CN101857859A - Preparation method of xylanase or lignin peroxidase and application thereof to industrial bleaching field - Google Patents
Preparation method of xylanase or lignin peroxidase and application thereof to industrial bleaching field Download PDFInfo
- Publication number
- CN101857859A CN101857859A CN 201010156197 CN201010156197A CN101857859A CN 101857859 A CN101857859 A CN 101857859A CN 201010156197 CN201010156197 CN 201010156197 CN 201010156197 A CN201010156197 A CN 201010156197A CN 101857859 A CN101857859 A CN 101857859A
- Authority
- CN
- China
- Prior art keywords
- zytase
- enzyme
- lignin peroxidase
- preparation
- lignin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Paper (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a preparation method of xylanase or lignin peroxidase, which comprises the following steps: (1) activating and culturing green Saccharomonospora strain to prepare mother liquor; (2) inoculating the mother liquid obtained in step (1) into Sauton culture medium for inducing to produce enzyme which contains induction substrate for culture; (3) centrifuging the bacteria liquid obtained in step (2) and then collecting clear liquid to obtain the crude extract of the enzyme; and (4) dialyzing the crude extract in step (3), and then carrying out chromatography and purification to obtain theenzyme solution of the xylanase or the lignin peroxidase. The mother liquid obtained in step (1) can be also inoculated into the lignin peroxidase containing the induction substrate prepared by post-fermentation and culture. The xylanase or the lignin peroxidase prepared by the method of the invention has lower cellulase activity, thereby preventing reduction of fiber strength and viscosity of thick liquid in the process of enzyme processing; and the prepared xylanase or the lignin peroxidase has certain heat-resistance and alkali resistance, can directly and effectively attack lignin and degrade hemicellulose, is applicable to pre-bleaching paper pulp in industrial production, and has excellent bleaching effect.
Description
Technical field
The invention belongs to bio-bleaching material field, be specifically related to a kind ofly utilize method that green sugared Zymomonas mobilis prepares zytase and lignin peroxidase and in the application of industrial bleaching field.
Background technology
Association with pulp bleaching is a necessary link in the paper industry.In chemistry or machinery pulping process, have 90% xylogen to remove from the cellulosic fibre of timber or other coarse raw materials approximately, but still residual 10%, this part residual xylogen can cause paper pulp to be brown, and reduction strength of paper (Chen Jiachuan etc., 1999).For bleached pulp must be removed remaining xylogen, traditional method is to use acid bleaching agent such as chlorine, dioxide peroxide etc. to remove the chemical bleaching method of xylogen, but this method can make the organic chloride that contains a large amount of difficult degradations, deleterious, carcinogenic strongly, teratogenesis in the paper waste, thereby cause the severe contamination (Feng Wenying etc., 2002) of environment.In order to address this problem, must reform bleaching process, develop new non-polluting bleachoing technology.Therefore traditional chlorine bleach method afterwards to a great extent by nearly chlorine-free bleaching (ElementalChlorine Free, ECF) and total chlorine free bleaching (TCF) (total chlorine-free TCF) replaces.The ECF bleaching has greatly reduced the formation of organic chloride, but has caused the increase that other pharmaceutical chemicalss consume simultaneously.If before chemical bleaching, with zymins such as zytase and lignin-degrading enzymes paper pulp is carried out pre-treatment, will make chemical bleaching agent such as chlorine be penetrated into the paper pulp the inside better, thereby significantly reduce the consumption (thank ring bright etc., 2003) of chemical bleaching agent.In the ascendant to the research of slurrying biotechnology both at home and abroad in recent years, wherein utilize the bleaching of microorganism enzymatic to become one of research topic the most promising in the current paper industry.Adopt enzyme preparation biological (in advance) bleaching, not only can reduce kappa number for pulps, suitably improve performance index such as the viscosity of paper pulp and whiteness, and can reduce follow-up ECF or TCF pharmaceutical chemicals consumption, the pollution load (Yao Chunli etc., 2003) of reduction bleaching effluent.
Xylan (xylan) is a kind of poly five-carbon sugar, it is the important component of plant hemicellulose, it accounts for 1/3rd of plant carbohydrates total amount, is the renewable physical resources (Katagiri etc., 2001) that content second enriches after Mierocrystalline cellulose at occurring in nature.Xylan mainly is present in the secondary wall of vegetable cell, is between xylogen and other saccharans, plays ligation.Xylan is a kind of heterozygosis poly molecule, and main chain is linked to each other by the wood sugar glycosidic bond by a plurality of xylopyranosyl.The substituting group of the weak point of the multiple different sizes of ining succession on the side chain mainly contains 0-ethanoyl, 4-0-methyl D-glucuronic acid residue, L-arabinose residue etc.Other several structural polysaccharides (as xylogen, Mierocrystalline cellulose, pectin, dextran etc.) are connected with key covalently or non-covalently in these side chains and the vegetable cell, form the important structure-cell walls of vegetable cell.Also just because the difference of these side chains makes that the structural changes scope of xylan is very big, by β-1, the polyxylan linear molecule that the 4-glycosidic link connects is to highly branched heterogeneous polysaccharide (Kavita etc., 2002) from only.
