CN104004735B - A kind of high temperature resistant restructuring 'beta '-mannase and application thereof - Google Patents
A kind of high temperature resistant restructuring 'beta '-mannase and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of high temperature resistant recombinant beta mannase and application thereof.By being cloned in yeast expression vector pPICZ α A according to the β mannase gene after Pichia sp. codon preference optimization from aspergillus niger, convert to Pichia sp. KM71H, the recombinant beta mannase of available high expressed amount.This recombinant beta mannase optimum temperature is 80 DEG C.The β mannase of the present invention being used for enzymolysis konjaku powder and produces manna oligosaccharide, concentration of substrate is high, and Product yields is high, and the response time is short, and conversion ratio is high, has bigger industrial production potential and using value.
Description
Technical field
The present invention relates to a kind of high temperature resistant restructuring 'beta '-mannase and application thereof, belong to technical field of bioengineering.
Background technology
Manna oligosaccharide be by mannose and glucose with β-1,4 glycosidic bond be connected the degree of polymerization formed be 2～10 functional
Oligosaccharide.Manna oligosaccharide has the propagation of the interior probiotics with bacillus bifidus as representative of promotion humans and animals intestinal, improves intestinal
The physicochemical property that in road, the functional oligose such as Bacterial community is common, the most also has removing free radical, enhancing body antioxidation
The ability of property, if manna oligosaccharide is applied to commercial development as functional food additives, application prospect is the most wide.
Rhizoma amorphophalli is the herbaceos perennial that the ground such as Yunnan Province of China, Sichuan, Shaanxi, Guizhou abound with, and contains in its taro tuber
Abundant mannan.At present, Rhizoma amorphophalli powder is mainly incorporated as edible crude fibre or as food additive, develops journey
Degree is not.Utilize 'beta '-mannase that Rhizoma amorphophalli hydrolysis is made manna oligosaccharide, can significantly widen the range of application of Rhizoma amorphophalli powder,
Improve raw material added value, there is economic benefit and social benefit widely.Enzyme process prepares the core technology of manna oligosaccharide
Use high vigor 'beta '-mannase and high concentration Konjac glucomannan solution.But at present fermentation enzyme vigor is low, cause enzyme production and
Use cost is high；Simultaneously because the viscosity of Konjac glucomannan aqueous solution is very big, the Konjac glucomannan of 40g/L, can in gel state
Stop the enzyme hydrolysis to it.Therefore the Konjac glucomannan enzyme hydrolysis process of domestic and foreign literature report all uses 10～30g/L
Konjac glucomannan solution, the post processing cost that result in enzymatic hydrolysate is high, production efficiency is low.Therefore solving enzyme process, to prepare manna low
Polysaccharide problem encountered, first has to obtain the 'beta '-mannase that vigor high stability is good, next to that optimize enzymolysis process
Improve concentration of substrate.
Summary of the invention
The technical problem to be solved is for the deficiencies in the prior art, it is provided that a kind of high temperature resistant recombinant beta-manna gathers
Carbohydrase.
The present invention also to solve the technical problem that being to provide above-mentioned 'beta '-mannase enzymolysis high concentration Rhizoma amorphophalli powder prepares manna
The application of oligosaccharide.
For solving above-mentioned technical problem, the technical solution used in the present invention is as follows:
A kind of high temperature resistant restructuring 'beta '-mannase, its aminoacid sequence is as shown in SEQ ID No:2.
The encoding gene of a kind of high temperature resistant restructuring 'beta '-mannase, is one of nucleotide sequence meeting following requirements:
(1) coding nucleotide sequence of aminoacid sequence as shown in SEQ ID No:2；
(2) nucleotide sequence as shown in SEQ ID No:1.
A kind of recombinant bacterial strain expressing high temperature resistant restructuring 'beta '-mannase, this bacterial strain is to incorporate claim on genome
The Pichia sp. of the encoding gene of the 'beta '-mannase described in 2.
Wherein, described Pichia sp. is secreting type pichia pastoris phaff (Pichia pastoris).
