CN107937378A - A kind of preparation method of neutrality inscribe transelminase - Google Patents

Abstract

The invention discloses a kind of preparation method of neutral inscribe transelminase, will cultivate ripe bacillus WENA703 and carries out second order fermentation to seeding tank, then to fermentation tank by eggplant bottle inclined plane inoculating, produces fermentation liquid；Above-mentioned fermentation liquid is by tubular type centrifugation, plate-frame filtering, ultrafiltration concentration, cardboard diatomite are degerming, standardization is prepared into neutral pectate lyase enzyme activity >=300000u/ml, and enzyme activity conservation rate (25 DEG C six months) is obtained as more than the 95% high vigor neutrality transelminase enzyme liquid of standard through preservative treatment；Wherein, bacillus (Bacillus cereus WENA703), is preserved in China typical culture collection center, deposit number is：CCTCC NO：M2017368.Technical solution using the present invention, the neutral pectate lyase enzyme system of generation can be adapted to preferably and regenerated papermaking technique.

Description

A kind of preparation method of neutrality inscribe transelminase

The present invention relates to biological material specimens preservation, the specific name of the biomaterial is：Bacillus (Bacillus Cereus WENA703), China typical culture collection center is preserved on June 23rd, 2017, address is military for China Chinese Wuhan Universitys, deposit number are：CCTCC NO：M2017368.

Technical field

The invention belongs to biological technical field, more particularly to a kind of preparation method of neutral inscribe transelminase.

Background technology

Though regenerated papermaking is using regenerated resources, the environmental pollution caused by slurrying, alkali collection is eliminated, no matter During with true qualities waste paper and cardboard or white waste paper production secondary stock, due to containing various useless impurity, equally can also discharge Contaminated wastewater environment.The pulping system that large-scale regenerated papermaking enterprise configures according to itself economic and production needs, is such as administered It is improper, also can generation environment pollution problem.As a whole, large-scale regenerated papermaking industry energy conservation emission reduction great efforts, effect are good, are producing Measure it is ever-increasing at the same time, COD discharge capacitys conversely decrease drastically.As COD discharge intensity, from the 0.17T/ ten thousand of 2001 Member drops to ten thousand yuan of the 0.025T/ of 2008, decreases by 85.3%, achieves noticeable achievement.National paper industry waste discharge in 2008 128.8 ten thousand T COD problems, mainly go out in 3081 small business of Zhan Zong enterprises number 88.18%, except some straw pulps Outside paper plant, there are a considerable amount of small-sized regenerated papermaking enterprises.It is right after wastewater discharge standard new GB3544-2008 performs The discharge of wastewater of wastepaper pulping and papermaking is single-row, it is proposed that the pollutant emission limit more more strict than common slurrying enterprise, it is special Very stringent regulation is not made that in terms of wastewater discharge, compared with old plant GB3544-2001, true qualities waste paper pulp-making The wastewater discharge of papermaking and white deinking pulping and paper-making is accordingly from the 210M3/T paper and 280M3/T paper of standard in 2001 Unified 20M3/T paper is reduced to, accordingly have compressed 190M3/T paper and 260M3/T paper, this can't but be to regenerated papermaking industry A severe challenge, in the case of current arid serious water shortage, water-saving, emission reduction, to subtract dirty be even more what regenerated papermaking industry faced One urgent and sacred mission.

There are drainage enzyme, the stickies control enzyme of certain influence power in the market at present, its main component is with Single Fiber element Based on enzyme and esterase, its mechanism of action is by the aqueous colloidal material and nothing in cellulase degradation selectivity paper pulp raw material With fiber fines, slurry is improved in wet end and the removal efficiency of press section free water, reaches the effect for improving drainage.It is being applied In have obvious negative effect, be embodied in：A, may occur broken hole in application process, increase web break number；B, when When the time of slurry storage is longer, enzyme reaction time lengthening, causes slurry weight in wet base to be decreased obviously, and paper pulp combination power weakens, and is drawn into paper Power substantially reduces, and paper machine breaks paper and rewinding termination showed increased, influences paper machine operation；C, paper lines is thick, feel is coarse, shadow Product quality is rung.Producer's use has great risk, and the decline of simultaneous product quality and quality may.Its extensive use More problem is still suffered from, the reality for causing enterprise selectively to use.

In addition, general its enzyme system produced at the same time of prior art strain enzyme-producing vigor cannot well with regenerated papermaking technique It is adapted.

Therefore for drawbacks described above present in currently available technology, it is really necessary to be studied, to provide a kind of scheme, Solve defect existing in the prior art.

The content of the invention

In view of this, it is necessory to provide a kind of complex enzyme formulation for flax fiber raw material degumming and preparation method thereof And flax fiber raw material Degumming method, cost is low, technique is simple and is suitable for industrial applications.

In order to overcome the shortcomings of the prior art, the present invention provides following technical scheme：

A kind of preparation method of neutrality inscribe transelminase, will cultivate ripe bacillus WENA703 by eggplant bottle Inclined plane inoculating carries out second order fermentation to seeding tank, then to fermentation tank, produces fermentation liquid；

Above-mentioned fermentation liquid is by tubular type centrifugation, plate-frame filtering, ultrafiltration concentration, cardboard diatomite are degerming, standardization preparation Into neutral pectate lyase enzyme activity >=300000u/ml, obtaining enzyme activity conservation rate (25 DEG C six months) through preservative treatment is More than the 95% high vigor neutrality transelminase enzyme liquid of standard；

Wherein, bacillus (Bacillus cereus WENA703), is preserved in China typical culture collection center, Deposit number is：CCTCC NO：M2017368.