Zytase can become xylan degrading oligose and wood sugar.The research of zytase was just begun as far back as the sixties, from the microorganism in difference source, be separated to the zytase (thank ring bright etc., 2004) of a large amount of dissimilar, difference in functionalitys.The nearest more than ten years, continuous development and progress along with biotechnology, particularly after the widespread use of genetic engineering technique and protein engineering, understanding to zytase is more deep, zytase all demonstrates wide application prospect in fodder industry, pulp and paper industry, foodstuffs industry, energy industry, caused the extensive concern (bosom Wen Hui etc., 2000) of scientists.
Sense stricto zytase is inscribe-β-1,4 zytase.Inscribe-β-1, the 4-zytase all exists great potential in many biological technology application.The β-1 of their main hydrolyzed xylan main chain backbones, 4 wood sugar glycosidic bonds, the lignocellulosic materials that will contain xylan is converted into oligomeric xylose and xylo-bioses etc., and then be converted into a series of biological products with high using value, as be applied to the bread manufacturing, beer and cheese fermentation etc. (Yao Chunli etc., 1998).Be used for inscribe-β-1,4 zytase that the zytase goods of pulp bio pre-bleaching mainly also are meant.Contain in the active centre of zytase a plurality of can with substrate bonded sublocus, the structure in these sites, size, quantity and they determine the kinetic parameter (Iqbal etc., 1994) of this enzyme to avidity, specificity, the mode of action and the enzyme reaction of different substrates with the spatial relation of catalysis group.
The generalized zytase is meant the general name of one group of enzyme that can the degradation of hemicellulose xylan, mainly comprise three classes: (1) inscribe-β-1,4-zytase (endo-β-1,4-xylanases), preferentially on different loci, act on xylan and long-chain wood oligose, from β-1, the inside cutting wood sugar glycosidic bond of 4-xylan backbone, thereby making xylan degrading is wood oligose, and its hydrolysate is mainly xylo-bioses and the above oligomeric xylose of xylo-bioses, and a spot of wood sugar and pectinose are also arranged.(2) circumscribed-β-1, (exo-β-1 4-xylanases), acts on the non-reducing end of xylan and wood oligose to the 4-zytase, produces wood sugar.(3) (β-xylosidases), this enzyme is by the terminal xylose residues (Jonathan etc., 2003) that discharges of cutting wood oligose for xylobiase.
Its catalysis characteristics of the zytase of different sources is also variant, is mainly reflected on different the optimal reaction pH value and thermostability.In recent years, people have extracted zytase from multiple microorganism.Most principal work concentrates on the research aspect of the enzymatic property of the zytase that extracts from bacterium and fungi.The existing at home and abroad more report of the enzymatic property of the zytase of relevant bacterium.Wherein the more pseudomonas (Pseudomonas) that has is carried out in research work, Nocardia bacteria (Nocardia), Flavobacterium (Flavobacterium), gas bacillus (Acromonas), more than ten reports (Suren etc., 1997) that belong to such as yellow sporangium (Xanthonona s).Data analysis shows that zytase that bacterium produces can be divided into two classes substantially: acidproof zytase of high-molecular weight and low-molecular-weight alkali resistant xylanase.But in fungi, do not have this difference, but the alkali resistance of lower molecular weight zytase but is a common.Bacteriogenic zytase protein subunit is more single, and molecular weight ranges is at 8～145kDa.Mycetogenetic zytase protein subunit is then complicated, and the molecular weight size variation is also bigger.Research to the fungi zytase mainly concentrates on mould go up (Hakan etc., 2004) of white-rot fungi, aspergillus and wood.No matter be from fungi or from bacterium, the internal cutting type xylanase optimum temperuture is generally between 40 ℃～60 ℃.General zytase that bacterium produces produces the zytase thermostability than fungi and becomes reconciled.In addition, more actinomyceticly belong to especially streptomycete, also can secrete zytase, and have better heat-resisting and alkali resistance and more and more be subjected to people's attention (Abdul etc., 1997).It generally is 3～10 that different microorganisms is produced the tolerant pH value scope of zytase.Optimum pH is generally 4～7.The iso-electric point variation range of different zytases is between 3～10.The great majority temperature range of certified endoxylanases between 45-75 ℃ shows high reactivity.Have only bacterium after the minority separation and purification and the endoxylanase of fungi showing maximum activity more than 80 ℃.Because the complex structure of Heteroxylan, xylan degrading enzyme can not be opened all xylan glycosidic bonds, so the hydrolysis of xylan needs overlapping but the multiple microbial xylanase acting in conjunction (Tuncer etc., 1999) of different qualities.
Because zytase expression level in natural materials is too low, be difficult to mass production, and some enzymic activitys of production cost costliness and zytase can not satisfy the application correlated condition fully.Therefore, up to the present zytase is not applied widely.Along with the continuous progress of biotechnology, people wish to come more in depth to study xylanase gene by genetically engineered and protein engineering, and develop the zytase product that meets people's requirement.The utilization genetic engineering technique carries out molecular modification to xylanase gene on molecular level, be expected to solve the applied defect of some xylanase activities such as resistance, pH value, thermostability etc., utilize bio-reactor then to be expected to hundreds and thousands of times of ground and improve its expression amounts.The genetically engineered research of zytase has become international and has studied one of focus.
