CN1304588C - Preparation of oligose from alpha-L-arabglycosidase - Google Patents
Preparation of oligose from alpha-L-arabglycosidase Download PDFInfo
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- CN1304588C CN1304588C CNB200510038237XA CN200510038237A CN1304588C CN 1304588 C CN1304588 C CN 1304588C CN B200510038237X A CNB200510038237X A CN B200510038237XA CN 200510038237 A CN200510038237 A CN 200510038237A CN 1304588 C CN1304588 C CN 1304588C
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Abstract
The present invention relates to a method for preparing oligoxylose, particularly to a method for preparing oligoxylose by alpha-L-arabinosidase. The technical scheme adopted by the present invention comprises the following steps that a, a xylan solution is prepared; b, xylanase and alpha-L-arabinosidase are added to the solution for hydrolyzation; c, a xylan water solution is decolorized, exchanged with ions and concentrated to be prepared into the oligoxylose after the enzymolysis of the xylan solution is carried out. The present invention has the advantage that the xylan is saccharified and decomposed into the oligoxylose by the combined function of the xylanase and the alpha-L-arabinosidase, so that the xylobiose content of a product is increased, and the quality and the purity of the product are improved.
Description
Technical field
The present invention relates to a kind of method for preparing xylo-oligosaccharide, especially a kind of α of application-L-arabinose glycosides enzyme prepares the method for xylo-oligosaccharide.
Background technology
Bifidus bacillus is a most important profitable strain in the human intestinal, the large intestine that mainly is present in human body, human body is had various health-care, improve intestinal environment, suppress pathogenic bacteria, regulate immunization, synthetic vitamin B group improves the absorption of calcium, reduces blood ammonia levels and cholesterol concentration, suppress the generation of cancer, effect such as the treatment tract function is disorderly and antitumor.Every studies show that, xylo-oligosaccharide are better than other active oligose, especially xylo-biosess as bifidus factor, have significant bifidus bacillus multiplication capacity, and not by digestibility, no carious tooth and promotion human body are to the characteristics such as absorption of calcium.Therefore, xylo-oligosaccharide becomes one of focus of foodstuffs industry research and development as a kind of new type functional oligose.China is a large agricultural country, and annual 5.7 hundred million tons of the various crop stalks that produce as if only burning to make fertilizer, are not only wasted resource but also cause environmental pollution.Therefore, utilize these biological wastes to prepare xylo-oligosaccharide and have certain economic benefits and social benefit.
The xylo-oligosaccharide production method comprises at present: acid-hydrolysis method, hot-water extraction, mould deep-fermentation and enzyme process etc., the xylanase hydrolysis method is the main method that xylo-oligosaccharide is produced, be about to corn cob sodium hydroxide solution lixiviate, with acid neutralization, filtration, filtrate is directly added zytase after treatment and is prepared oligose then.But, at corn cob, about 94% hemicellulose is a pectinose glucuronic acid xylan in the agriculture and forestry organic waste material such as bagasse and stalk, this class xylan be one with β-1, the xylan backbone that 4 glycosidic links link, have 1 above, 3-pectinose or 1, the side shoot that the 2-glucuronic acid constitutes, when utilizing this class xylan of single xylanase hydrolysis to prepare xylo-oligosaccharide, these side shoots then produce the space barrier to the effect of zytase, make zytase can not in conjunction with and decompose its contiguous xylan, this not only influences the efficient of enzymatic hydrolysis, and influences the quality of xylo-oligosaccharide.
Summary of the invention
The low deficiency of xylobiose content when overcoming the prior art for preparing xylo-oligosaccharide, the invention provides a kind of xylo-oligosaccharide preparation method that can improve xylobiose content, promptly utilize the combined action of α-L-arabinose glycosides enzyme and zytase, by adding α-L-arabinose glycosides enzyme when the xylanase hydrolysis xylan, remove the pectinose side shoot on the xylan, to reduce the effect generation steric restriction of these side shoots to zytase, make the big fragment that has the pectinose side shoot in the enzymolysis product obtain further hydrolysis, improve the output and the purity of final xylo-oligosaccharide product, solved problem effectively.
The technical solution adopted for the present invention to solve the technical problems may further comprise the steps:
A. prepare xylan solution;
B. adding zytase and α-L-arabinose glycosides enzyme are hydrolyzed in above-mentioned solution;
C. behind the enzymolysis xylan digest get final product through decolouring, ion-exchange, after concentrating xylo-oligosaccharide.
