CN104004735A - High-temperature resistant recombined beta-mannase and application thereof - Google Patents
High-temperature resistant recombined beta-mannase and application thereof Download PDFInfo
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Abstract
The invention discloses high-temperature resistant recombined beta-mannase and the application of the high-temperature resistant recombined beta-mannase. A beta-mannase gene from aspergillus niger is cloned to a pichia pastoris expression vector pPICZ alpha A and converted into pichia pastoris KM71H so that the recombined beta-mannase with high expression quantity can be obtained, wherein the beta-mannase gene is optimized according to the preference of the pichia pastoris codon. The optimum temperature of the recombined beta-mannase is 80 DEG C. The beta-mannase is applied to enzymolysis konjac powder for producing mannan oligosaccharide, the concentration of a substrate is high, the yield of products is high, the reaction time is short, the conversion rate is high and the high-temperature resistant recombined beta-mannase has high industrial potential productivity and application 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 is to take by seminose and glucose the functional oligosaccharide that the polymerization degree that β-Isosorbide-5-Nitrae glycosidic link is connected to form is 2～10.Manna oligosaccharide have promote to take in humans and animals enteron aisle the probiotics that bifidus bacillus is representative propagation, improve in enteron aisle the common physicochemical properties of functional oligose such as flora structure, also there is the ability of removing free radical, enhancing body oxidation-resistance simultaneously, if manna oligosaccharide is applied to commercial exploitation as functional food additives, application prospect is very wide.
Konjaku is the per nnial herb that the ground such as Yunnan Province of China, Sichuan, Shaanxi, Guizhou abound with, and in its taro stem tuber, contains abundant mannosans.At present, Rhizoma amorphophalli powder is mainly used as edible robust fibre or as foodstuff additive, exploitation degree is inadequate.Utilize 'beta '-mannase that konjaku hydrolysis is made to manna oligosaccharide, can greatly widen the range of application of Rhizoma amorphophalli powder, improve raw material added value, there is economic benefit and social benefit widely.The core technology that enzyme process is prepared manna oligosaccharide is to adopt high vigor 'beta '-mannase and high density konjak gum solution.But fermenting enzyme vigor is low at present, causes the production of enzyme and use cost high; Because the viscosity of the konjak gum aqueous solution is very large, the konjak gum of 40g/L has been gel state, can stop the hydrolytic action of enzyme to it simultaneously.Therefore the konjak gum enzyme hydrolysis process of domestic and foreign literature report all adopts the konjak gum solution of 10～30g/L, has caused the aftertreatment cost of enzymolysis product high, production efficiency is low.Therefore solving enzyme process prepares the problem that manna oligosaccharide runs into, and first will obtain the 'beta '-mannase that vigor high stability is good, is secondly to optimize enzymolysis process to improve concentration of substrate.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, and a kind of high temperature resistant restructuring 'beta '-mannase is provided.
The technical problem that the present invention also will solve is to provide the application that above-mentioned 'beta '-mannase enzymolysis high density Rhizoma amorphophalli powder is prepared manna oligosaccharide.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A high temperature resistant restructuring 'beta '-mannase, its aminoacid sequence is as shown in SEQ ID No:2.
An encoding gene for high temperature resistant restructuring 'beta '-mannase is one of nucleotide sequence meeting following requirement:
(1) nucleotide sequence of coding aminoacid sequence as shown in SEQ ID No:2;
(2) nucleotide sequence as shown in SEQ ID No:1.
Express a recombinant bacterial strain for high temperature resistant restructuring 'beta '-mannase, this bacterial strain is on genome, to have integrated the pichia spp of the encoding gene of 'beta '-mannase claimed in claim 2.
Wherein, described pichia spp is secretor type pichia pastoris phaff (Pichia pastoris).
The method that builds above-mentioned recombinant bacterial strain, it comprises the steps:
(1) apply conventional construction of recombinant plasmid method and construct the codon optimized plasmid pPICZ α A – man of restructuring, this plasmid be take AOX1 as promotor, and contains the beta-mannase gene shown in SEQ ID No:1 and Zeocin resistant gene;
(2) will recombinate codon optimized plasmid pPICZ α A – man with after Sac I linearization for enzyme restriction, electric shock is transformed in pichia spp Host Strains, forms the pichia spp recombinant bacterial strain of expressing 'beta '-mannase;
(3) by recombinant bacterial strain after Methanol Utilization Phenotype screening, the screening of foreign gene multi-copy integration and abduction delivering screening, filter out the recombinant bacterial strain of overexpression 'beta '-mannase.