International studies show that, some bacterial strain in the actinomycetes also can be passed through its excretory extracellular enzyme (zytase and peroxidase) degradation of hemicellulose and xylogen.The peroxidase AliP-P3 of reported first actinomycetes-green spore streptomycete (Streptomycesviridosporus) excretory lignin degradings such as Ramachandra in 1988, and enzyme carried out preliminary evaluation (Ramachandra etc., 1988).Ball and McCarthy in 1988 also once report can produce zytase and peroxidase outside the born of the same parents but the actinomycetes (Ball etc. of cellulose-less enzymic activity, 1988), report other actinomycetes-after 1994 in succession as the purple streptomycete of heat (S.thermoviolacaceus), deinsectization streptomycete (S.avermilitis UAH30), streptomyces albus (S.albus), thermomonospora fusca (Thermomonospora fusca BD25) etc. can be secreted high zytase of thermostability and peroxidase.
Xylogen (lignin) is that a class is formed by connecting, is only second to the abundantest and most important cellulosic natural aromatic high molecular polymer by phenylpropyl alcohol alkane unit by ehter bond and carbon bond in vegitabilia, be distributed widely in and have in the fascicular higher plant, be gymnosperm and angiosperm special chemical component (Hammel K E, Jensen K A, Mozuch M D, et al., 1993).Xylogen in wood fibre except that being present in primary cell wall and secondary cell wall; also surrounding Mierocrystalline cellulose and hemicellulose is filled between the microfibril together; combine closely with the cell wall polysaccharides composition; constitute one; strengthen the intensity and the hardness of plant tissue; prevent too much moisture and objectionable impurities infiltration cell simultaneously, plant is had support and provide protection (Cheng Yanjun, 1994).
Studies show that the degraded fully of xylogen is fungi, bacterium and the coefficient result of corresponding microorganism group.Occurring in nature, fungi are considered to play leading role (Higuchi T, 1990 in the degraded of xylogen; Ball A S, B Godden, P Helvenstein, et al., 1990).According to the rotten type of fungi, it can be divided into whiterot fungi, brown rot fungus and soft-rot bacterium three classes (Tuomela M., Vikman M., Hatakka A, etal., 2000) to different components in the lignocellulose.Whiterot fungi, brown rot fungus all belong to Basidiomycetes (Basidiomycetes), and soft-rot bacterium belongs to capsule Gammaproteobacteria (Ascomycetes) or imperfect fungi (Fungi Imperfect).
Many investigators find that in succession bacterium also has the function of secretion lignin-degrading enzymes, and bacterium can breed in being added with the soil of Vinsol, and this molecule of can slowly degrading.The bacterium class that wherein works mainly is bacterial strain (the Tuomela M. of acinetobacter (Acinetobacter sp.), Flavobacterium (Flavobacterium sp.), micrococcus sp (Micrococcussp.), Rhodopseudomonas (Pseudomonas sp.) and xanthomonas (Xanthomonas sp.), Vikman M.Hatakka A, et al., 2000; Carol A C, 1996; De Boer H A, Zhang Y Z, Collins C, et al., 1987; Nie G, ReadingN S, Aust S D, 1998).In these bacteriums, Rhodopseudomonas is the most effective degraded person (Lokesh K V, 2001).Certainly, the katalysis of lignin-degrading enzymes in degradation by bacteria xylogen process at present, discover that bacterium can the lignin degrading low molecular weight part and the degraded product of xylogen, therefore infer that they may work in the final stage of lignin degradation, and the degradation by bacteria xylogen occurs in the elementary metabolism stage, the generated time of lignin-degrading enzymes be the logarithmic phase of bacterial growth and stationary phase (Nie G, Reading N S, Aust S D, 1998).
Actinomycetes not only can be at elementary metabolism stage lignin degrading, make lignocellulosic substance generation modification at the initial stage of Degradation, also can be at low molecular weight substance (Mohamed S A, the Sami S of Degradation later stage metabolism fungus degrading xylogen generation, Ali G, 1999).Actinomycetes in the lignin degradation process by increasing the water-soluble of xylogen and then promoting its degraded, simultaneously owing to can penetrate insoluble matrix such as lignocellulose, thereby in neutrality, slight alkalinity soil or compost, actinomycetes also participate in organic initial degraded and humify (Tuomela M., Vikman M., Hatakka A, et al., 2000; George P C, Susan K K, Tawnya M B, 2000).
In the world the research of some bacterial strains in the streptomyces in the actinomycetes (Streptomyces) Pseudonocardia (Pseudonocardia) is shown strong interest (Alan C andDavid B W, 1983 in recent years; Joshua S, Diana I and David BW et al., 1997), some bacterial strains can produce heat-staple plurality of enzymes and be, participate in lignocellulose degradation, wherein the thread fungus lignin degrading of strepto-genus reaches as high as 20% (Tuomela M., Vikman M., HatakkaA, et al., 2000).Discover simultaneously actinomycetes than the white-rot fungi that belongs to Basidiomycotina have growth rapidly, nutritional condition is simple, be difficult for polluting, be easy to large-scale application and produce characteristics (Antonopoulos VT such as the enzyme resisting basic is strong, Hernandez M and Arias ME, et al., 2001).Therefore, xyloid degraded is had more commercial value to actinomycetes and potentiality are used, and in recent years its research begun to obtain investigator's attention, but still are in the initial stage.
From Boruff in 1934 and Buswell find first can the microbial population of lignin degrading after, people have carried out big quantity research to the biodegradable enzyme system of xylogen.Nineteen eighty-three Glenn and Tien etc. find that simultaneously Phanerochaete chrysosporium produces the outer peroxidase of born of the same parents system--lignin peroxidase (Lignin peroxidases) and manganese peroxidase (Manganese peroxidases) in the white-rot fungi; The Jitian of Japan finds laccase (Laccase) (Buswell J.A, E.Odier, 1985) first in raw lacquer.The main enzyme of the biological degradation system that finally studies show that xylogen is made up of these three kinds of enzymes.