The concrete steps of the inventive method are:
A. the damping fluid compound concentration with pH5-7 is the xylan solution of 1-25%;
B. add zytase and α-L-arabinose glycosides enzyme in above-mentioned solution, hydrolysis is 5-18 hour in the time of 60-100 ℃; Wherein the zytase consumption is 8-200 (a U/g xylan), and α-L-arabinose glycosides enzyme dosage is 5-50 (a U/g xylan);
C. behind the enzymolysis xylan digest get final product through decolouring, ion-exchange, after concentrating xylo-oligosaccharide.
That xylan solution concentration is recommended among the step a of the present invention is 3-20%, preferably 20%.Temperature of reaction is recommended 80-100 ℃ among the step b, preferably 90 ℃, specifically can decide according to the thermostability of different sources enzyme; Hydrolysis time was recommended 12 hours; Every gram xylan substrate recommends to use zytase 50-150U, preferably 200 (U/g xylans); α-L-arabinose glycosides enzyme dosage is recommended 5-50 (U/g xylan); 10 (U/g xylans) preferably.
Being used for the damping fluid of the xylan solution prepared among the present invention can be citrate buffer solution, phosphoric acid buffer.
Prepare in the method for xylo-oligosaccharide with α-L-arabinose glycosides enzyme in the present invention, when hydrolyzed xylan, add α-Pu Taotang aldehydic acid enzyme again, can remove the side shoot of xylan better, improve the content of xylo-bioses in the product; Wherein the consumption of α-Pu Taotang aldehydic acid enzyme is recommended 6-60 (U/g xylan).
Because the thermostability enzyme has risk of pollution, the fast reaction speed of minimizing, can improve zymolyte solubleness and higher advantages such as anti-chemical modification are arranged in biological treatment process, can improve the technology and the economic feasibility of xylan hydrolysis process.So the preferred thermotolerance zytase of zytase among the present invention, the α-preferred thermotolerance α of L-arabinose glycosides enzyme-L-arabinose glycosides enzyme.
In the present invention, xylan source cork and grass hemicellulose as raw material, xylan preparation can be with reference to method (the Pellerin P of Pellerin P etc., Gosselin M, Lepoutre JP, Samain E and Debeire P (1991) Enzymic production ofoligosaccharides from corncob xylan.Enzyme Microb Technol 132:617-621) extracts from vegetable fibres such as corn cob, straw and bagasse with alkali, as the enzymatic conversion substrate.Also can buy from Sigma company.
The preparation of zytase among the present invention and α-L-arabinose glycosides enzyme can be made by the microbial liquid fermentation process that produces xylan degrading enzyme system, comprise bacterium and fungi, for example, Thermotoga maritima Thermotoga maritima, thermophilicly separate sugared anaerobic bacillus(cillus anaerobicus) Thermoanaerobacteriumsaccharolyticum JW/SL-YS485, Trichodermareesei Trichoderma reesei, straw mushroom bacterium Volvariella volvacea etc., can be from U.S. type culture collection center, Chinese microorganism strain preservation center and edible mushrooms institute buy, the zytase that the mentioned microorganism fermentation produces and α-L-arabinose glycosides enzyme can be through drainage column, molecular sieve or ion exchange resin are prepared into partially purified zymin.Zytase also can be bought from Sigma company.
The thermotolerance enzyme of mentioning among the present invention also can be by utilizing gene recombination technology with zytase in the thermophilic microorganism and the high expression level acquisition in appropriate host cell respectively of α-L-arabinose glycoside enzyme gene.Thermotoga maritima is that a kind of 55-90 of being grown in ℃ submarine volcano mouth is neighbouring, the bacterium of strictly anaerobic, provides the important source of high reactivity and thermostability hemicellulase.The invention provides a kind of method for preparing the thermotolerance enzyme: natural zytase and α-L-arabinose glycoside enzyme gene is the primer according to the gene 5 ' end of the utmost point thermotolerance zytase of Thermotoga maritima coding and α-L-arabinose glycosides enzyme and suitable 5 ' end of 3 ' terminal sequence institute synthetic and 3 ' end, from Thermotoga maritima, obtain by pcr amplification, subsequently this dna fragmentation is inserted in the plasmid, reorganization pET-20b-xynB and pET-20b-ara that structure is finished, with conversion appropriate host cell comprises intestinal bacteria and makes its expression subsequently.Afterwards, the reorganization bacterium of having expressed is passed through centrifugal collecting cell, use damping fluid (50mmol/L, pH 6.8) re-suspended cell subsequently and make it broken, after heat treatment the centrifuging and taking supernatant liquor is enzyme liquid.