Preferably, culture medium prescription is：D-sorbite 8%, wheat bran 3%, whey powder 1%, corn pulp 1.6%, sodium chloride 0.5%th, corn plumule powder 1.5%, water 84.4%, wherein the sum of described each component is 100%.

Preferably, seed tank culture condition：35 DEG C ± 1 DEG C of seed tank culture temperature, 120r/min, 8 during speed of agitator 0 When 160r/min, tank pressure 0.1MPa, 1 during throughput 0：0.20th, 8 when 1：0.33, when cultivation cycle 12 is small, seed maturity standard is Thalline more than 90% is in monomer, even dyeing, and neutral pectinesterase enzyme activity is not higher than 200u/ml, pH value 6.7 or so.

Preferably, fermentation tank culture condition：33 DEG C ± 1 DEG C during fermentation tank culture temperature 0,6 when 34 DEG C ± 1 DEG C, stirring turn 180r/min when 120r/min, 8 during speed 0,140r/min when 32, tank pressure 0.1MPa, 1 during throughput 0：0.16th, 8 when 1：0.28、 1 when 32：0.2, when fermentation period 38 is small ± 4 it is small when；Putting tank condition is：PH value rises to 7.0+, and enzyme activity is not further added by, thalline 90% gemma formed above simultaneously has part that autolysis occurs.

Compared with prior art, the neutral pectate lyase enzyme system that technical solution using the present invention produces can be more preferable Adaptation and regenerated papermaking technique, possess following technique effect in regenerated papermaking technique：

1st, using transelminase, cellulase and zytase can direct hydrolysis pectic substance, xylan, Portugal gather The class material such as sugar, heteroglycan, so as to discharge fibre bundle so that bast and xylem are easily isolated, and recycle laccase hydrolysis separation Lignin afterwards；

2nd, using hydrogen peroxide bleaching process, while reaching Baidu's requirement of spinning process requirement, the high pH of oxygen bleaching is utilized The operational characteristic of value and high temperature, further removes residue glue, so as to achieve the purpose that flax fiber raw material degumming；

3rd, bleaching liquor dosage more than 20% during reduction bleaching flax fibers；

4th, amount of caustic soda more than 40% during reduction bleaching flax fibers；

5th, flax fiber fracture strength increase by more than 5%, fineness increase by more than 20%, elongation at break increase by 30% with On, fibre strength is uniform so that the spinnability of fiber is obviously improved, so as to increase economic efficiency, reduces production cost；

6th, the color of waste water is improved as present light brown or yellow by original black or dark brown, COD reduce by 30% with On.

Embodiment

The invention will be further described below.

There are drainage enzyme, the stickies control enzyme of certain influence power in the market at present, its main component is with Single Fiber element Based on enzyme and esterase, its mechanism of action is by the aqueous colloidal material and nothing in cellulase degradation selectivity paper pulp raw material With fiber fines, slurry is improved in wet end and the removal efficiency of press section free water, reaches the effect for improving drainage.But it is being answered There is obvious negative effect in, still suffer from more problem in practical applications.In addition, prior art strain enzyme-producing vigor one As at the same time its produce enzyme system cannot be adapted well with regenerated papermaking technique.

The defects of in order to overcome the prior art, applicant further investigate regenerated papermaking technique, are lived by studying The fibre composition used with paper forms, it is found that a small amount of pectic substance and hemicellulose are to hinder fiber to absorb water in short term, moisten the pass risen Key factor.Therefore, applicant attempts to be coupled into a work system by multienzyme, the synergistic effect by a variety of enzyme systems to fiber, Solve above-mentioned technical problem.However, general its enzyme system function of producing at the same time of prior art strain enzyme-producing vigor is unable to reach reality Border application requirement.For this reason, applicant is to cultivating, producing enzyme vigor is high and enzyme system adapts to be goed deep into regenerated papermaking technique bacterial strain Research.

Based on the studies above, technical solution of the present invention is proposed, it is specific as follows：

1st, neutral inscribe transelminase is the key for solving above-mentioned technical problem of the present invention, and applicant is by passing through reason The selection and breeding studied high vigor neutrality pectate lyase enzyme system and produce bacterial strain are combined by with practice.Our company technical staff is according to this The method in field, Dongguan, Guangdong, Shandong Weifang, Henan Luohe, Chongqing etc. using regenerated papermaking plant emissions sewage and By gathering bacterium sample naturally at the trench of factory's pulping system, screen to obtain one plant through shaking flask from nearly hundred plants of bacterial strains and go out bacterium germination Strain, and by using nitrosoguanidine and ultraviolet radiation mutagenesis combination gene recombination technology to obtain nearly ten thousand plants of changes again under cellular level Different bacterial strain, carries out enzymatic productivity screening by flat-plate bacterial colony form primary dcreening operation combination producing enzyme shaking flask, obtains industrialized producing Bacterial strain, takes orthogonal experiment method to carry out medium optimization, obtains optimal seed culture and culture medium.Eventually pass through shaking flask Screening obtains the bacterial strain that one plant of laboratory numbering is WENA703, i.e. bacillus (Bacillus cereus WENA703), its Biological characteristics is：Thalline is in rod-shaped, even dyeing, and G+, facultative aerobic form brood cell, and brood cell does not protrude thalline, thalline both ends More smooth, majority is in catenation.The bacterial strain has been carried out for proprietary program in China typical culture collection center Culture collection is accepted, and deposit number is：CCTCC NO：M2017368.