Lignin peroxidase is a series of Fe of containing of representative
3+, porphyrin ring (IX) and the attached base of protoheme isozyme, the kind and the physico-chemical property of the enzyme that is produced by different microorganisms have nothing in common with each other.Lignin peroxidase is the lignin-degrading enzymes of finding from Phanerochaete chrysosporium (Phanerochaetechrysosporium) at first, plays key effect in lignin degradation, is the main component of the peroxidase of lignin degrading.Lignin peroxidase substrate scope is wider, not only can oxidative lignin and xylogen model thing, can also direct oxidation have the aromatics of methyl oxygen, phenols or the non-phenols of high oxidation reduction potential, and big other organic double compound of class.
The molecular structure of manganese peroxidase is similar to lignin peroxidase, also is the outer peroxidase of born of the same parents that a kind of H202 of dependence has glycosyl, and enzymatic reaction needs mn ion.Mostly the main generation bacterium of manganese peroxidase is some white-rot fungis, belongs to Basidiomycotina, Holobasidiomycetidae, the polyporaceae of aptychus Zoopagales.The characteristics of manganese peroxidase are can only oxidation phenol type xylogen, in the process of oxidation of phenol, manganese peroxidase under the startup of H202, oxidation Mn
2+Be Mn
3+, Mn then
3+Oxidation of phenol generates benzene oxygen residue.This mode with the lignin peroxidase oxidation of phenol has obviously different (Wang Hailei, Li Zongyi, 2003).
Laccase is an effectively degrading lignocellulosic of a class, extensively is present in the biological enzyme in the secretory product of various plants and mushroom.In fungi, laccase is distributed in (Duran N, Ferrer I, Rodriguez J, 1987) in basidiomycetes (Basidimycetes), pore fungus (Polyporus), the Podospora (Podospora) mostly.Recently, discover that bacterium also can produce laccase,, in addition, in some animal kidneys and serum, also found laccase (Zhang Jiayao, Gong Liping, Luo Yuxuan etc., 2002) as giving birth to fat azospirillum (Azospirillum lipoferum).
Though utilize the bio-bleaching research of zytase to carry out the time of a suitable fixed length, but still need update to adapt to requirements of actual production.The activity that often contains cellulase in the general zytase.Particularly the hyperactivity of endo cellulase can cause in the enzyme treating processes that fibre strength, slurry viscosity descend, and therefore the selection to the enzyme source is very crucial.
In nearly 20 years, warm enzyme (pH8-9,65 ℃) and alkaline high temperature resistant enzyme (pH9-10,70 ℃) three phases (Milagres etc., 2004) in acid cold-adapted enzyme (pH5-6,55 ℃), the meta-alkalescence have been experienced in the development of zytase.The zytase optimum pH of early development in acid range, must be regulated slurry pH value during owing to pulping bleaching mostly, makes troubles to operation.In order to adapt to the alkaline hot conditions of paper pulp after the alkaline process chemical pulp oxygen delignification, the zytase of exploitation recent years generally is neutral alkali partially, and optimum temperature more also has raising.Such enzyme need not to regulate pH value of slurry and temperature when being used for association with pulp bleaching, be beneficial to bleaching operation, and after enzyme is handled, can extract the back and recycle being dissolved in xylogen in the washes, xylan etc., (Zhang Yong etc., 2003) thereby such as the colourity of reduction waste water and COD.The alkali-resistant body acidic xylanase of as seen researching and developing the cellulose-less enzymic activity has crucial meaning.For the very flourishing country of some paper-making industries such as the U.S., Canada, Sweden, be main raw material mostly with timber, the scale great majority of slurry factory are at hundreds of tons every day, replace or partly replace chemistry route to carry out association with pulp bleaching beginning just to be subjected to favoring widely with biological approach from the seventies, and the application in paper industry of the zytase goods through improveing has realized industrialization (Qasim etc., 2000) in recent years.In China, the nearest more than ten years of research that are used for the microbial enzyme product of bio-bleaching just begin, basically also be limited to analytically screening, zytase purifying and the structure thereof of producing the natural strain excellent of zytase, character, only cloned the minority xylanase gene, the industrialization of utilization genetic engineering means is produced the zytase product and be yet there are no report.Therefore, screening from the strain excellent extracellular enzyme, separation and evaluation are applicable to that the alkaline-resisting heat resistant xylanase of pulping bleaching has great importance.
And lignin peroxidase has huge practical application potentiality at aspects such as biotechnology and environmental engineering such as bio-pulping, bio-bleaching, soil organisms scrubbing, the investigator has carried out a large amount of research to it, but what exist in actual applications is still perplexing the researchdevelopment of this enzyme such as aspect problems such as economy, feasibility and adaptability.
At first, its performance of most of lignin peroxidase of having reported can not satisfy the actual requirement of industrial application, require not only heat-resisting but also the alkaline-resisting and cellulose enzymic activity not of used zymin in pulp and paper industry, therefore, good product lignin peroxidase bacterial strain is very important.Secondly, the lignin peroxidase production cost is higher at present, and it is lower that its reason mainly is that lignin peroxidase is produced bacterial strain output, is badly in need of improving bacterial strain unit's fermenting enzyme vigor, with problems such as the scale operation that solves enzyme and industrial applications.Have, the enzymatic property of lignin peroxidase zymin and application characteristic also are necessary basic data and theoretical foundations in the practical application again.