In step (1), described recombinant plasmid pPICZ alpha A – man be codon preference optimization afterwards synthetic goal gene man be inserted into the recombinant plasmid forming in plasmid pPICZ α A.Concrete grammar is referring to the Multi-Copy Pichia Expression Kit operational manual of Invitrogen company.
In step (2), after the codon optimized plasmid pPICZ α A – man that will recombinate is with Sac I linearization for enzyme restriction, before electric shock is transformed in pichia spp Host Strains, codon optimized plasmid pPICZ α A – man can be imported to intestinal bacteria and increase.
The preparation method of above-mentioned high temperature resistant restructuring 'beta '-mannase, by above-mentioned recombinant bacterium in BMGY substratum 30 ℃ be cultured to cell density OD
600nm=2～6, the centrifugal collection thalline of room temperature, with BMMY liquid nutrient medium, cell is resuspended to OD
600nmbe 1.0 with abduction delivering, 28 ℃ are continued to cultivate 72h, it is 1v/v% that every 24h adds pure methyl alcohol to final concentration to substratum, by the recombinant bacterium that completes cultivation through the centrifugal thalline of removing, collect supernatant liquor, the super filter tube that the molecular weight of take is 10KDa, the albumen of elimination small component and salt, through molecular sieve Superdex75PrepGrade purifying, use citric acid-Sodium phosphate dibasic damping fluid of 100mmol/L pH5.0, flow velocity 0.3ml/min wash-out, distribute and collect the sample in elution peak, through enzyme activity determination, determine the collection tube at object sample place, obtain electrophoretically pure target protein, this restructuring 'beta '-mannase enzyme is lived as 1168IU/mg, optimal reactive temperature is 80 ℃, optimal pH is 5.0.
Above-mentioned high temperature resistant restructuring 'beta '-mannase is prepared the application in manna oligosaccharide at enzyme process.
Preferably, the biomass that are rich in mannosans of take are prepared manna oligosaccharide as material, enzyme method, and described biomass are one or more of Rhizoma amorphophalli powder, sesbania gum, Viscogum BE and guar gum
What more have choosing is, take the high temperature resistant restructuring 'beta '-mannase addition of 10-50IU/g, under the concussion of pH4.0-6.0,50-90 ℃ or agitation condition, enzymolysis concentration of substrate is 30-200g/L biomass solution, the centrifugal residue that goes of lowering the temperature after enzymolysis 0.5-8h, supernatant is manna oligosaccharide.
Most preferably, concentration of substrate is the high-concentration raw material of 100-200g/L, enzyme dosage is 10-50IU/g, disposable adding of enzyme starting stage, substrate adds (that is to say the total addition level that first calculates biomass according to the concentration of 100-200g/L, then add several times) several times, adds the timed interval at 1-3h, the cooling rear centrifugal residue that goes after common enzymolysis 4-12h under the concussion of pH4.0-6.0,50-90 ℃ or agitation condition, supernatant is manna oligosaccharide.
Beneficial effect: the present invention compared with prior art, has following advantage:
(1) the restructuring 'beta '-mannase output in the present invention is high, energetic, good stability.
(2) the efficient zymohydrolysis of konjaku flour of the restructuring 'beta '-mannase in the present invention, the optimal proportions of 'beta '-mannase and Rhizoma amorphophalli powder is 50IU/g, lower than report in the past.During enzymolysis 50g/L Rhizoma amorphophalli powder, manna oligosaccharide yield is 78%, and manna oligosaccharide concentration is 39g/L, and contents of monosaccharides is very low.
(3) in the present invention, the enzymolysis process after optimization and Rhizoma amorphophalli powder feed supplement adding technology can improve the enzymolysis efficiency of restructuring 'beta '-mannase to high concentration substrate greatly, and the highest manna oligosaccharide concentration is 81g/L, higher than report in the past.
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 the optimization of Rhizoma amorphophalli powder Enzymatic Hydrolysis Process hydrolysis temperature.