After the WENA703 bacterial strains are by above-mentioned mutagenesis, its enzymatic productivity more original starting strain vigor (neutral inscribe pectin Acid cleavage enzyme) 50 times are improved, and stablized by 8 passages more than generation, performance.The strain enzyme-producing vigor is high, and stability is good, Its operative temperature (15 degrees Celsius -70 degrees Celsius) and action pH value (5.5-9.5) can keep more than 80% all than broad Enzymatic activity.

2nd, Spawn incubation.Described bacillus WENA703 cultures containing such as lower inclined plane (including test tube slant and eggplant bottle it is oblique Face) on culture medium, high enzymatic productivity can be kept, strain passes on 8 generation above enzymatic productivities through the slant medium to be stablized, inclined-plane Culture medium prescription is：Beef extract 1%, yeast extract 1%, peptone 1%, glucose 0.5%, sodium chloride 0.5%, 1.5-2.0% Agar, pH value 7.2 ± 0.1,0.1MPa sterilize 30 minutes, and blank inclined-plane is made, and are inoculated with the strain, 34 DEG C ± 1 DEG C, cultivate 20- 24 it is small when, it is spare.Its neutral transelminase activities of the enzyme systems that ferments is up to 267000u/ml, average level 224000 u/ml。

3rd, the optimization of culture medium and shake flask culture conditions.The bacillus WENA703 bacterium pass through shake flask test, difference To different carbon source (glucose, starch, sucrose, corn flour, cellulosic powder, wheat bran, orange peel powder, megasse, pomace etc.), nitrogen source (ammonium sulfate, corn pulp, ammonium nitrate, ammonium chloride, urea, peptone, yeast extract, beancake powder, groundnut meal etc.), inorganic salts (chlorine Change sodium, magnesium sulfate, dipotassium hydrogen phosphate, sodium nitrate etc.), liquid amount, shaking speed, initial ph value, cultivation temperature, cultivation cycle into Go experiment, obtain the optimum medium and shake flask culture conditions of high yield pectin enzyme system, this culture medium and shake flask culture conditions It is：Culture medium prescription (%) D-sorbite 8, wheat bran 3, whey powder 1, corn pulp 1.6, sodium chloride 0.5, corn plumule powder 1.5, water 84.4, wherein the sum of described each component is 100%, it is 6.0-6.5 with sodium carbonate tune pH value before sterilizing, 0.1MPa sterilizes 30 points Clock, condition of culture：32 DEG C ± 1 DEG C, rotating speed 220-240r/min, when culture 28-32 is small.Trained in above culture medium and condition of culture Obtained enzyme liquid to be supported, pectinase activity is tested according to DNS colorimetric methods, an enzyme activity unit is defined as under determination condition, by Enzyme amount needed for substrate release 1mg galacturonic acids per minute.

4th, the preparation of the high vigor neutrality transelminase enzyme liquid of standard.To cultivate ripe bacillus WENA703 by Eggplant bottle inclined plane inoculating is to seeding tank to fermentation tank, and second order fermentation, it is crude enzyme liquid to produce fermentation liquid.Its culture medium prescription is (%)) D-sorbite 8, wheat bran 3, whey powder 1, corn pulp 1.6, sodium chloride 0.5, corn plumule powder 1.5, water 84.4, wherein institute It is 100% to state the sum of each component, is 6.0-6.5 with sodium carbonate tune pH value before sterilizing, 0.1MPa sterilizes 30 minutes；Condition of culture： 35 DEG C ± 1 DEG C of seed tank culture temperature, 160r/min when 120r/min, 8 during speed of agitator 0, tank pressure 0.1MPa, during throughput 0 1：0.20th, 8 when 1：0.33, when cultivation cycle 12 is small, seed maturity standard is in monomer for thalline more than 90%, even dyeing, in Property pectinesterase enzyme activity is not higher than 200u/ml, pH value 6.7 or so；33 DEG C ± 1 DEG C during fermentation tank culture temperature 0,6 when 34 DEG C ± 1 DEG C, 180r/min when 120r/min, 8 during speed of agitator 0,140r/min when 32, tank pressure 0.1MPa, 1 during throughput 0： 0.16th, 8 when 1：0.28th, 32 when 1：0.2, when fermentation period 38 is small ± 4 it is small when, when preceding 8 when 4 is small, the later stage is when 2 is small Detect pH value, thalli morphology, enzyme activity once, putting tank condition is：PH value rises to 7.0+, and enzyme activity is not further added by, thalline 90% gemma formed above simultaneously has part that autolysis occurs.Its pectin activities of the enzyme systems that ferments is up to 267000u/ml, puts down Equal level is 224000u/ml.Above-mentioned fermentation liquid process, tubular type centrifugation, plate-frame filtering, ultrafiltration concentration, cardboard diatomite remove Bacterium, standardization are prepared into neutral pectate lyase enzyme activity >=300000u/ml, and enzyme activity conservation rate (25 is obtained through preservative treatment DEG C six months) the high vigor neutrality transelminase enzyme liquid of standard that is more than 95%.