At present, the bacterial classification research of producing lignin peroxidase is still concentrated on Mycophyta both at home and abroad, in fact the Degradation and Transformation of xylogen never only is the result of basidiomycetes effect, and the effect of many actinomycetes and bacterium can not be ignored.Actinomycetes not only can make lignocellulosic substance generation modification at the initial stage of Degradation at elementary metabolism stage lignin degrading, also can be at the low molecular weight substance of Degradation later stage metabolism fungus degrading xylogen generation.And the extracellular enzyme research that actinomycetes produce is mainly concentrated on zytase, lignin peroxidase research aspect therein, its secretion generation mechanism of indivedual bibliographical informations, fermentation condition are only arranged, partly purify and assess its potential use in the wood fibre degraded, compare with the research of whiterot fungi excretory lignin-degrading enzymes, the lignin peroxidase in actinomycetes source is in its purification and evaluation and study report less (Sunna A and Antranikian G, 1997) aspect the degraded molecule mechanism of xylogen.Report to the sugared sporangium secretion of green extracellular enzyme class, especially lignin peroxidase does not appear in the newspapers both at home and abroad as yet, does not see green sugared sporangium lignin peroxidase purifying and characteristic research yet.Therefore be necessary that aspects such as the purifying of enzyme and enzymatic property are furtherd investigate to this bacterium lignin peroxidase excretory mechanism.
The zytase and the lignin peroxidase of the green sugared sporangium preparation of the inventive method utilization have certain alkaline-resisting thermotolerance, can adapt to the requirement of industrial bleaching, therefore can be used for the research of association with pulp bleaching effect.
Summary of the invention
The objective of the invention is for a kind of making method with zytase and lignin peroxidase of alkali-resistant is provided, utilize the zytase of this method preparation and lignin peroxidase can adapt to the alkaline hot conditions of paper pulp after the alkaline process chemical pulp oxygen delignification.
Realize that above-mentioned purpose technical scheme of the present invention is, the preparation method of zytase or lignin peroxidase is characterized in that, this preparation method may further comprise the steps, and (1) makes mother liquor with the sugared sporangium strain activation and culture of green; (2) mother liquor that (1) step is made is inoculated in to contain and induces inducing in the product enzyme Sang Tasi substratum of substrate to cultivate; (3) stillness of night is collected in the centrifugal back of bacterium liquid that (2) step is made, and promptly gets the crude extract of enzyme; (4) will carry out chromatography after (3) step crude extract dialysis and purifying obtains zytase or lignin peroxidase liquid.
Among the preparation method of above-mentioned zytase or lignin peroxidase, the preparation method of the mother liquor in the step (1) is the green sugared sporangium bacterial classification that 4 ℃ are preserved down, an amount of mycelia of picking or spore under aseptic condition, be inoculated on the Sang Tasi solid culture base plane, cultivate 72h down in 45 ℃, then the green sugared sporangium after above-mentioned activation culture, the an amount of mycelia of picking under aseptic condition, be connected in the bottled 50mL liquid Sang Tasi substratum of 150mL triangle, shaking culture 3 days is as mother liquor under 45 ℃, 120rpm condition.
Among the preparation method of above-mentioned zytase or lignin peroxidase, used Sang Tasi substratum (STS) consist of soy peptone 10.0g, yeast powder 2.0g, enzymic hydrolysis casein 2.0g, sodium-chlor 6.0g, glucose 10.0g, distilled water 1000.0mL.
Among the preparation method of above-mentioned zytase or lignin peroxidase, being used to prepare the used substrate of inducing of zytase in the step (2) is one of the following: cotton yarn, xylan, xylan and cotton yarn mixture, poplar powder, poplar powder and cotton yarn mixture, pine powder or pine powder and cotton yarn mixture.
Among the preparation method of above-mentioned zytase or lignin peroxidase, being used to prepare the used substrate of inducing of lignin peroxidase in the step (2) is one of the following: Stem or leaf of Shrub Lespedeza, wheat straw, Cortex Populi Tomentosae, pine powder, Straw Pulp, rugose wood element, phenylcarbinol, veratryl alcohol, two or four chlorophenesic acid or guaiacin.
Among the preparation method of above-mentioned zytase or lignin peroxidase, in the step (2), mother liquor is 2% (1mL/50mL) in the inoculum size that inoculation contains when inducing inducing of substrate to produce enzyme Sang Tasi substratum.
Among the preparation method of above-mentioned zytase or lignin peroxidase, in the step (3), adopt CM Sephadex C-50 medium in return, pH 6.0 phosphoric acid buffers carry out cation-exchange chromatography as the experiment damping fluid to crude extract.
Among the preparation method of above-mentioned zytase or lignin peroxidase, the mother liquor that makes of (1) step can be inoculated in to contain to induce and carry out fermentation culture behind the substrate and make lignin peroxidase.
Utilize the zytase or the lignin peroxidase of method for preparing can be used for industrial bleaching field.
Technical scheme of the present invention has the following advantages, and utilizes in (1) the inventive method in the zytase liquid that green sugared Zymomonas mobilis produces, and the activity of its cellulase is lower, can prevent to cause in the enzyme treating processes decline of fibre strength, slurry viscosity; (2) utilize the zytase of the inventive method preparation, have certain alkali-resistant, show high reactivity in 7.0 times reactions of pH, simultaneously 90 ℃ handle 3h down after enzyme live and residually reach 63.55%, adapt to the requirement of industrial condition; (3) also utilize green sugared sporangium fermentation mass production lignin peroxidase liquid in the inventive method, and can directly effectively attack xylogen and produce bleaching effect; (4) present method is beneficial to the lignin peroxidase that green sugared sporangium produces, has certain alkali-resistant, reaction shows high reactivity in pH7.0～8.0 time, simultaneously 70～90 ℃ handle 1h down after enzyme live to original 61%, adapt to the requirement of industrial condition.