The method building above-mentioned recombinant bacterial strain, it comprises the steps:
(1) the conventional construction of recombinant plasmid method of application constructs the codon optimized plasmid pPICZ α A man of restructuring, this matter
Grain is with AOX1 as promoter, and contains the beta-mannase gene shown in SEQ ID No:1 and Zeocin resistance base
Cause；
(2) will recombinate after codon optimized plasmid pPICZ α A man Sac I linearization for enzyme restriction, electroporated enter complete
In red yeast host bacterium, i.e. constitute the Pichia sp. recombinant bacterial strain expressing 'beta '-mannase；
(3) by recombinant bacterial strain through Methanol Utilization Phenotype screening, the screening of exogenous gene multi-copy integration and abduction delivering
After screening, i.e. filter out the recombination engineering of overexpression 'beta '-mannase.
In step (1), described recombinant plasmid pPICZ alpha A man is the purpose base of synthesis after codon preference optimizes
Because man is inserted in plasmid pPICZ α A the recombiant plasmid formed.Concrete grammar sees Invitrogen company
Multi-Copy Pichia Expression Kit workbook.
In step (2), after codon optimized plasmid pPICZ α A man Sac I linearization for enzyme restriction of recombinating,
Electroporated enter in Pichia sp. Host Strains before, codon optimized plasmid pPICZ α A man can be imported escherichia coli
Expand.
The preparation method of above-mentioned high temperature resistant restructuring 'beta '-mannase, by above-mentioned recombinant bacterium in BMGY culture medium 30 DEG C
Cultivate to cell density OD600nm=2～6, the centrifugal thalline of collecting of room temperature, with BMMY fluid medium, cell is resuspended
To OD600nmBeing 1.0 with abduction delivering, 28 DEG C are continued to cultivate 72h, every 24h and add pure methanol to the denseest to culture medium
Degree is 1v/v%, and the recombinant bacterium completing to cultivate is removed thalline by centrifugation, collects supernatant, with molecular weight as 10KDa
Super filter tube, filter off the albumen of small component and salt, through molecular sieve Superdex75PrepGrade purification, use 100
The citrate-phosphate disodium hydrogen buffer of mmol/L pH5.0, flow velocity 0.3ml/min eluting, distribution is collected in eluting peak
Sample, through enzyme activity determination, determine the collecting pipe at purpose sample place, obtain electrophoretically pure destination protein, this restructuring
It is 1168IU/mg that 'beta '-mannase enzyme is lived, and optimal reactive temperature is 80 DEG C, and optimum pH is 5.0.
The application in enzyme process prepares manna oligosaccharide of the above-mentioned high temperature resistant restructuring 'beta '-mannase.
Preferably, to prepare manna oligosaccharide rich in the biomass of mannan for material, enzyme method, described biomass are
One or more of Rhizoma amorphophalli powder, sesbania gum, locust bean gum and guar gum
More have choosing, with the high temperature resistant restructuring 'beta '-mannase addition of 10-50IU/g, in pH4.0-6.0,
Under the concussion of 50-90 DEG C or stirring condition, enzymolysis concentration of substrate is 30-200g/L biomass solution, drops after enzymolysis 0.5-8h
Temperature is centrifugal goes residue, supernatant to be manna oligosaccharide.
Most preferably, concentration of substrate is the high-concentration raw material of 100-200g/L, and enzyme dosage is at the beginning of 10-50IU/g, enzyme
Stage beginning disposably adds, and substrate adds several times (that is first according to the concentration calculating biomass of 100-200g/L
Total addition level, add the most several times), add time interval at 1-3h, the concussion of pH4.0-6.0,50-90 DEG C or
Being centrifuged after cooling after being total to enzymolysis 4-12h under stirring condition goes residue, supernatant to be manna oligosaccharide.
Beneficial effect: the present invention compared with prior art, has the advantage that
(1) the restructuring 'beta '-mannase yield in the present invention is high, energetic, good stability.
(2) restructuring 'beta '-mannase in the present invention can efficiently zymohydrolysis of konjaku flour, 'beta '-mannase and Rhizoma amorphophalli powder
Optimal proportion is 50IU/g, less than previously reported.During enzymolysis 50g/L Rhizoma amorphophalli powder, manna oligosaccharide yield is 78%, sweet
Dew concentration of oligosaccharide is 39g/L, and contents of monosaccharides is the lowest.
