A kind of method of polyacrylic resin immobilized enzyme continuous degradation xylogenTechnical field
The present invention relates to a kind of method of polyacrylic resin immobilized enzyme continuous degradation xylogen, belong to xylogen enzymolysis technical field.
Background technology
Xylogen is extensively present in the bulk raw materials such as various grain straws, due in the biorefinery process of lignocellulose, xylogen is the primary structure barrier of restriction transformation efficiency, at present, and the industrial xylogen often utilized in chemical reaction removing lignocellulose.But chemical treatment method pollutes comparatively greatly usually, energy consumption is higher, and as the current bleaching technology based on chlorination generally adopted in China, the pollution problem caused manifests day by day, has been strictly prohibited or limits use in developed country.In addition, in chemical degradation xylogen process, due to the reaction conditions of high temperature, also can there is the reaction of some negative senses in xylogen, produce adverse influence to treating processes.And the zymolysis technique of xylogen has environmental friendliness, less energy-consumption, advantage that reaction specificity is strong, is subject to the extensive concern of investigator in recent years.
Compared with traditional chemical catalyst, enzyme as a class biological catalyst, have that catalytic efficiency is high, specificity is strong, enzyme live controlled, reaction conditions is gentle, the advantage such as pollution-free, the field such as be widely used in pharmacy, environmental protection, food, brewage.But enzyme also exists many defects in actual applications, as stable not to heat, strong acid, highly basic, organic solvent etc., be difficult to reclaim from reaction system, pollution products also brings difficulty etc. to purification of products, thus causes the raising of production cost.In order to address these problems, enzyme immobilization technology arises at the historic moment.So-called enzyme immobilization to refer to solid material by enzyme constraint or be limited in certain area, makes it still can carry out catalyzed reaction, and a recyclable and reusable class technology.Such as, Liu Lukuan (Liu Lukuan, Su Zhongliang, Li Lu, Deng the preparation of. amino functional SBA-15 molecular sieve and to Mierocrystalline cellulose enzyme immobilizatio [J]. Qingdao University of Science and Technology's journal (natural science edition), 2013,34 (6): 579-584) people such as, amino functional SBA-15 mesopore molecular sieve makes carrier to have adopted copolycondensation legal system standby, carry out cellulase immobilization, compared with resolvase, immobilized enzyme improves all greatly to the tolerance of soda acid, thermostability, operational stability, package stability.In patent CN102392013A, contriver discloses a kind of magnetic immobilized cross-linked cellulase aggregate and preparation method thereof and application, organically combined by magnetic harden monitoring technology and cross-linked enzyme aggregate technology, obtain the magnetic immobilized cross-linked lipase aggregate that not only there is high enzyme vigor but also have good operation performance, as biological enzyme agent widespread use, extensive enzyme reactor can be specially adapted to.In a word, selecting suitable solid support material to be raising activity of the immobilized enzyme is substantially the most also most important approach.
Method at present about polyacrylic resin immobilized enzyme continuous degradation xylogen have not been reported.
Summary of the invention
The method of a kind of polyacrylic resin immobilized enzyme continuous degradation xylogen that the present invention proposes, adopt solvable-soluble carrier (S-IS) polyacrylic resin, the solvability of carrier is changed by adjust ph, enable immobilized enzyme enzymolysis xylogen under dissolved state, under insoluble state, make immobilized enzyme precipitate recycling, reach the object such as utilization ratio, reduction production cost, shortening production cycle improving enzyme.
The method of a kind of polyacrylic resin immobilized enzyme of the present invention continuous degradation xylogen, specifically comprises the following steps:
(1) take polyacrylic resin and put into deionized water, be configured to the solution that mass concentration is 4 ~ 8%, then the sodium hydroxide solution adding 1 ~ 3mol/L adjusts pH to be 10 ~ 11, makes it dissolve, adjust pH to 8 ~ 9 with the acetic acid of 1 ~ 3mol/L again, namely obtain carrier soln.
(2) be that the enzyme liquid of 0.25 ~ 2mg/mL adds in carrier soln by concentration, again linking agent is added in carrier soln by the concentration of 1 ~ 3mg/mL, stir 3 ~ 6h, drip 1 ~ 3mol/L acetic acid adjust ph again to 3 ~ 4, immobilized enzyme is separated out, leave standstill 20 ~ 40min, then centrifugal treating is carried out, the speed of centrifugation is 4000 ~ 6000rpm, time is 5 ~ 15min, finally throw out is washed 3 ~ 5 times with containing the sodium-chlor of 0.5 ~ 2mol/L and 0.01 ~ 0.03mol/L acetic acid solution of 0.5 ~ 2mol/L calcium chloride, being i.e. fixed enzyme.
