan enzyme gene GNGlu according to claim 2, it is characterised in that its nucleotide sequence such as SEQ ID
NO. shown in 2.
4. include the recombinant vector of beta glucan enzyme gene GNGlu described in claim 2.
5. recombinant vector according to claim 4, it is characterised in that the recombinant vector is pPICz α A-GNGlu, its
In, the EcoR I and NotI that beta glucan enzyme gene GNGlu described in claim 2 is inserted on plasmid pPICz α A are restricted
Between restriction enzyme site, the nucleotide sequence is located at the downstream of AOX1 promoters and is regulated and controled by it, obtain recombinant vector for pPICz
αA-GNGlu。
6. the recombinant bacterial strain containing beta glucan enzyme gene GNGlu described in claim 2.
7. a kind of method that fermentation prepares 1,4 beta-glucanase NGlu, it is characterised in that comprise the following steps：
1）Host cell X33 is converted with the recombinant vector described in claim 5, recombinant bacterial strain is obtained；
2）Recombinant bacterial strain is cultivated, new 1,4 beta-glucanase NGlu expression is induced；And
3）Reclaim and purify expressed new 1,4 beta-glucanase NGlu.
8. the method that fermentation according to claim 7 prepares 1,4 beta-glucanase NGlu, it is characterised in that step 2）It is divided into three
The individual stage：
First stage is the glycerine or glucose of thalline cultivation stage, stream plus sterilized 2L 50%, is cultivated 20-24 hours, stream
Terminate after glycerol adding or glucose are complete；
Second stage is the hungry stage, glycerine or glucose feeding it is complete after, rise i.e. table when dissolved oxygen rises to more than 80%, pH
The bright stage terminates；
Phase III is induced expression stage, stream plus inducing culture, and keeps dissolved oxygen more than 20%, whole fermented and cultured
About 190 hours time.
9. 1,4 beta-glucanase NGlu described in claim 1 is used for the application of hydrolysing p-glucosidic bonds.
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