(3) enzymolysis process and Rhizoma amorphophalli powder feed supplement adding technology after optimizing in the present invention are greatly improved recombinant beta-manna and gather
The carbohydrase enzymolysis efficiency to high concentration substrate, the highest manna oligosaccharide concentration is 81g/L, higher than previously reported.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE collection of illustrative plates of 'beta '-mannase.
Fig. 2 is that Rhizoma amorphophalli powder Enzymatic Hydrolysis Process pH optimizes.
Fig. 3 is Rhizoma amorphophalli powder Enzymatic Hydrolysis Process hydrolysis temperature optimization.
Fig. 4 is Rhizoma amorphophalli powder Enzymatic Hydrolysis Process concentration of substrate optimization.
Fig. 5 is Rhizoma amorphophalli powder Enzymatic Hydrolysis Process enzyme dosage optimization.
Fig. 6 is Rhizoma amorphophalli powder Enzymatic Hydrolysis Process enzymolysis time optimization.
Fig. 7 is beta-mannase enzymatic degradation konjaku powder chromatography of ions testing result.
Detailed description of the invention
Below in conjunction with specific embodiment, the operational approach of the present invention is expanded on further.But these embodiments are only used in detail
The present invention is described, rather than limits the scope of the present invention.
Embodiment 1: the structure of recombiant plasmid.
Recombinant plasmid pPICZ alpha A man is the beta-mannase gene after optimizing according to Pichia sp. codon preference
(as shown in SEQ ID No:1) is inserted in plasmid pPICZ α A formation.
Embodiment 2: the preparation of target DNA and the electricity conversion of Pichia sp..
Obtained E. coli transformant is cultivated, extracts plasmid, use Sac I to carry out enzyme action, pass through purified reagent
Box purification obtains linear target DNA；Produce Pichia pastoris GS115, KM71H, SMD1168, X 33 respectively
Electricity turn competent cell；80 μ l electricity are turned the linearisation DNA mixing of competent cell and 10 μ l, proceeds to 2mm
Electricity converts in cup and shocks by electricity, and shock parameters is: 1500V, 25 μ F, 200 Ω.
Embodiment 3: the screening of transformant.
By electricity convert after Pichia pastoris GS115, KM71H, SMD1168, X 33 competent cell coat containing
On the YPDS flat board of 100 μ g/mL bleomycin, cultivating until transformant occurs under the conditions of 30 DEG C, this transformant is
Pichia sp. recombinant bacterial strain.
Four kinds of different monoclonals of picking, are seeded in 25ml BMGY culture medium 30 DEG C and cultivate to cell density
OD600nm=2～6, room temperature is centrifugal collects thalline, with BMMY fluid medium by resuspended for cell to OD600nmIt is 1.0
With abduction delivering.28 DEG C are continued to cultivate 96h, every 24h and add 100% methanol extremely final concentration of 1v/v% to culture medium.
The pichia yeast genetic engineering bacteria completing to cultivate is removed thalline by centrifugation, collects supernatant, measure crude enzyme liquid enzyme and live.When
When host is GS115 and SMD1168, bacterial strain produced beta-mannase enzyme activity is on the low side；And KM71H and X 33
Preferable as host's enzymatic productivity, methanol slowly utilizes the KM71H host of type the most prominent, bacterium after methanol induction 96h
Strain fermentative activity reaches 252IU/mL.
Enzyme activity determination method and enzyme unit definition alive: use DNS fixed sugar method to measure the activity of 'beta '-mannase.By 0.9
ML5.0g/L locust bean gum substrate solution (pH5.0 citric acid phosphoric acid disodium hydrogen buffer) with 0.1mL through the dilutest
The enzyme liquid mix homogeneously released, 80 DEG C of reaction 10min, add 3.0mL DNS reagent immediately, boil 7min, after cooling
It is settled to 25mL, shakes up.Sample liquid absorbance is measured at 540nm.Mannan enzyme activity unit is defined as often
Enzyme amount needed for minute hydrolysis substrate produces the mannose of 1 μm ol is defined as a unit of activity (1IU).