Since the original strain that the present invention uses is in the sewage and soil of regenerated papermaking enterprise, use WENA703 bacterial strains carry out enzymatic production, its neutral pectate lyase enzyme system produced can be adapted to preferably and regenerated papermaking work Skill.

Based on the studies above basis, the present invention proposes the complex enzyme formulation for regenerated papermaking technique, this meets enzyme preparation Development process it is as follows：Strain breeding thereof, Spawn incubation, enzymatic production, flocculating polymer, plate-frame filtering, ultrafiltration concentration, cardboard silicon Diatomaceous earth is degerming, preservative treatment, standardization prepare, standard enzyme liquid compounding, regenerated papermaking enzyme preparation product.It is it is characterized in that, compound Enzyme preparation each component and mass percentage are as follows：

Neutral inscribe transelminase (enzyme activity >=20000u/ml)：25%-35%；

Endo cellulase (enzyme activity >=5000u/ml)：20%-30%；

Zytase (enzyme activity >=2000u/ml)：15%-25%；

Lipase (enzyme activity >=200u/ml)：10%-20%；

Laccase (enzyme activity >=20u/ml)：5%-10%；

Stabilizer：15%-20%；

Wherein, neutral inscribe transelminase is fermented by bacillus (Bacillus cereus WENA703) and made Standby, which is preserved in China typical culture collection center, deposit number For：CCTCC NO：M2017368.

In above-mentioned technical proposal, by according to " multienzyme coupling ... enzyme-chemically coupling ... non-aqueous enzymatic catalysis technology " design Thinking is more by neutral pectase, neutral xylanase, neutral cellulase and β-glucosyl enzym etc. by related coupling and technology Kind enzyme is coupled into a work system, the synergistic effect by a variety of enzyme systems to fiber：A, efficiently separate fiber and open fiber Lumen pore, effective softening fibre cell membrane, increase the permeability of fiber, the profit that fully absorbs water beneficial to fiber rises, so as to improve mashing effect Fruit, reduces beating energy consumption.B, fiber cohesion is reduced so that fiber becomes more soft, beneficial to the fibrillating of fiber so that Fiber obtains more longitudinal splitting chances, reduces transversely cutting, effectively protects fiber.C, broken fibre primary wall, is conducive to The stripping of outer layer of secondary wall, makes fiber obtain abundant wire-dividing broom purification, and combining power between fiber strengthens.D, defibrination is reduced to fiber Loss, continue to act on the noncrystalline domain of microfibre using cellulase, it is exposed non reducing end, be effectively increased fibre Contact area between dimension, improves into the intensity of paper, effectively reduces fiber fines and is lost in.E, rationally the content of control hemicellulose exists Between 5%~10%, the content of hemicellulose is very big to the influence with reference to power, because the strand of hemicellulose is shorter than cellulose, Have that many keys are misaligned, without crystalline texture, its hydrophily is very strong, and when mashing easily absorbs water swollen and fibrillating, increases The surface area of fiber is added, dissociate more hydroxyls, is conducive to improve the intensity of paper, especially initial beating-up period is to bursting strength Raising with tensile strength becomes apparent.

Complex enzyme formulation each component concrete function is as follows：

A, neutral inscribe-transelminase (Endo-pectate lyase), numbering EC 4.2.2.2.System is entitled more Poly- (Isosorbide-5-Nitrae-α-D- galacturonic acids) lyases, also known as pectic acid transeliminase.Act on pectic acid and low methoxy pectin, production The raw unsaturated oligogalacturonans assembled by unsaturated dimer and unsaturated tripolymer.

In wastepaper as raw material, pectic substance is combined together with cuticula fibrous outer, although only accounting for the 1% of raw material weight, But seriously affect water imbibition.Conventional method is to use alkaline scouring, removes the pectin, wax and pigment of raw fibre, but can not be removed The impurity such as seed.Pectic substance is removed by kiering enzyme-alkaline pectase, can improve the water imbibition of fiber, keeps fiber soft Soft style；At the same time, moreover it is possible to handled at a temperature of 5O DEG C or so, be the excellent process of energy-saving and environmental protection.Hydrocellulose The material being adhered between hemicellulose, cellulose and cellulose, efficiently separates fiber, and effective softening fibre cell membrane, increase Add the permeability of fiber, the profit that fully absorbs water beneficial to fiber rises, so as to improve fiber water retention value；

B, neutral endo-cellulase, numbering EC 3.2.1.4

That is inscribe-Isosorbide-5-Nitrae -1,4 beta-glucanase (Endo-1,4- β-glucanase), is the Cx enzymes in early enzyme concept.System Unite entitled 1,4- (1,3；1,4)-callose 4- glucan hydrolases.It is typical β-dextranase.Catalytic cellulose point Isosorbide-5-Nitrae-β-D- glucoside bonds hydrolyze in subchain, in the lichenin and cereal beta-D-glucans quasi-molecule that also hydrolyze the key Han 1,3- 1,4- keys.Also there is different degrees of vigor in xylan, xyloglucan, beta glucan and various artificial substrates.Make cellulose Inflated polymer, forms short chain cell-oligosaccharide (i.e. cellodextrin, also known as powder-beta-dextrin).There is the different component of several random degree, its One of be probably act on first crystallization or high ordered fiber element enzyme, product for 2-5 glucose units glucan.At random Effect is mainly reflected on CMC, phosphoric acid expansion cellulose and short chain cell-oligosaccharide, and primary product is cellobiose and trisaccharide.No Act on cellobiose.Enzyme activity assay shows glucan Isosorbide-5-Nitrae-β-glucosyl enzym and Isosorbide-5-Nitrae-β-glucosyl enzym, has an impact to it, especially Its CMC forms method for substrate using viscosity descent method or reduction end base.