Fig. 4 is the optimization of Rhizoma amorphophalli powder Enzymatic Hydrolysis Process concentration of substrate.
Fig. 5 is the optimization of Rhizoma amorphophalli powder Enzymatic Hydrolysis Process enzyme dosage.
Fig. 6 is the optimization of Rhizoma amorphophalli powder Enzymatic Hydrolysis Process enzymolysis time.
Fig. 7 is beta-mannase enzyme liberating konjaku powder chromatography of ions detected result.
Embodiment
Below in conjunction with specific embodiment, further set forth working method of the present invention.But these embodiment only, for describing the present invention in detail, limit the scope of the invention and be not used in.
Embodiment 1: the structure of recombinant plasmid.
Recombinant plasmid pPICZ alpha A – man is inserted in plasmid pPICZ α A and forms according to the beta-mannase gene (as shown in SEQ ID No:1) after the optimization of pichia spp codon preference.
Embodiment 2: the preparation of target DNA and the electricity of pichia spp transform.
Resulting intestinal bacteria transformant is cultivated, extracted plasmid, adopt Sac I to carry out enzyme and cut, by purification kit purifying, obtain linear target DNA; The electricity of producing respectively Pichia pastoris GS115, KM71H, SMD1168, X – 33 turns competent cell; 80 μ l electricity are turned to competent cell and mix with the linearizing DNA of 10 μ l, proceed to 2mm electricity and transform electric shock in cup, shock parameters is: 1500V, 25 μ F, 200 Ω.
Embodiment 3: the screening of transformant.
Pichia pastoris GS115, KM71H, SMD1168, X – 33 competent cells after electricity is transformed are coated on the YPDS flat board that contains 100 μ g/mL bleomycins, cultivate until transformant occurs under 30 ℃ of conditions, and this transformant is pichia spp recombinant bacterial strain.
Four kinds of different mono-clonals of picking, are seeded in 25ml BMGY substratum 30 ℃ and are cultured to cell density OD
600nm=2～6, the centrifugal collection thalline of room temperature, with BMMY liquid nutrient medium, cell is resuspended to OD
600nmbe 1.0 with abduction delivering.28 ℃ are continued to cultivate 96h, and it is 1v/v% that every 24h adds 100% methyl alcohol to final concentration to substratum.The pichia yeast genetic engineering bacteria that completes cultivation, through the centrifugal thalline of removing, is collected to supernatant liquor, measure crude enzyme liquid enzyme and live.When host is GS115 and SMD1168, it is on the low side that bacterial strain produces beta-mannase enzyme activity; And KM71H and X – 33 are better as host's enzymatic productivity, methyl alcohol slowly utilizes the KM71H host of type more outstanding, and after methanol induction 96h, strain fermentation vigor reaches 252IU/mL.
Enzyme activity determination method and enzyme unit definition alive: adopt the fixed sugared method of DNS to measure the activity of 'beta '-mannase.0.9mL5.0g/L locust bean gum substrate solution (pH5.0 Ning Meng Suan – Sodium phosphate dibasic damping fluid) is mixed through the enzyme liquid of suitably dilution with 0.1mL, 80 ℃ of reaction 10min, add 3.0mL DNS reagent immediately, boil 7min, after cooling, be settled to 25mL, shake up.In 540nm place working sample liquid absorbancy.Mannosans enzyme activity unit is defined as the required enzyme amount of seminose that per minute hydrolysis substrate produces 1 μ mol and is defined as a unit of activity (1IU).
Embodiment 4: the high density fermentation of restructuring 'beta '-mannase.
Picking transforms the mono-clonal of the recombinant yeast pichia pastoris bacterium KM71H there is recombinant plasmid pPICZ alpha A – man, is seeded in BMGY substratum 30 ℃ and is cultured to cell density OD
600nm=2～6, the inoculum size of volume fraction 10% of take is inoculated in the BMS that glycerol content is 40g/L carries out fermentation culture.Fermentor tank volume is 3L, and liquid amount 1.5L cultivates under the condition of 30 ℃, pH5.0 (28% ammoniacal liquor regulation and control).Recombination yeast is behind fermentor cultivation 24h left and right, and when glycerine exhausts, weight in wet base is at 90 – 150g/L, and the speed with 18.15mL/h/L flows glycerol adding 4h then.The hungry 1h that cultivates after stopping flowing glycerol adding and again rebounding to DO, when the wet amount of cell reaches after 180 – 220g/L, starts stream and adds methanol induction and cultivate.After methanol induction 96h, strain fermentation vigor reaches 2319IU/mL.