Embodiment 4: the high density fermentation of restructuring 'beta '-mannase.
Picking converts the monoclonal of the recombinant yeast pichia pastoris bacterium KM71H having recombinant plasmid pPICZ alpha A man, is seeded to
BMGY culture medium is cultivated to cell density OD for 30 DEG C600nm=2～6, it is inoculated in sweet with the inoculum concentration of volume fraction 10%
Oil content be 40g/L BMS in carry out fermentation culture.Fermenter volume is 3L, liquid amount 1.5L, 30 DEG C, pH
Cultivate under conditions of 5.0 (the ammonia regulation and control of 28%).After recombination yeast is about fermentor cultivation 24h, when glycerol consumes
To the greatest extent, weight in wet base is at 90 150g/L, then with the speed stream glycerol adding 4h of 18.15mL/h/L.Stop stream glycerol adding to DO
Hungry cultivation 1h after bounce-back again, after cell moisture reaches 180 220g/L, starts stream and adds methanol induction cultivation.First
After alcohol-induced 96h, strain fermentation vigor reaches 2319IU/mL.
Embodiment 5: the purification of restructuring 'beta '-mannase.
Fermentation liquid is centrifuged gained supernatant and carries out ultrafiltration by the super filter tube that molecular weight is 10KDa, filters off the albumen of small component
And salt, take the liquid 2mL of ultra filtration, through molecular sieve Superdex75PrepGrade purification, use 100mmol/L
The citrate-phosphate disodium hydrogen buffer of pH5.0, flow velocity 0.3ml/min eluting, the sample in distribution collection eluting peak,
Through enzyme activity determination, determining the collecting pipe at purpose sample place, obtain electrophoretically pure destination protein, enzyme work is 1168
IU/mg.As it is shown in figure 1, be the SDS-PAGE collection of illustrative plates of purified enzyme liquid.Wherein M is standard molecular weight protein,
1 be the recombinant yeast pichia pastoris KM71H fermented supernatant fluid containing pPICZ α A plasmid as comparison, 2 for containing
The recombinant yeast pichia pastoris KM71H fermented supernatant fluid of pPICZ α A-man plasmid, 3 is the restructuring 'beta '-mannase of purification.
Embodiment 6: the characterization analysis of restructuring 'beta '-mannase.
The zymologic property of analysis 'beta '-mannase after purification, the optimum pH of 'beta '-mannase is 5.0, at pH
Time between 4～6, this enzyme all has the relative enzyme more than 95% to live, and keeps 70h, still have in the range of pH3～7
The enzyme activity of more than 90%.The optimal reactive temperature of 'beta '-mannase is 80 DEG C, still has up to 76.8% when 90 DEG C
Relative enzyme live.It is incubated 70h at 60 DEG C and remains in that the enzyme activity of more than 90%.
Embodiment 7: the process optimization of Rhizoma amorphophalli powder enzymatic hydrolysis condition.
(1) enzymolysis pH: with the enzyme liquid addition of 125IU/g Rhizoma amorphophalli powder, in 80 DEG C, under 170rpm, different pH
(3.0～6.0) enzymolysis 50g/L Konjac glucomannan solution, after 2h, HPLC detects the pH impact on manna oligosaccharide yield,
Result shows that when (Fig. 2) enzymolysis pH is 3.0, Rhizoma amorphophalli powder absorbs water into solid-state in buffer, does not occur enzymatic hydrolysis reaction,
Enzymolysis pH is in the range of 4.0～5.0, and the enzymolysis efficiency of Rhizoma amorphophalli powder is higher.
(2) hydrolysis temperature: with the enzyme liquid addition of 125IU/g Rhizoma amorphophalli powder, in pH5.0,170rpm, different temperatures
Under (50～90 DEG C) enzymolysis 50g/L Konjac glucomannan solution, after 2h HPLC detect the temperature impact on manna oligosaccharide yield,
Result show (Fig. 3) hydrolysis temperature in the range of 50～90 DEG C, the enzymolysis efficiency of Rhizoma amorphophalli powder is the highest.