Description of drawings
Fig. 1 is to be the product xylanase activity analytical results of comparison needle to not isogeneous induction substrate with the STS substratum;
Among the figure, k, STS substratum; K+, to induce substrate be the substratum of cotton yarn; M, to induce substrate be the xylan substratum; M+, to induce substrate be the substratum of xylan and cotton yarn mixture; Y, to induce substrate be poplar powder substratum; Y+, to induce substrate be the substratum of poplar powder and cotton yarn mixture; S, to induce substrate be the pine powder substratum; S+, to induce substrate be the substratum of pine powder and cotton yarn mixture.
Fig. 2 is to be the product lignin peroxidase activation analysis result of comparison needle to not isogeneous induction substrate with the STS substratum;
Fig. 3 is a fermentor tank medium green sugar colour sporangium thalli growth curve;
Fig. 4 is dissolved oxygen and a pH change curve in the fermenting process;
Fig. 5 is that inoculum size influences enzymatic production in the fermenting process;
Fig. 6 be in the fermention medium carbon nitrogen source ratio to the influence of enzymatic production;
Fig. 7 is the influence of stirring velocity to enzymatic production;
Fig. 8 is that different air flows are to producing the result of enzyme influence;
Fig. 9 is the product enzyme time-histories of zytase;
Figure 10 is the alkali resistance analytical results of zytase;
Figure 11 carries out bio-bleaching to the Triploid of Populus Tomentosa sulfate pulp, analyzes whiteness, the viscosity of floating back paper pulp and changes;
Figure 12 carries out bio-bleaching to the Triploid of Populus Tomentosa sulfate pulp, analyzes the kappa value that floats back paper pulp and changes;
Whiteness and viscosity after Figure 13 paper pulp Sav bleaching change;
Kappa value after Figure 14 paper pulp Sav bleaching changes.
Embodiment
Below technical scheme of the present invention is specifically described green sugared sporangium (Saccharomonospora viridis (Schuurmans etal) Nonmura﹠amp that the present invention is used; Ohara4.1089) bacterial classification is available from DSMZ of Institute of Microorganism, Academia Sinica, for Ministry of Health's pharmaceutical biological product calibrating is introduced from Poland.Material therefor is commercially available getting in the following embodiment.
1.1 the activation culture of bacterial classification
With 4 ℃ of green sugared sporangium bacterial classifications of preserving down, an amount of mycelia of picking or spore under aseptic condition are inoculated on the Sang Tasi solid culture base plane, cultivate 72h down in 45 ℃.
1.2 the preparation of substratum
Green sugared sporangium induces product enzyme substratum to induce substrate for the Sang Tasi substratum adds.The composition of Sang Tasi substratum (STS) is: soy peptone 10.0g, yeast powder 2.0g, enzymic hydrolysis casein 2.0g, sodium-chlor 6.0g, glucose 10.0g, distilled water 1000.0mL.(solid medium adds agar 15.0～20.0g/1000.0ml), pH:7.2.
The substrate of inducing that produces zytase is one of following material: cotton yarn (4cm2*3), xylan 8g, xylan and cotton yarn mixture, poplar powder 16g, poplar powder and cotton yarn mixture, pine powder 16g or pine powder and cotton yarn mixture.
The substrate of inducing that produces lignin peroxidase is one of following material: Stem or leaf of Shrub Lespedeza, wheat straw, Cortex Populi Tomentosae, pine powder, Straw Pulp, rugose wood element, phenylcarbinol, veratryl alcohol, two or four chlorophenesic acid, guaiacin.
1.3 produce the screening of enzyme substratum
Be provided with 7 kinds of different substrates of inducing, simultaneously in contrast with the STS substratum.With postvaccinal product enzyme substratum at 45 ℃, after 12h is cultivated in concussion under the 120rpm condition, every interval 12h sampling, the preparation crude enzyme liquid is measured zytase, cellulase activity, lignin peroxidase, determine to live at the product enzyme peak and the highest enzyme of every kind of substratum, screening can be induced the higher zytase of generation vigor and the best product enzyme substratum of cellulose-less enzymic activity, lignin peroxidase.
1.4 the preparation of mother liquor and crude enzyme liquid
With an amount of mycelia of picking under aseptic condition of the green sugared sporangium after the activation culture, be connected in the bottled 50mL liquid Sang Tasi substratum of 150mL triangle, shaking culture 3 days is as mother liquor under 45 ℃, 120rpm condition.The mother liquor for preparing is inoculated in to induce produces in the enzyme substratum, inoculum size is 2% (1mL/50mL).Shaking culture under 45 ℃, 120rpm.Get cultured bacterium liquid at 4 ℃, centrifugal 8min under the 10000g collects supernatant, and promptly the crude extract of enzyme can be preserved standby down in-20 ℃.