It is particularly widely used in paper industry in waste paper pulp-making workshop section, reduces fiber cohesion, makes Obtaining fiber becomes more soft, beneficial to the fibrillating of fiber so that fiber obtains more longitudinal splitting chances, reduces laterally Cut-out, effectively protects fiber, and the fiber fines that increasing has utility value are kept, and improves retention；

C, zytase：

Endoxylanase 1,3- xylobiases (Xylan endo-1,3- β xylosidase) numbering EC 3.2.1.32, Also known as zytase and inscribe -1,3- beta-xylanases.The entitled 1,3- β-D- xylan xylanohydrolase enzymes of system.Random hydrolysis 1,3- β-D- xylan 1,3- β-D- xylose glycosidic bonds.

At present, the non-wood pulp in whole world paper pulp accounts for more than 60% in China, and wherein China's straw pulp of rice and wheat then accounts for the whole world More than the 85% of straw pulp of rice and wheat.10-the 30% of hemicellulose (predominantly this glycan) reachable straw pulp component, hemicellulose in straw pulp Cellulose content is higher to be so that the hygroscopic expansion of straw pulp, causes straw pulp drainability poor, and sticky net roll banding is low into the well-illuminated embrittlement of paper, intensity One of the main reason for, thus straw pulp is simply possible to use in the production of low-grade paper.China's Forest Resources shortage, paper making raw material wood pulp lack It is weary, it is impossible to meet the needs of, thus the quality-improving of a large amount of straw pulps is extremely important China's paper industry.For grass The key of the quality-improving work of slurry is to remove hemicellulose, in the current paper industry in China, mainly using sulphite Method slurrying, its labor intensity is big, and efficiency is low, of high cost, and causes serious environmental pollution.The resistance to neutrality of cellulose-less enzymatic activity Zytase can will partly or entirely substitute traditional chemical Treatment straw pulp, while effectively removal half fiber is maintained, and not It can cause the damage to straw pulp fiber so that the lignin in straw pulp is easier to come off, and not only improves association with pulp bleaching degree and application Class, and the dosage of chemical substance can be substantially reduced.Meanwhile be enzymatically treated mild condition, efficient, cost is low, it is poly- to digest wood The product xylose of sugar recyclable other purposes of work, and effectively reduce the environmental pollution in paper mill again.In addition, the enzyme is in alkaline bar Used under part, and the straw pulp treatment process in traditional paper industry can be coordinated, use is very convenient.

It continuingly acts on the noncrystalline domain of microfibre in waste paper pulp, it is exposed non reducing end, is effectively increased Interfibrous contact area and felt power, so that sludge can effective reuse.

D, laccase：

Laccase (laeease, Ec1.10.3.2) be a kind of cupric polyphenol oxidase (polyphehenoi oxidases, PPO), it makes the oxidation of the materials such as hydroquinone, polyphenol, p-phenylenediamine, ascorbic acid by obtaining O2 catalysis, is allowed to generate phase The benzoquinones and water answered.

Laccase has wide spectrum degradability, can be widely used in pesticide, dyestuff, preservative, herbicide, insecticide and fungicide Deng the degraded of the chlorophenols compound in chemicals and aniline substituent.Industrial " three Waste ", chemical pesticide often contain when decomposing Toxic phenol or arylamine.The immobilization laccase processing paper plant waste water such as Bollag, removes effectively sylvan.It is with diatomite etc. Carrier immobilized Laccase Catalyzed pesticide decomposes the oxidation of 2,4- Dichlorophenols, generates insoluble oligomer and removes.Savolainen etc. With laccase Anaerobic Treatment debarking waste water, laccase is to removing the lignin derivative in waste water, tannin, phenolic compound etc. Noxious material has good effect.Laccase can be additionally used in the decoloration of dyestuff, Chao Wei-Liang etc. and be separated with rotting in timber The fungi SM77 arrived carries out the decoloration of azo dyes (such as Orange G), and SM77 is fixed with algae gastral cavity acid sodium.Research shows SM77 pairs The decoloration of orange G is mainly completed by laccase.The beer for the high tannin acid content of laccase treatment that Yague etc. is produced using whiterot fungi Wine waste water, the results showed that the product of polyphenol substance greatly reduces.White-rot fungi can be thorough by synthetic dyestuffs using the laccase of its secretion Bottom is degraded to CO：And H：0, and there is good decolorizing effect.The principle of coupled reaction can occur with many kinds of substance using laccase, Fix it on the filter membrane of sewage-treating reactor, the residue of most of insecticides tested in sewage can be coupled into volume it is big, Material that is not soluble in water, being obstructed by filter membrane, so as to achieve the purpose that environmental protection.

Laccase can be catalyzed lignin degradation in wastepaper as raw material, can increase the yield of fiber, improve fibrous binding force, so that Improve the physical index of paper.