Embodiment 5: the purifying of restructuring 'beta '-mannase.
The super filter tube that the centrifugal gained supernatant of fermented liquid is 10KDa by molecular weight carries out ultrafiltration, the albumen of elimination small component and salt, get the liquid 2mL of ultra filtration, through molecular sieve Superdex75PrepGrade purifying, use citric acid-Sodium phosphate dibasic damping fluid of 100mmol/LpH5.0, flow velocity 0.3ml/min wash-out, distribute and collect the sample in elution peak, through enzyme activity determination, determine the collection tube at object sample place, obtain electrophoretically pure target protein, enzyme is lived as 1168IU/mg.As shown in Figure 1, be the SDS-PAGE collection of illustrative plates of purified enzyme liquid.Wherein M is standard molecular weight protein, 1 for the recombinant yeast pichia pastoris KM71H fermented supernatant fluid that contains pPICZ α A plasmid in contrast, 2 is the recombinant yeast pichia pastoris KM71H fermented supernatant fluid that contains pPICZ α A-man plasmid, the 3 restructuring 'beta '-mannases that are purifying.Embodiment 6: the zymologic property analysis of restructuring 'beta '-mannase.
The zymologic property of analyzing the 'beta '-mannase after purifying, the optimal pH of 'beta '-mannase is 5.0, when pH is between 4～6, this enzyme all has the relative enzyme work that is greater than 95%, in the scope of pH3～7, keeps 70h, still has more than 90% enzyme activity.The optimal reactive temperature of 'beta '-mannase is 80 ℃, still has up to 76.8% relative enzyme and live in the time of 90 ℃.At 60 ℃, be incubated 70h and still keep more than 90% enzyme activity.
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, under 80 ℃, 170rpm, different pH (3.0～6.0) enzymolysis 50g/L konjak gum solution, after 2h, HPLC detects the impact of pH on manna oligosaccharide yield, result shows that (Fig. 2) enzymolysis pH is that 3.0 o'clock Rhizoma amorphophalli powders absorb water into solid-state in damping fluid, there is not enzymatic hydrolysis reaction, enzymolysis pH is in 4.0～5.0 scopes, and the enzymolysis efficiency of Rhizoma amorphophalli powder is higher.
(2) hydrolysis temperature: with the enzyme liquid addition of 125IU/g Rhizoma amorphophalli powder, under pH5.0,170rpm, differing temps (50～90 ℃) enzymolysis 50g/L konjak gum solution, the impact of HPLC detected temperatures on manna oligosaccharide yield after 2h, result shows that (Fig. 3) hydrolysis temperature is within the scope of 50～90 ℃, and the enzymolysis efficiency of Rhizoma amorphophalli powder is all higher.
(3) concentration of substrate: with the enzyme liquid addition of 125IU/g Rhizoma amorphophalli powder, under pH5.0,80 ℃, 170rpm enzymolysis concentration of substrate at the konjak gum solution of 2.5～50g/L, the impact of HPLC detection substrate concentration on manna oligosaccharide yield after 2h, result shows that (Fig. 4) is along with the increase of concentration of substrate, the corresponding increase of manna oligosaccharide output, does not have the obvious mass transfer phenomenon of being obstructed.
(4) enzyme dosage: the konjak gum solution of 50g/L of take is substrate, enzymolysis under pH5.0,80 ℃, 170rpm, enzyme liquid addition is 12.5～125IU/g Rhizoma amorphophalli powder, after 2h, HPLC detects the impact of enzyme dosage on manna oligosaccharide yield, result shows that (Fig. 5) is when enzyme dosage is brought up to 50IU/g from 12.5IU/g, along with enzyme dosage increases, manna oligosaccharide output improves constantly, but manna oligosaccharide output increases not obvious while continuing to be increased to a certain degree from 50IU/g.