(3) concentration of substrate: with the enzyme liquid addition of 125IU/g Rhizoma amorphophalli powder, in pH5.0,80 DEG C, enzyme under 170rpm
Solving the Konjac glucomannan solution that concentration of substrate is 2.5～50g/L, after 2h, HPLC detection substrate concentration is to manna oligosaccharide yield
Impact, result does not shows that (Fig. 4) increases accordingly along with the increase of concentration of substrate, manna oligosaccharide yield, not substantially
Mass transfer be obstructed phenomenon.
(4) enzyme dosage: with the Konjac glucomannan solution of 50g/L as substrate, at pH5.0,80 DEG C, enzymolysis under 170rpm,
Enzyme liquid addition is 12.5～125IU/g Rhizoma amorphophalli powders, and after 2h, HPLC detects the enzyme dosage impact on manna oligosaccharide yield,
Result show (Fig. 5) when enzyme dosage brings up to 50IU/g from 12.5IU/g, along with enzyme dosage increase, manna is oligomeric
Candy output improves constantly, but manna oligosaccharide yield increases inconspicuous when 50IU/g continues to increase to a certain degree.
(5) enzymolysis time: with the enzyme liquid addition of 50IU/g Rhizoma amorphophalli powder, in pH5.0,80 DEG C, enzyme under 170rpm
Solving the Konjac glucomannan solution that concentration of substrate is 50g/L, different time points sampling in the range of 0～4h, HPLC detects enzymolysis
The time impact on manna oligosaccharide yield, result shows that (Fig. 6) is extremely strong due to Rhizoma amorphophalli water absorption, presents solid-state during 0h,
As time goes on, mannase generation enzyme hydrolysis effect, substrate constantly liquefies, and substantially liquefies after reaction 2h
Completely, enzymolysis has tended to stopping, and manna oligosaccharide content the most no longer rises.
Embodiment 8: the 'beta '-mannase degradation analysis to Rhizoma amorphophalli powder of recombinating under optimum enzymatic hydrolysis condition.
With the enzyme liquid addition of 50IU/g Rhizoma amorphophalli powder, in pH5.0,80 DEG C, under 170rpm enzymolysis concentration of substrate as 50g/L
Konjac glucomannan solution, be heated up to 100 DEG C after enzymolysis 2h and keep 10min to make enzyme-deactivating, centrifugal after cooling remove residue, on
Clear for HPLC and ion chromatography.HPLC result shows, manna oligosaccharide concentration is 39g/L, and manna is oligomeric
Sugar yield is 78%.Fig. 7 is beta-mannase enzymatic degradation Rhizoma amorphophalli powder chromatography of ions testing result, it can be seen that this is heavy
Group 'beta '-mannase enzymolysis konjaku powder primary product is mannobiose and manna six sugar, and mannobiose content is 45.5%,
Manna six sugar is 34.3%, and manna one sugar, mannotriose, mannotetrose content are respectively 7.5%, 8.8%, 3.4%.
Embodiment 9: manna oligosaccharide prepared by restructuring 'beta '-mannase enzymolysis locust bean gum.
With the enzyme liquid addition of 30IU/g locust bean gum, in pH5.0,70 DEG C, enzymolysis concentration of substrate is under 170rpm
The Konjac glucomannan solution of 30g/L, is heated up to 100 DEG C and keeps 10min to make enzyme-deactivating after enzymolysis 2h, after cooling centrifugal go residual
Slag, supernatant is used for HPLC and ion chromatography.HPLC result shows, manna oligosaccharide concentration is 25.8g/L, sweet
Dew yield of oligosaccharide is 78%.
Embodiment 10: manna oligosaccharide prepared by 'beta '-mannase enzymolysis high concentration Rhizoma amorphophalli powder of recombinating under enzymatic hydrolysis condition.