E, lipase

Lipase (Lipase, glycerol ester hydrolase) is under the jurisdiction of carboxylic ester hydrolase class, can be progressively by triglycerides It is hydrolyzed into glycerine and aliphatic acid.Lipase is present in animal and plant and microorganism (such as mould, bacterium) tissue containing fat In.

It acts on acetate, acrylate, synthetic rubber and other special gelatin water in wastepaper as raw material, is allowed to become to have Have water miscible lower-molecular substance such as alcohols etc. so that cause the quality of paper machine and paper decline the problem of effectively solved.

The present invention has following distinguishing feature from the principle of various enzymes：

1) acetate, acrylate, synthetic rubber and other special gelatin water, are effectively hydrolyzed, is allowed to become that there is water solubility Lower-molecular substance such as alcohols etc.；

2) fiber cohesion, is reduced so that fiber becomes more soft, beneficial to the fibrillating of fiber so that fiber obtains More longitudinal splitting chances are obtained, reduce transversely cutting, effectively protect fiber, the fiber fines that increasing has utility value are kept, Improve retention；

3), using enzyme system the characteristics of, the noncrystalline domain of microfibre is continuingly acted in slurry, it is exposed irreducibility end End, is effectively increased interfibrous contact area and felt power, so that sludge can effective reuse；

4) process water systems such as machine water, are continuingly acted on by biology enzyme, hydrolysis is adhered material and paper machine is remaining Colloidal solid, eliminates the sticky aggegation of the fiber fines and partial particulate material that thus bring, will have by the effect of retention agent With fiber flocculation reuse；

5) it is changed into multiple spot, by the synergistic effect of biology enzyme, between the pulp fiber and fiber before online to couple, its is netted Structure more crypto set, fiber fines and sludge can be kept in paper.

Action effect of the above-mentioned complex enzyme formulation in regenerated papermaking technique is presented as：

1) paper strength, is improved：10%~30% is improved into paper bursting strength；Folding strength improves 10%~40%；Into paper ring Pressure improves 10%-30%.

2) steam consumption, is reduced:Ton paper reduces by 0.1 ton~0.15 ton steam.

3), being lost in reduces：Machine water concentration significantly reduces；Retention improves；

4) paper machine operation efficiency, is improved:Page wet strength is improved, so as to reduce the shutdown frequency, improves paper machine operation efficiency.

5), for a long time using the whole approach system of paper machine can be made to become more to clean, paper machine color spot etc. caused by corruption is starched is avoided Paper defects and disconnected paper phenomenon, reduce the wash number of forming net and woollen blanket.

6th, the technology is applied safe and reliable in technology for making daily used paper：Will not because of in production process due to have a power failure, Slurry resting period in stock tank is long more than when small (such as 24) caused by equipment is shut down caused by the abnormal factors, produces Reduce the side effect of pulp strength.

The preparation method of complex enzyme formulation of the present invention is：By raw material enzyme preparation respectively through pre-treatment, then mix in proportion Uniformly, then add the protective agent of appropriate preservative and enzyme, carried out by diatomite filter it is degerming, then lead to measure enzyme activity, Quantitative package, becomes product.

The invention also discloses a kind of regenerated papermaking technique, connected in the water conservancy pulper in waste paper pulp-making by metering pump Continuous to add complex enzyme formulation, which adds according to 100 grams of standards of over dry raw material per ton；

The component and mass percentage of the complex enzyme formulation are as follows：

Neutral inscribe transelminase (enzyme activity >=20000u/ml)：25%-35%；

Endo cellulase (enzyme activity >=5000u/ml)：20%-30%；

Zytase (enzyme activity >=2000u/ml)：15%-25%；

Lipase (enzyme activity >=200u/ml)：10%-20%；

Laccase (enzyme activity >=20u/ml)：5%-10%；

Stabilizer：15%-20%；

Wherein, neutral inscribe transelminase is fermented by bacillus (Bacillus cereus WENA703) and made Standby, which is preserved in China typical culture collection center, deposit number For：CCTCC NO：M2017368.

Using above-mentioned technical proposal, had the following technical effect that in regenerated papermaking technique：

1) retention, is improved.Machine water concentration is reduced, reduces online concentration, retention aid and filtering aid, is conducive to sheet forming, Paper-forming evenness is improved, reduces quantitative difference of banner, reduces disconnected paper, so as to improve speed, stablizes paper machine operation and improves production capacity.

2) anionic trash, is eliminated.Increase wire-dividing broom purification and improve fibrous binding force, intensity can be improved, improve tensile strength That is pulling force, improves ring pressure and bursting strength, is improved in retention, on the basis of reducing mud discharging, had to paper intensity and degree of sizing Improve.

3) the whole pulp-water system of papermaking, is cleaned.Felt and forming net service life can be extended, reduce and replace downtime, So as to improve production efficiency；

4) cost, energy conservation and environmental protection, are reduced.Sludge discharge is reduced, middle-stage wastewater COD is reduced, waste paper pulp will be reduced Usage amount, saves material, and reaches the effect of energy-saving and emission-reduction.

5) first-past retention of paper machine and total retention, are improved.System anion rubbish is reduced, sewage disposal can be sent to some other department Sludge significantly reduces, while reduces middle-stage wastewater import COD, reduces sewage disposal difficulty and processing cost.

6) product quality, is lifted.Quantitative difference of banner is reduced, improves into paper ring pressure, bursting strength, folding strength, improves degree of sizing, Comprehensive Improvement Of Product Quality.