(5) enzymolysis time: take the enzyme liquid addition of 50IU/g Rhizoma amorphophalli powder, the konjak gum solution that enzymolysis concentration of substrate is 50g/L under pH5.0,80 ℃, 170rpm, different time points sampling within the scope of 0～4h, HPLC detects the impact of enzymolysis time on manna oligosaccharide yield, result shows that (Fig. 6) is because konjaku water-absorbent is extremely strong, during 0h, present solid-state, As time goes on, mannase generation enzymic hydrolysis effect, substrate constantly liquefies, after reaction 2h, basic liquefaction completely, enzymolysis has been tending towards stopping, and manna oligosaccharide content also no longer rises.
Embodiment 8: the degradation analysis of 'beta '-mannase to Rhizoma amorphophalli powder of recombinating under optimum enzymatic hydrolysis condition.
Take the enzyme liquid addition of 50IU/g Rhizoma amorphophalli powder, the konjak gum solution that enzymolysis concentration of substrate is 50g/L under pH5.0,80 ℃, 170rpm, after enzymolysis 2h, heating to 100 ℃ keeps 10min to make enzyme-deactivating, the cooling rear centrifugal residue that goes, supernatant is for HPLC and ion chromatography.The demonstration of HPLC result, manna oligosaccharide concentration is 39g/L, manna oligosaccharide yield is 78%.Fig. 7 is beta-mannase enzyme liberating Rhizoma amorphophalli powder chromatography of ions detected result, as we know from the figure, this restructuring 'beta '-mannase enzymolysis konjaku powder primary product is mannobiose and sweet dew six sugar, mannobiose content is 45.5%, sweet dew six sugar are 34.3%, sweet dew one sugar, mannotriose, mannotetrose content are respectively 7.5%, 8.8%, 3.4%.
Embodiment 9: restructuring 'beta '-mannase enzymolysis Viscogum BE is prepared manna oligosaccharide.
Take the enzyme liquid addition of 30IU/g Viscogum BE, the konjak gum solution that enzymolysis concentration of substrate is 30g/L under pH5.0,70 ℃, 170rpm, after enzymolysis 2h, heating to 100 ℃ keeps 10min to make enzyme-deactivating, the cooling rear centrifugal residue that goes, supernatant is for HPLC and ion chromatography.The demonstration of HPLC result, manna oligosaccharide concentration is 25.8g/L, manna oligosaccharide yield is 78%.
Embodiment 10: the 'beta '-mannase enzymolysis high density of recombinating under enzymatic hydrolysis condition Rhizoma amorphophalli powder is prepared manna oligosaccharide.
The Rhizoma amorphophalli powder of 150g/L is disposable whole interpolations when 0h, 'beta '-mannase when 0h by the disposable whole interpolations of 50IU/g Rhizoma amorphophalli powder (in 150g/L Rhizoma amorphophalli powder), at pH, be 5.0, temperature is enzymolysis under 80 ℃, the rotating speed condition that is 170rpm, after 6h, enzymolysis finishes, enzyme liquid is heated to 100 ℃ and kept 10min to make enzyme-deactivating, the cooling rear centrifugal residue that goes, supernatant is analyzed for HPLC, and result shows that manna oligosaccharide concentration is 56.6g/L.
Embodiment 11: the Rhizoma amorphophalli powder of restructuring 'beta '-mannase enzymolysis portion-wise addition is prepared manna oligosaccharide.
The Rhizoma amorphophalli powder of 150g/L divides three interpolations, and the interpolation time is respectively 0h, 2h and 4h.'beta '-mannase when 0h by the disposable whole interpolations of 50IU/g Rhizoma amorphophalli powder (in 150g/L Rhizoma amorphophalli powder).At pH, be 5.0, temperature is enzymolysis under 80 ℃, the rotating speed condition that is 170rpm, after 6h, enzymolysis finishes, and enzyme liquid is heated to 100 ℃ and kept 10min to make enzyme-deactivating, the cooling rear centrifugal residue that goes, supernatant is analyzed for HPLC, and result shows that manna oligosaccharide concentration is 81g/L.
Claims (12)
1. a high temperature resistant restructuring 'beta '-mannase, is characterized in that, its aminoacid sequence is as shown in SEQ ID No:2.
2. an encoding gene for high temperature resistant restructuring 'beta '-mannase, is characterized in that, is one of nucleotide sequence meeting following requirement:
(1) nucleotide sequence of coding aminoacid sequence as shown in SEQ ID No:2;
(2) nucleotide sequence as shown in SEQ ID No:1.