The invention has the beneficial effects as follows, adopt the combined action of zytase and α-L-arabinose glycosides enzyme, the xylan saccharification is decomposed, make it to generate xylo-oligosaccharide, improved xylobiose content in the product, improved the quality and the purity of product.To carry out the thin-layer chromatography stratographic analysis by the xylan enzymolysis liquid that the inventive method obtains and show (seeing Fig. 2, Fig. 3), compare with existing single xylan enzyme process, after α-L-arabinose glycosides enzyme added, xylo-bioses and wood sugar obviously increased, and xylotriose prolongs in time also minimizing.Further adopting the HPLC high pressure liquid chromatography to detect shows, in zytase and α-symphyogenetic xylan digest of L-arabinose glycosides enzyme, the content of xylo-bioses is significantly improved, xylotriose is almost degraded fully simultaneously, the above wood oligose of trisaccharide also obviously reduces, and produces (seeing Fig. 3, Fig. 4) with pectinose.This shows that the corn cob xylan more helps xylanase hydrolysis xylan skeleton after α-L-arabinose glycosides enzyme has been removed the Arabinoside side shoot.The xylo-oligosaccharide analysis on Content is used Sugarpakl on Waters HPLC 246-E high pressure liquid chromatograph, 6.5mm * 300mm sugar post analysis.Chromatographic condition: column temperature: 85 ℃; Moving phase: water; Flow velocity: 0.5mL/min; Differential refraction detector sensitivity: 4, external standard method is demarcated, and sample size is 10 μ L.Xylo-bioses and xylotriose standard model are purchased the company in Sigma.
Description of drawings
The present invention is further described below in conjunction with drawings and Examples.
Fig. 1 is the SDS-PAGE collection of illustrative plates of utmost point thermotolerance α-L-arabinose glycosides enzyme purification process, M among the figure: protein standard specimen; 1:E.coli JM109 (DE3)/pET-20b; 2:E.coliJM109 (DE3)/the full cell of pET-20b-ara; 3:E.coli JM109 (DE3)/pET-20b-ara thermal treatment; The pure enzyme of 4:Ara.
Fig. 2 is the TLC collection of illustrative plates of different time enzymic hydrolysis corn cob xylan. among the figure 2,4,6,8,10: xylanase hydrolysis 1.5h, 3h, 5h, 8h, 12h; 3,5,7,9,11: zytase and α-L-arabinose glycosides enzyme associating hydrolysis 1.5h, 3h, 5h, 8h, 12h; 1,12: the xylo-oligosaccharide standard specimen.
Fig. 3 is the chromatography of ions figure of xylanase hydrolysis corn cob xylan product. 9.543min among the figure: xylotriose; 11.408min: xylo-bioses; 14.039min: wood sugar.
Fig. 4 is the chromatography of ions figure of zytase and α-L-arabinose glycosides enzyme associating hydrolysis of corncob xylan product. 9.822min among the figure: xylotriose; 11.435min: xylo-bioses; 14.052min: wood sugar; 16.114min: pectinose.
Embodiment
Embodiment 1:(1) getting particle diameter is air-dry corn cob fiber about 3mm, soak the centrifugal moisture of removing in back with clear water earlier, add sodium hydroxide solution by solid-to-liquid ratio 7～10: 1, final concentration is 1%～3%, at room temperature or lixiviate under the high temperature, collect filtrate then and be neutralized to subacidity, promptly get xylan with the long-pending ethanol sedimentation of triploid then with acid.
(2) preparation thermotolerance enzyme
According to xynB, two pairs of primers of ara sequences Design among the Genbank, be that template is carried out pcr amplification with the genomic dna of T.maritima bacterium.Tm-xymB-N end primer be (5 '-CCGTTCCATGGACTACAGGATGTGC-3 '), and it is (5 '-CCGCTCGAGCGGATATATCTTTCTTCCCTT-3 ') that Tm-xymB-C holds primer.EcoRV restriction enzyme site and initiator codon are arranged in the Tm-ara-N:5-GGGGGTACCATGTCCTACAGGATAGTG-3; XhoI restriction enzyme site in the Tm-ara-C:5-AACTGCAGTCACTCGAGCAATTCTACCTCAAT-3.Behind zytase and α-L-arabinose glycosides enzyme PCR product insertion plasmid pET-20b, make up recombinant expression plasmid pET-20b-xynB and pET-20b-ara.