Embodiment 1

Start on July 11st, 2017 to have tried out the complex enzyme formulation of the present invention, July in Dongguan City Xu Feng Paper Co., Ltd The morning 11 on the 11st：00, which starts addition, uses, and September end on probation on the 21st, by the application test of more than 60 days, specific test data was such as Shown in following table：

1 test data of table

Wherein：Since retention agent adds failure of pump during 12 days-September of September 16 days, including during which data do not count.

1 test data of upper table shows：

1st, concentration of surfing the Internet reduces, wet end load reduction；

2nd, machine water concentration reduces, and retention rate is obviously improved, beneficial to sludge reuse；

3rd, waterline has shortening, and drainability enhancing, is saved beneficial to steam.

2 finished paper ash content of table compares

2 as shown by data of upper table：Finished paper ash content preceding data relatively on probation are obviously improved 3%~5%, and sludge reuse effect is bright It is aobvious.

Table 3 uses the ton paper Economic and Efficiency Analysis (estimating) after complex enzyme formulation

Note：Papermaking enzyme is counted by 8.8 ten thousand yuan/ton (not tax included price).

From above-mentioned data analysis, complex enzyme formulation of the present invention can reach following effect：

1st, wet end speed difference is decreased obviously, and wet end dehydration is obviously improved, and waterline shortens, and dehydration is more uniform, and waterline is neater It is whole.Be conducive to the improvement of paper industry evenness, it is less to be conducive to quantitative difference of banner；

2nd, with speed, labial lamina opening is raised, and fan pump frequency improves, and wet end load declines obvious, high vacuum electric current phase It should reduce；Forming net abrasion can be mitigated, prolonged the service life；

3rd, white water consistency is averagely reduced to May by the April and is declined slightly, and retention accordingly improves, and white-water system is more Add cleaning, production run efficiency improves；

4th, finished paper ash content preceding data relatively on probation are obviously improved 3%~5%, sludge reuse positive effect.

5th, long-time service, beneficial to system clean, improves production efficiency.

Embodiment 2

Start on April 28th, 2017 to have tried out serial papermaking provided by the invention in Guangzhou Huadu Changxing Paper Co., Ltd Enzyme product ---, end June 2, tested by the continuous applications of 35 days, specific test data such as following table：

4 test data of table contrasts

Table 5：Use the ton paper Economic and Efficiency Analysis (estimating) after papermaking enzyme

Note：Papermaking enzyme is counted by 80,000 yuan/ton (not tax included price).

From above-mentioned data analysis, complex enzyme formulation of the present invention can reach following effect：

1st, gluing thing significantly reduces, and drying portion gluing thing substantially reduces, and the disconnected paper situation thus influenced is reduced；

2nd, wet end speed difference is decreased obviously, and wet end dehydration is obviously improved, and waterline shortens, and dehydration is more uniform, and waterline is neater It is whole.Be conducive to the improvement of paper industry evenness, it is less to be conducive to quantitative difference of banner；

3rd, with speed, labial lamina opening is raised, and fan pump frequency improves, and wet end load declines obvious, high vacuum electric current phase It should reduce；Forming net abrasion can be mitigated, prolonged the service life；

4th, white water consistency is averagely reduced to May by the April and is declined slightly, and retention accordingly improves, and white-water system is more Add cleaning, production run efficiency improves；

5th, long-time service, beneficial to system clean, improves production efficiency.

Embodiment 3

2 months 25-April 30 in 2017 have tried out provided by the invention multiple in Zhaoqing Cologne packaging limited company Synthase formulation products, Zhaoqing Cologne packaging limited company are located in Zhaoqing City of Guangdong Province great Wang new and high-tech development zones, mainly Product is 300 tons of brown paper of daily output, totally one production line.It is as follows by the interim application test of 42 days, specific test data Table：

6 slurrying workshop section of table (being analyzed by taking stretchable paper AD202 as an example)

7 slurrying workshop section of table (being analyzed by taking stretchable paper AMD303 as an example)

8 AD202 of table tries out front and rear into paper data comparison (2 months)

9 AD202 of table tries out front and rear into paper data comparison (April)

10 AMD303 of table tries out front and rear into paper data comparison

It could see by above-mentioned test data, complex enzyme formulation of the present invention produces significant economic effect in regenerated papermaking technique Benefit is (with stretchable paper AD202；Analyzed exemplified by 95g/m2)：

1st, the Economic and Efficiency Analysis produced：

(1), moisten the defibrination electric current that the effect that rises is brought to decline, ton paper saves 16.5 degree of electricity, according to every 0.7 yuan of meter of the degree electricity charge Calculate, 11.55 yuan of ton paper power cost saving；

(2), fiber wire-dividing broom purification is strengthened, and interfibrous cohesiveness enhancing, the lifting of intertexture power, the retention rate brought improve, The production brought according to every raising one percentage point increases by 1 kilogram, according to the lifting values of 6.8% retention rate, improves yield 6.8 kilograms × 2 yuan=13.8 yuan (according to 2000 yuan of meters of manufacture cost)；

(3), enzyme preparation additive amount is 0.075 kilogram of ton paper, according to 108 yuan/kilogram (without tax, containing freight charges) meter, is needed altogether Want 0.075 × 108=8.1 first；

It is above-mentioned total：Ton paper it is cost-effective=11.55+13.8-8.1 member=17.25 yuan.