Wherein, be one or more in laccase, peroxidase and violent peroxidase in the enzyme liquid described in step (2), linking agent is any one in 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide, glutaraldehyde and tannic acid.
(3) be in the lignin liquor of 10 ~ 100mg/mL by 10 ~ 100mmol/L Acetic acid-sodium acetate buffer concentration, immobilized enzyme is put into lignin liquor, immobilized enzyme concentration is made to reach 10 ~ 100IU/mL, then at 30 ~ 50 DEG C, pH3 ~ 5 time hydrolysis 12 ~ 48h, the structural changes of xylogen in filtrate is detected after filtering, then filter residue is put into new xylogen, repeat hydrolysis 8 ~ 15 times.
Xylogen recited above is any one in sourwood quality, alkali lignin, enzymolysis xylogen, sulfonated lignin, Milled wood lignin.
The invention has the advantages that:
1, the method for a kind of polyacrylic resin immobilized enzyme of the present invention continuous degradation xylogen, is recycling in process, and the xylogen of fixing chemical fibre enzyme also likely in residue come off is combined, and such enzyme and carrier adsorption are cross-linked, and is easy to reclaim.
2, the method for a kind of polyacrylic resin immobilized enzyme of the present invention continuous degradation xylogen, after each hydrolysis, remaining residue participates in hydrolysis next time, and decrease the loss of enzyme, immobilized enzyme shows good operational stability, is conducive to suitability for industrialized production.
3, the method for a kind of polyacrylic resin immobilized enzyme of the present invention continuous degradation xylogen, immobilized enzyme still maintains the activity of more than 85% after reusing through 10 times, and resolvase can only use 1 time, and this makes enzymolysis efficiency significantly improve.
Accompanying drawing explanation
Fig. 1: the schema of the method for a kind of polyacrylic resin immobilized enzyme continuous degradation xylogen that the present invention proposes.
Embodiment
Below in conjunction with the drawings and specific embodiments in detail technical characterstic of the present invention is described in detail.
The method of a kind of polyacrylic resin immobilized enzyme of the present invention continuous degradation xylogen, as shown in Figure 1, specifically comprises the following steps:
(1) take polyacrylic resin and put into deionized water, be configured to the solution that mass concentration is 4 ~ 8%, then the sodium hydroxide solution adding 1 ~ 3mol/L adjusts pH to be 10 ~ 11, makes it dissolve, adjust pH to 8 ~ 9 with the acetic acid of 1 ~ 3mol/L again, namely obtain carrier soln.
(2) be that the enzyme liquid of 0.25 ~ 2mg/mL adds in carrier soln by concentration, again linking agent is added in carrier soln by the concentration of 1 ~ 3mg/mL, stir 3 ~ 6h, drip 1 ~ 3mol/L acetic acid adjust ph again to 3 ~ 4, immobilized enzyme is separated out, leave standstill 20 ~ 40min, then centrifugal treating is carried out, the speed of centrifugation is 4000 ~ 6000rpm, time is 5 ~ 15min, finally throw out is washed 3 ~ 5 times with containing the sodium-chlor of 0.5 ~ 2mol/L and 0.01 ~ 0.03mol/L acetic acid solution of 0.5 ~ 2mol/L calcium chloride, being i.e. fixed enzyme.
Wherein, be one or more in laccase, peroxidase and violent peroxidase in the enzyme liquid described in step (2), linking agent is any one in 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide, glutaraldehyde and tannic acid.
(3) be in the lignin liquor of 10 ~ 100mg/mL by 10 ~ 100mmol/L Acetic acid-sodium acetate buffer concentration, immobilized enzyme is put into lignin liquor, immobilized enzyme concentration is made to reach 10 ~ 100IU/mL, then at 30 ~ 50 DEG C, pH3 ~ 5 time hydrolysis 12 ~ 48h, the structural changes of xylogen in filtrate is detected after filtering, then filter residue is put into new xylogen, repeat hydrolysis 8 ~ 15 times.
Xylogen recited above is any one in sourwood quality, alkali lignin, enzymolysis xylogen, sulfonated lignin, Milled wood lignin.
embodiment 1:
The method of a kind of polyacrylic resin immobilized enzyme of the present invention continuous degradation xylogen, specifically comprises the following steps:
(1) take polyacrylic resin and put into deionized water, be configured to the solution that mass concentration is 6%, then the sodium hydroxide solution adding 3mol/L adjusts pH to be 11, makes it dissolve, then adjust pH to 9 with the acetic acid of 3mol/L, namely obtain carrier soln.
(2) be that the enzyme liquid of 1mg/mL adds in carrier soln by concentration, again linking agent is added in carrier soln by the concentration of 2mg/mL, stir 4h, then drip 1mol/L acetic acid adjust ph to 4, immobilized enzyme is separated out, leave standstill 40min, then carry out centrifugal treating, the speed of centrifugation is 5000rpm, and the time is 10min, finally throw out is washed 3 times, being i.e. fixed enzyme with containing the sodium-chlor of 1mol/L and the 0.02mol/L acetic acid solution of 1mol/L calcium chloride.