3. express a recombinant bacterial strain for high temperature resistant restructuring 'beta '-mannase, it is characterized in that, this bacterial strain is on genome, to have integrated the pichia spp of the encoding gene of 'beta '-mannase claimed in claim 2.
4. recombinant bacterial strain according to claim 3, is characterized in that, described pichia spp is secretor type pichia pastoris phaff (Pichia pastoris).
5. build the method for recombinant bacterial strain claimed in claim 3, it is characterized in that, it comprises the steps:
(1) apply conventional construction of recombinant plasmid method and construct the codon optimized plasmid pPICZ α A – man of restructuring, this plasmid be take AOX1 as promotor, and contains the beta-mannase gene shown in SEQ ID No:1 and Zeocin resistant gene;
(2) will recombinate codon optimized plasmid pPICZ α A – man with after Sac I linearization for enzyme restriction, electric shock is transformed in pichia spp Host Strains, forms the pichia spp recombinant bacterial strain of expressing 'beta '-mannase;
(3) by recombinant bacterial strain after Methanol Utilization Phenotype screening, the screening of foreign gene multi-copy integration and abduction delivering screening, filter out the recombinant bacterial strain of overexpression 'beta '-mannase.
6. construction process according to claim 5, is characterized in that, in step (1), described recombinant plasmid pPICZ alpha A – man be codon preference optimization afterwards synthetic goal gene man be inserted into the recombinant plasmid forming in plasmid pPICZ α A.
7. construction process according to claim 5, it is characterized in that, in step (2), after the codon optimized plasmid pPICZ α A – man that will recombinate is with Sac I linearization for enzyme restriction, before electric shock is transformed in pichia spp Host Strains, codon optimized plasmid pPICZ α A – man can be imported to intestinal bacteria and increase.
8. the preparation method of high temperature resistant restructuring 'beta '-mannase claimed in claim 1, is characterized in that, by recombinant bacterium claimed in claim 3 in BMGY substratum 30 ℃ be cultured to cell density OD
600nm=2～6, the centrifugal collection thalline of room temperature, with BMMY liquid nutrient medium, cell is resuspended to OD
600nmbe 1.0 with abduction delivering, continue to cultivate 72h for 28 ℃, it is 1v/v% that every 24h adds pure methyl alcohol to final concentration to substratum, by the recombinant bacterium that completes cultivation through the centrifugal thalline of removing, collect supernatant liquor, through ultrafiltration and molecular sieve purification, prepare high temperature resistant restructuring 'beta '-mannase.
9. high temperature resistant restructuring 'beta '-mannase claimed in claim 1 is prepared the application in manna oligosaccharide at enzyme process.
10. application according to claim 9, is characterized in that, the biomass that are rich in mannosans of take are prepared manna oligosaccharide as material, enzyme method, and described biomass are one or more of Rhizoma amorphophalli powder, sesbania gum, Viscogum BE and guar gum.
11. application according to claim 10, it is characterized in that, take the high temperature resistant restructuring 'beta '-mannase addition of 10-50IU/g, under the concussion of pH4.0-6.0,50-90 ℃ or agitation condition, enzymolysis concentration of substrate is 30-200g/L biomass solution, the centrifugal residue that goes of lowering the temperature after enzymolysis 0.5-8h, supernatant is manna oligosaccharide.
12. application according to claim 11, it is characterized in that, concentration of substrate is the high-concentration raw material of 100-200g/L, enzyme dosage is 10-50IU/g, disposable adding of enzyme starting stage, substrate adds several times, adds the timed interval at 1-3h, the cooling rear centrifugal residue that goes after common enzymolysis 4-12h under the concussion of pH4.0-6.0,50-90 ℃ or agitation condition, supernatant is manna oligosaccharide.
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| CN111424048A (en) * | 2020-06-09 | 2020-07-17 | 北京挑战农业科技有限公司 | Gene for expressing acidic β -mannase, vector and application thereof |
| CN111424048B (en) * | 2020-06-09 | 2020-10-02 | 北京挑战农业科技有限公司 | Gene for expressing acidic beta-mannase, vector and application thereof |
| CN116463320A (en) * | 2022-07-13 | 2023-07-21 | 中南大学 | Beta-mannase derived from mine drainage metagenome, gene, enzyme preparation and application thereof |
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