2nd, potential economic benefit：

(1), into the lifting of paper index, the appropriate ratio for reducing pulpboard can be brought；

(2), white-water system cleans, and the SS values that the elevator belt of retention rate comes in water system can significantly reduce.

Embodiment 4

28 days-May 31 April in 2017 has tried out complex enzyme provided by the invention in Jiangmen star Paper Co., Ltd Formulation products, were tested, the morning 8 April 28 by the continuous applications of 34 days：45, which start addition, uses, and cut-off has been tried out May 31 Into during which since Equipment disables 2 days enzyme preparation products.

Specific test data such as following table：

The floating sludge contrast situation of 11 wet slag of table slurry

Two workshops conveying, the one floating contrast table of workshop wet slag slurry

Note：It it is 1-April 27 April before on probation；Trial employment period is 28-May 24 April.

12 slurry of table contrasts situation

Note：It it is 1-April 27 April before on probation；28-May 31 trial employment period in my April.

13 speed of table contrasts situation

Note：It it is 1-April 27 April before on probation；(wherein 16-5 in the May months 28 28-May 31 trial employment period in my April Since equipment fault reason influences during day, statistics is not entered into).

Table 14 uses the ton paper Economic and Efficiency Analysis (estimating) after enzyme preparation

Note：1st, papermaking enzyme is counted by 80,000 yuan/ton (not tax included price).

2nd, the unit price of ton paper material unit consumption is the manufacture cost-price differential (trial employment period average daily two of kraft inner paper and sand tube paper Workshop screenings decrement is 342.72 tons, calculates, is back to use in craft paper for totally 34.72 tons according to solid content 10%).

By above-mentioned test data, following conclusion (of pressure testing) can be drawn：

1st, May 16 due to there is equipment fault, caused to produce unstable, contrast cut-off 16 day operation data in May system Meter, it is 33.9% that 560 meters/min of speed, which accounts for total time ratio,；

2nd, two workshops conveying, the one workshop floating amount of wet slag slurry is reduced to the per day of May by April per day 765M3 419.27M3, daily reduces 342.73M3, reduces 44.8% on year-on-year basis；

3rd, online concentration reduces (starch adhesive, lining slurry, core slurry) 6.4%, 2.6%, 0.27% on year-on-year basis, and drainability is accelerated, and is beneficial to Speed improves；

4th, machine water system more cleans, and stablizes for a long time using beneficial to production system；

5th, since wet slag starches the reduction of floating amount, economic benefits.

The explanation of above example is only intended to help to understand method and its core concept of the invention.It should be pointed out that pair For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out Some improvement and modification, these are improved and modification is also fallen into the protection domain of the claims in the present invention.

The foregoing description of the disclosed embodiments, enables professional and technical personnel in the field to realize or use the present invention. A variety of modifications to these embodiments will be apparent for those skilled in the art, as defined herein General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, it is of the invention The embodiments shown herein is not intended to be limited to, and is to fit to and the principles and novel features disclosed herein phase one The most wide scope caused.

Claims (4)

1. a kind of preparation method of neutrality inscribe transelminase, it is characterised in that ripe bacillus will be cultivated WENA703 carries out second order fermentation by eggplant bottle inclined plane inoculating to seeding tank, then to fermentation tank, produces fermentation liquid；

Above-mentioned fermentation liquid is by tubular type centrifugation, plate-frame filtering, ultrafiltration concentration, cardboard diatomite are degerming, standardize and be prepared into Property pectate lyase enzyme activity >=300000u/ml, through preservative treatment obtain enzyme activity conservation rate (25 DEG C six months) for 95% with On the high vigor neutrality transelminase enzyme liquid of standard；

Wherein, bacillus (Bacillus cereus WENA703), is preserved in China typical culture collection center, preservation Numbering is：CCTCC NO：M2017368.

2. the preparation method of neutrality inscribe transelminase according to claim 1, it is characterised in that culture medium prescription For：D-sorbite 8%, wheat bran 3%, whey powder 1%, corn pulp 1.6%, sodium chloride 0.5%, corn plumule powder 1.5%, water 84.4%, wherein the sum of described each component is 100%.

3. the preparation method of neutrality inscribe transelminase according to claim 1, it is characterised in that seed tank culture Condition：35 DEG C ± 1 DEG C of seed tank culture temperature, 160r/min when 120r/min, 8 during speed of agitator 0, tank pressure 0.1MPa, ventilation 1 when measuring 0：0.20th, 8 when 1：0.33, when cultivation cycle 12 is small, seed maturity standard is in monomer for thalline more than 90%, and dyeing is equal Even, neutral pectinesterase enzyme activity is not higher than 200u/ml, pH value 6.7 or so.

4. the preparation method of neutrality inscribe transelminase according to claim 1, it is characterised in that fermentation tank culture Condition：33 DEG C ± 1 DEG C during fermentation tank culture temperature 0,6 when 34 DEG C ± 1 DEG C, 180r/min when 120r/min, 8 during speed of agitator 0, 140r/min when 32, tank pressure 0.1MPa, 1 during throughput 0：0.16th, 8 when 1：0.28th, 32 when 1：0.2, ± 4 when fermentation period 38 is small Hour；Putting tank condition is：PH value rises to 7.0+, and enzyme activity is not further added by, 90% gemma formed above of thalline and has part hair Raw autolysis.