After getting 0.01 μ g recombinant expression plasmid pET-20b-xynB and pET-20b-ara electricity respectively and being transformed in 50 μ L e. coli jm109s (DE3) or BL21-CodonPlus (the DE3)-RIL competent cell, shaking culture 1h in the SOC substratum of 500 μ L, get 100 μ L bacterium liquid and coat LB/Amp (100 μ g/mL) or Kna (30 μ g/mL) flat board, 37 ℃ of overnight incubation, thus genetic engineering bacterium E.coli BL21-CodonPlus (the DE3)-RIL/pET-20b-xynB of zytase (XynB) and the genetic engineering bacterium E.coli JM109/pET-20b-ara of α-L-arabinose glycosides enzyme (Ara) obtained.
The cultivation of Thermotoga maritima is that substratum is driven oxygen, fills nitrogen, adds reductive agent Na after the cooling through heating
2S 0.5g/L, and being sub-packed in the 100mL serum bottle that fills nitrogen in advance, seal and the cooling of sterilizing after, insert kind of a liquid with syringe by 0.5% inoculum size, 80 ℃ leave standstill and cultivate 8～10h and get final product.
Used culture medium prescription is (1L): 10g Tryptones, 5g yeast powder, 27g NaCl, 1mg resin reddish black (resazurin), 15ml trace element trace (Difco Maritima Brothmedium), 0.5g Na
2S, pH 7.0.The preparation of trace element: by following prescription, Nitrilotriacetic acid 1.5g, MgSO
47H
2O 3.0g, MnSO
4H
2O 500.0mg, NaCl 1.0g, FeSO
47H
2O 100.0mg, Co (NO
3)
26H
2O 100.0mg, CaCl
2(anhydrous) 100.0mg, ZnSO
47H
2O 100.0mg, CuSO
45H
2O 10.0mg, AlK (SO
4)
2(anhydrous) 10.0mg, Boric acid 10.0mg, Na
2MoO
42H
2O 10.0mg, Na
2SeO
3(anhydrous) 1.0g.Earlier Nitrilotriacetic acid is dissolved in 500mL water, pH is modulated 6.5 with the KOH of 2～3M.Add other compositions again, be settled to 1L.
Single colony inoculation of picking genetic engineering bacterium E.coli BL21-CodonPlus (DE3)-RIL/pET-20b-xynB and JM109 (DE3)/pET-20b-ara contains the LB liquid nutrient medium of Amp resistance in 5mL, 37 ℃ of incubated overnight, insert LB resistance liquid nutrient medium again with 1% inoculum size then, rotating speed 200r/min, 37 ℃ of shaking culture are to OD
600Reach 0.8～1.0 and make liquid seeds, be linked in the fermentor tank.
The LB substratum is formed: 1% Tryptones, 0.5% yeast extract, 1%NaCl, transfer pH 7.0, and after sterilization is cooled to 37 ℃, adds and insert 0.01%Amp, liquid amount 60%.The initial pH 7.0 of fermentor tank, final pH 7.9, air flow 1: 1, temperature is 30 ℃, rotating speed 250r/min.Be cultured to OD
600Reach 0.6～0.8, stream adds IPTG to concentration 0.8mmol/L, continues to cultivate 6h, puts jar.
With 4, the centrifugal 10min collecting cell of 800 * g, resuspended with 150mL potassium phosphate buffer (50mmol/L, pH 6.8), through ultrasonic wave or broken instrument (the French Pressure of high pressure cell, Thermo) smudge cells, the centrifugal 20min of 9,600 * g, get supernatant behind 70 ℃ of following thermal treatment 20min, the centrifugal 30min of 9,600 * g, supernatant liquor is enzyme liquid.The SDS-PAGE collection of illustrative plates of its purge process as shown in Figure 1.
(3) with the alkali extracting to xylan be made into 25% concentration with 60mmol/L pH5.5 citrate buffer solution after, add thermotolerance zytase 200U/g, thermotolerance α-L-arabinose glycosides enzyme 10U/g, 100 ℃ of hydrolysis temperatures, hydrolysis 8 hours.Behind the enzymolysis xylan digest get final product through decolouring, ion-exchange, after concentrating the high-load xylo-oligosaccharide of xylo-bioses.
Embodiment 2: substantially the same manner as Example 1, difference is, 95 ℃ of hydrolysis temperatures, and xylan hydrolysis 12 hours, respectively at hydrolysis 1.5h, 3h, 5h, 8h, 12h sampling carrying out TLC analyzes, as shown in Figure 2; 12 hours products therefroms of hydrolysis carry out ion chromatography as shown in Figure 4.
Embodiment 3: substantially the same manner as Example 1, difference is, only add the thermotolerance zytase and carried out xylan hydrolysis 12 hours in 95 ℃, and respectively at hydrolysis 1.5h, 3h, 5h, 8h, 12h sampling carrying out TLC analyzes, as shown in Figure 2; 12 hours products therefroms of hydrolysis carry out ion chromatography as shown in Figure 3.