1.5 green sugared sporangium enzymatic production top condition
1.5.1 fermentor tank medium green sugar colour sporangium thalli growth curve and bacterium liquid optical density(OD) OD660
In the fermentor cultivation process, the every 6h sampling in inoculation back is to fermentation ends.With with under the condition not inoculation culture liquid be blank, incubation time is an X-coordinate, the OD660 absorbance of bacterium liquid is that ordinate zou is drawn the growth curve of green sugared sporangium in fermentor tank.
Get quantitative culture liquid centrifugal 10min under the 3000r/min condition, the clear liquid that inclines is used distilled water wash mycelium 2 times, dries to constant weight under 105 ℃, measures green sugared sporangium different fermentations cell concentration in period in fermentor tank.
1.5.2 optimal conditions of fermentation is explored
On the experimental result basis in front, the experiment of this group is adopted Sang Taisi substratum+Straw Pulp to induce as the best and is produced the lignin peroxidase substratum.The sugared sporangium mother liquor of green is inoculated in this substratum.Determined the suitableeest product enzymatic process condition according to dissolved oxygen, pH, inoculum size, carbon nitrogen source ratio, stirring velocity, air flow.
1.6 fermentation back enzyme is lived and the protein content time-history analysis
1.7 result
1.7.1.1 produce the best inducing culture of zytase
With the STS substratum is blank, after 8 kinds of different liqs culture medium culturing, induces and produces the enzyme result as shown in Figure 1.7 kinds add the substratum of inducing substrate and induce product enzyme result to compare with control medium, induce the equal difference of effect of producing zytase little, wherein poplar powder substratum induces the product xylanase activity the highest, pine powder and cotton yarn mixture substratum take second place, be respectively 46.89U/mL and 45.14U/mL, (39.62U/mL) compares no significant difference with blank.Therefore can not screen the best by the height that produces xylanase activity and induce product enzyme substratum.Induce the result as can be known from cellulase activity simultaneously, between the 5.47U/mL, to compare level lower with the activity of zytase at 0.08U/mL in enzyme work.Wherein pine powder and cotton yarn mixture substratum induce that cellulase-producing is active to be 0.08U/mL, compare significant difference (p≤0.05) with blank (5.40U/mL), and other several substratum are compared with blank and be there is no significant difference.Comprehensive two kinds of enzyme work are induced the result and are adapted to the association with pulp bleaching requirement of actual application, and selecting this substratum is that the best induces green sugared sporangium to produce the zytase substratum.
1.7.1.2 produce the lignin peroxidase optimal medium
As seen from Figure 2, compared with the control, Stem or leaf of Shrub Lespedeza powder, barley seeding food, Cortex Populi Tomentosae powder, pine powder, Straw Pulp etc. can promote the generation of three kinds of wood fibre degrading enzymes preferably, and wherein Straw Pulp induces effect best (LiP 0.27U/mL, Xylanase 5.14U/mL, Cellulase 1.10U/mL).And slightly carry xylogen, and phenylcarbinol, veratryl alcohol, 2,4-DCP, methyl catechol etc. do not have obvious facilitation to the generation of wood fibre degrading enzyme.This shows that with respect to single lignin structure analogue, wood powder class material can be more
The generation of promotion wood fibre degrading enzyme, its reason may be since at wood powder class material except containing xylogen, Mierocrystalline cellulose, outside the materials such as hemicellulose, also contain a spot of resin, fat, wax, solubility tannin, pigment and ash and grade, the composition relative complex, some components wherein can promote the generation of several degrading enzymes respectively, several enzymes have synergy, and the intermediate product of degraded can play certain inducing action to other enzyme.
1.7.2 the lignin peroxidase optimal conditions of fermentation is produced in green sporangium fermentation
1.7.2.1 fermentor tank medium green sugar colour sporangium thalli growth curve and bacterium liquid optical density(OD) OD660
Show as Fig. 3 result, green sugared sporangium has experienced about 5 hours lag phase in fermentor tank after, enter logarithmic phase very soon, but variation along with environment such as thalli growth, nutritive substance consumption, meta-bolites accumulation and pH, the growth of unsuitable bacterium gradually, cause growth velocity to reduce gradually, ferment that thalline enters stationary phase after 66 hours, this moment, cell concentration reached 19.08g/L.By Fig. 3 simultaneously as can be seen, bacterium liquid light absorption value trend over time is consistent with bacterial concentration, illustrates that there are certain positive correlation in bacterial concentration and absorbance.Therefore, available bacterium liquid light absorption value substitutes the growth curve of green sugared sporangium, judges bacterium liquid fermentation stage of living in according to the variation of absorbance, the exact grasp fermentation time, and this method is not only easy but also can reduce personal errors, and is very great to the scale operation meaning.
1.7.2.2 dissolved oxygen and pH change the influence to enzymatic production in the fermenting process
As shown in Figure 4, under the identical situation of other culture condition, because green sugared sporangium is an aerobic microbiological, at a large amount of oxygen of process of growth consumption, cause dissolved oxygen parameter (DO) lasting obviously decline after cultivating beginning for some time in the fermenting process, but suitable stirring velocity and air input all remain on more than 35% DO, this basic guarantee the demand of bacterial metabolism activities such as the quick growth and breeding of this bacterial strain cell in aerobic enzymatic production process and enzyme product be synthetic to dissolved oxygen.