The Rhizoma amorphophalli powder of 150g/L the most all adds when 0h, and 'beta '-mannase presses 50IU/g Rhizoma amorphophalli when 0h
Powder (in terms of 150g/L Rhizoma amorphophalli powder) is disposable all to be added, pH be 5.0, temperature be 80 DEG C, rotating speed be 170rpm
Under conditions of enzymolysis, after 6h, enzymolysis terminates, enzyme liquid is heated up to 100 DEG C keep 10min make enzyme-deactivating, after cooling from
The heart removes residue, and supernatant is analyzed for HPLC, and result display manna oligosaccharide concentration is 56.6g/L.
Embodiment 11: manna oligosaccharide prepared by the Rhizoma amorphophalli powder that restructuring 'beta '-mannase enzymolysis is added batch-wise.
The Rhizoma amorphophalli powder of 150g/L adds in three times, and the interpolation time is respectively 0h, 2h and 4h.'beta '-mannase is at 0h
Time all add by 50IU/g Rhizoma amorphophalli powder (in terms of 150g/L Rhizoma amorphophalli powder) is disposable.PH be 5.0, temperature be 80 DEG C,
Rotating speed is enzymolysis under conditions of 170rpm, and after 6h, enzymolysis terminates, and enzyme liquid is heated up to 100 DEG C and keeps 10min to make enzyme
Inactivation, is centrifuged after cooling and removes residue, and supernatant is analyzed for HPLC, and result display manna oligosaccharide concentration is 81g/L.
Claims (1)
1. the high temperature resistant restructuring 'beta '-mannase application in enzyme process prepares manna oligosaccharide；
Concentration of substrate is the high-concentration raw material of 100-200g/L, and enzyme dosage is 10-50IU/g, and the enzyme starting stage is disposable
Adding, substrate adds several times, adds time interval at the concussion of 1-3h, pH 4.0-6.0,50-90 DEG C or stirring condition
Being centrifuged after cooling after being total to enzymolysis 4-12h down goes residue, supernatant to be manna oligosaccharide；Described biomass be Rhizoma amorphophalli powder,
One or more of sesbania gum, locust bean gum and guar gum；
Wherein, described high temperature resistant restructuring 'beta '-mannase prepares as follows:
A builds recombinant bacterial strain:
(1) the conventional construction of recombinant plasmid method of application constructs the codon optimized plasmid pPICZ α A man of restructuring, this matter
Grain is with AOX1 as promoter, and contains the beta-mannase gene shown in SEQ ID No:1 and Zeocin resistance base
Cause；Described recombinant plasmid pPICZ alpha A man is that the genes of interest man of synthesis is inserted into after codon preference optimization
The recombiant plasmid formed in plasmid pPICZ α A；
(2) will recombinate after codon optimized plasmid pPICZ α A man Sac I linearization for enzyme restriction, by codon optimized
Plasmid pPICZ α A man imports escherichia coli and expands, more electroporated enter in Pichia sp. Host Strains KM71H,
I.e. constitute the Pichia sp. recombinant bacterial strain expressing 'beta '-mannase；
(3) by recombinant bacterial strain through Methanol Utilization Phenotype screening, the screening of exogenous gene multi-copy integration and abduction delivering
After screening, i.e. filter out the recombination engineering of overexpression 'beta '-mannase；
The recombinant bacterium that step A is built by B in BMGY culture medium 30 DEG C cultivate to cell density OD600nm=2～6,
Room temperature is centrifugal collects thalline, with BMMY fluid medium by resuspended for cell to OD600nmIt is 1.0 with abduction delivering, 28 DEG C
Continue to cultivate 72h, every 24h and add pure methanol extremely final concentration of 1%v/v to culture medium, the recombinant bacterium cultivated will be completed
Remove thalline by centrifugation, collect supernatant, prepare high temperature resistant restructuring 'beta '-mannase through ultrafiltration and molecular sieve purification.
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| CN107365756A (en) * | 2017-08-01 | 2017-11-21 | 天津大学 | A kind of method for preparing β mannases using the fermentation of mannosan type natural plant gum liquid hydrolyzate |
| CN107760700B (en) * | 2017-09-30 | 2020-11-06 | 武汉轻工大学 | Beta-mannase gene, recombinant expression vector, strain, beta-mannase and preparation method and application thereof |
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