Wherein, the enzyme liquid described in step (2) is laccase, and linking agent is 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide.
(3) be in the lignin liquor of 50mg/mL by 50mmol/L Acetic acid-sodium acetate buffer concentration, immobilized enzyme is put into lignin liquor, immobilized enzyme concentration is made to reach 50IU/mL, then 45 DEG C, be hydrolyzed 36h under pH4, the structural changes of xylogen in filtrate is detected after filtering, then filter residue is put into new xylogen, repeat hydrolysis 10 times.
Xylogen recited above is alkali lignin.
Result shows, macromole xylogen is cracked into small molecules xylogen or lignin monomer, and in xylogen, total hydroxy radical content increases, and phenyl ring isocyclic compound content reduces, and in addition, immobilized enzyme still maintains the activity of more than 85% after reusing through 10 times.
embodiment 2:
The method of a kind of polyacrylic resin immobilized enzyme of the present invention continuous degradation xylogen, specifically comprises the following steps:
(1) take polyacrylic resin and put into deionized water, be configured to the solution that mass concentration is 4%, then the sodium hydroxide solution adding 2mol/L adjusts pH to be 10, makes it dissolve, then adjust pH to 8 with the acetic acid of 1mol/L, namely obtain carrier soln.
(2) be that the enzyme liquid of 0.5mg/mL adds in carrier soln by concentration, again linking agent is added in carrier soln by the concentration of 3mg/mL, stir 6h, then drip 2mol/L acetic acid adjust ph to 3, immobilized enzyme is separated out, leave standstill 30min, then carry out centrifugal treating, the speed of centrifugation is 4000rpm, and the time is 15min, finally throw out is washed 4 times, being i.e. fixed enzyme with containing the sodium-chlor of 2mol/L and the 0.01mol/L acetic acid solution of 2mol/L calcium chloride.
Wherein, the enzyme liquid described in step (2) is laccase, peroxidase and violent peroxidase, and linking agent is glutaraldehyde.
(3) be in the lignin liquor of 30mg/mL by 80mmol/L Acetic acid-sodium acetate buffer concentration, immobilized enzyme is put into lignin liquor, immobilized enzyme concentration is made to reach 80IU/mL, then 35 DEG C, be hydrolyzed 24h under pH5, the structural changes of xylogen in filtrate is detected after filtering, then filter residue is put into new xylogen, repeat hydrolysis 15 times.
Xylogen recited above is sulfonated lignin.
Result shows, macromole xylogen is cracked into small molecules xylogen or lignin monomer, and in xylogen, total hydroxy radical content increases, and phenyl ring isocyclic compound content reduces, and in addition, immobilized enzyme still maintains the activity of more than 60% after reusing through 15 times.
embodiment 3:
The method of a kind of polyacrylic resin immobilized enzyme of the present invention continuous degradation xylogen, specifically comprises the following steps:
(1) take polyacrylic resin and put into deionized water, be configured to the solution that mass concentration is 7%, then the sodium hydroxide solution adding 1mol/L adjusts pH to be 11, makes it dissolve, then adjust pH to 8 with the acetic acid of 2mol/L, namely obtain carrier soln.
(2) be that the enzyme liquid of 1.5mg/mL adds in carrier soln by concentration, again linking agent is added in carrier soln by the concentration of 1mg/mL, stir 3h, then drip 3mol/L acetic acid adjust ph to 4, immobilized enzyme is separated out, leave standstill 20min, then carry out centrifugal treating, the speed of centrifugation is 6000rpm, and the time is 5min, finally throw out is washed 5 times, being i.e. fixed enzyme with containing the sodium-chlor of 1.5mol/L and the 0.03mol/L acetic acid solution of 1.5mol/L calcium chloride.
Wherein, the enzyme liquid described in step (2) is laccase, and linking agent is glutaraldehyde.
(3) be in the lignin liquor of 80mg/mL by 40mmol/L Acetic acid-sodium acetate buffer concentration, immobilized enzyme is put into lignin liquor, immobilized enzyme concentration is made to reach 40IU/mL, then 40 DEG C, be hydrolyzed 48h under pH3.5, the structural changes of xylogen in filtrate is detected after filtering, then filter residue is put into new xylogen, repeat hydrolysis 8 times.
Xylogen recited above is alkali lignin.
Result shows, macromole xylogen is cracked into small molecules xylogen or lignin monomer, and in xylogen, total hydroxy radical content increases, and phenyl ring isocyclic compound content reduces, and in addition, immobilized enzyme still maintains the activity of more than 72% after reusing through 8 times.