Embodiment 4: will be from straw with the alkali extracting to xylan be made into 4% concentration with 10mmol/L pH6 citrate buffer solution after, add zytase 50U/g, α-L-arabinose glycosides enzyme 5U/g, 90 ℃ of hydrolysis temperatures, hydrolysis 4 hours.Behind the enzymolysis xylan digest get final product through decolouring, ion-exchange, after concentrating the high-load xylo-oligosaccharide of xylo-bioses.
Embodiment 5: will be from bagasse with the alkali extracting to xylan be made into 20% concentration with 30mmol/L pH7 citrate buffer solution after, add zytase 150U/g, α-L-arabinose glycosides enzyme 20U/g, 75 ℃ of hydrolysis temperatures, hydrolysis 15 hours.Behind the enzymolysis xylan digest get final product through decolouring, ion-exchange, after concentrating the high-load xylo-oligosaccharide of xylo-bioses.
Embodiment 6: with alkali extracting corn cob xylan.With straw mushroom bacterium (Volvariella volvacea) behind liquid fermenting, obtain supernatant with the nylon membrane filtration, through 60～80% ammonium sulfate precipitations, make precipitation resuspended with pH 6.8 phosphoric acid buffers again, get supernatant and be prepared into partially purified α-L-arabinose glycosides enzyme through Phenyl-agarose drainage column, TOSOHASS-HW-55S molecular sieve and HTP hydroxyapatite column chromatography.
With the alkali extracting to xylan be made into 1% concentration with 50mmol/L pH5 citrate buffer solution after, add zytase 8U/g, α-L-arabinose glycosides enzyme 50U/g, 55 ℃ of hydrolysis temperatures, hydrolysis 18 hours.Behind the enzymolysis xylan digest get final product through decolouring, ion-exchange, after concentrating the high-load xylo-oligosaccharide of xylo-bioses.
Embodiment 7: substantially the same manner as Example 6, difference is corn cob xylan 20mmol/L pH6.5 Na
2HPO
4-NaH
2PO
4Damping fluid is made into 3% concentration, enzyme dosage: thermotolerance zytase 200U/g, α-L-arabinose glycosides enzyme 15U/g, 60 ℃ of hydrolysis temperatures, hydrolysis 5 hours.
Embodiment 8: substantially the same manner as Example 1, difference is, has also added α-Pu Taotang aldehydic acid enzyme 6-60U/g during xylan hydrolysis, 80 ℃ of hydrolysis temperatures.
Claims (5)
1, a kind of method for preparing xylo-oligosaccharide with α-L-arabinose glycosides enzyme may further comprise the steps:
A. the damping fluid compound concentration with pH5-7 is the xylan solution of 1-25%;
B. add zytase and α-L-arabinose glycosides enzyme in above-mentioned solution, hydrolysis is 5-18 hour in the time of 60-100 ℃; Wherein the zytase consumption is 8-200 (a U/g xylan), and α-L-arabinose glycosides enzyme dosage is 5-50 (a U/g xylan);
C. behind the enzymolysis xylan digest get final product through decolouring, ion-exchange, after concentrating xylo-oligosaccharide.
2, according to claim 1ly prepare the method for xylo-oligosaccharide, it is characterized in that with α-L-arabinose glycosides enzyme:
Xylan solution concentration is 3%-20% among the step a;
The zytase consumption is the 50-150U/g xylan among the step b, and α-L-arabinose glycosides enzyme dosage is the 5-50U/g xylan.
3, according to claim 1ly prepare the method for xylo-oligosaccharide, it is characterized in that with α-L-arabinose glycosides enzyme: temperature of reaction described in the step b is 80-100 ℃.
4, according to claim 3ly prepare the method for xylo-oligosaccharide, it is characterized in that with α-L-arabinose glycosides enzyme:
A. with pH6.5 damping fluid preparation xylan solution, strength of solution is 20%;
B. add zytase and α-L-arabinose glycosides enzyme in above-mentioned solution, hydrolysis is 12 hours in the time of 90 ℃; Wherein the zytase consumption is the 200U/g xylan, and α-L-arabinose glycosides enzyme dosage is the 10U/g xylan.
5, according to claim 1ly prepare the method for xylo-oligosaccharide, it is characterized in that also added α-Pu Taotang aldehydic acid enzyme among the step b, the consumption of said α-Pu Taotang aldehydic acid enzyme is 6-60 (a U/g xylan) with α-L-arabinose glycosides enzyme.
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