Fermented liquid pH value changes and can probably be divided into 3 stages in the fermenting process, can see that in the fs pH is reducing gradually, its reason may be by in the thalli growth process to the consumption of nitrogenous source and utilize carbon source to produce organic acid and cause; Along with the increase of fermentation time, thalline carries out elementary metabolism, secondary metabolism, and exoprotein increases sharply, and consumes nitrogenous source simultaneously and the ammonium material that accumulates also causes the rising gradually of pH; And producing enzyme latter stage, and pH and yield of enzyme no longer increase, prolong to produce the enzyme time and can find, because the exhausting of nitrogenous source, the death of thalline, self-dissolving, the release of a large amount of alkaline matters causes pH to rise once more again.
1.7.2.3 inoculum size is to the influence of enzymatic production in the fermenting process
Under the identical situation of other culture condition, add the different ratios seed liquor and ferment, the result as shown in Figure 5, when insert the bacterium liquid measure be the substratum add-on 10% the time, the thalline yield of enzyme is the highest.Inoculum size is 5% o'clock, because cell concentration is low excessively in the fermention medium, thereby is unfavorable for a large amount of breedings of thalline, and then influences its enzymatic productivity; On the contrary, inoculum size is 15% o'clock, and thalline is bred in a large number, causes that oxygen content and nutritive substance descend rapidly in the substratum, and thalli growth is restricted, and is unfavorable for that also it produces enzyme.
1.7.2.4 the carbon nitrogen source ratio is to the influence of enzymatic production in the fermention medium
The size of carbon-nitrogen ratio not only directly influences the pH value of substratum as shown in Figure 6, causes pathways metabolism and meta-bolites to change, and has a strong impact on the growth of thalline and produces enzyme, also causes the waste of raw material simultaneously easily.Ferment with different carbon-nitrogen ratios, the result shows, is 1: 3 o'clock in carbon-nitrogen ratio, and the yield of enzyme of thalline is the highest, exceeds closely 70% than (C/N is 1) relative activity before not optimizing, and it is relevant with the restriction of carbon nutrition to illustrate that this bacterium produces enzyme.
1.7.2.5 stirring velocity is to the influence of enzymatic production
As shown in Figure 7, under the identical situation of other culture condition, when the fermentor tank mixing speed is 150rpm, bacterium liquid dissolved oxygen is relatively low, cause thalli growth relatively poor, the delay of mixing speed 250rpm condition hypothallus entered stationary phase in 16 hours relatively, and the peak period of producing enzyme prolongs and enzyme activity decline; When mixing speed was 350rpm, thalli growth was very fast relatively, but can cause excessive shearing force to mycelium equally, caused the sex change inactivation of zymoprotein, and under the high-speed stirring situation, the foam in the fermentor tank increases, and also is unfavorable for producing enzyme simultaneously; So select 250r pm be the suitableeest mixing speed, make that on the one hand dissolved oxygen increases in the substratum, make nutritive substance and somatic cells uniform contact on the other hand, while diluting cells metabolite on every side helps cell metabolism.Illustrate that thus suitable stirring velocity improved the dissolved oxygen situation of fermentation system well, reduced unnecessary power consumption and too much strong mechanical stirring, thereby avoided excessive oxygen and shearing action that thalli growth and enzyme activity are produced detrimentally affect.
1.7.2.6 air flow is to the influence of enzymatic production
As shown in Figure 8, in the fermenting process, different air flows to the result that produces the enzyme influence as shown in the figure, when air flow was 2.5L/min, the dissolved oxygen situation was relatively poor relatively in the substratum, thalli growth is slow, causes producing the enzyme time lag, enzymic activity is relatively low; When air flow increases to 5L/min, oxygen dissolving value can maintain more than 35% in the fermentor tank, and thalli growth is comparatively fast more vigorous, produces the enzyme peak period and also shifts to an earlier date to some extent, and enzyme activity improves; When air flow continued to increase to 7.5L/min, thalline produces the enzyme situation did not all have obvious improvement, but foam is a lot of in the fermentor tank.So take all factors into consideration, it is comparatively suitable that air flow is chosen 5L/min, under this condition, oxygen dissolving value in the fermentor tank can maintain 35% above level, both has been unlikely to because of air flow hangs down excessively oxygen dissolving value to be descended rapidly, and the growth of thalline is impacted, make bacterial metabolism accumulation, inhibitory enzyme synthetic; Can not make fermentor tank produce too much foam because of air flow is too high again.
1.7.3 optimal conditions of fermentation
The sugared sporangium of green has been carried out the fermentation of inducing of lignin peroxidase with the 16L fermentor tank, determined the suitableeest product enzymatic process condition: inoculum size is 10%, the carbon nitrogen source ratio is 1: 3, stirring velocity is 250r/min, air flow is 5L/min, by control air flow and adjustment mixing speed, dissolved oxygen is maintained more than 35%, under this condition green sugared sporangium than shake flat experiment in advance nearly 24h reach and produce the enzyme peak, enzyme work reaches as high as 0.41U/mL.
The active time-history analysis of extracellular enzyme
Green sugared sporangium produces the enzymic change rule and sees Fig. 9, xylanase activity significantly increases before 48h, gathers way slowly from the 48h activity, reaches the maximum enzyme peak of producing during to 156h, enzyme is lived and is 45.14U/mL, and this produces the enzyme peak than the late appearance of streptomyces albus 12 hours.From 156h to 192h, xylanase activity sharply reduces, and drops to the lower-most point (14.62U/mL) of whole product enzyme time-histories when 192h.
Meanwhile by the result as can be known, compare with xylanase activity, the cellulase activity level is extremely low, and (0.08～2.33U/mL), its product enzyme peak occurs when 96h, has shifted to an earlier date 60 hours than zytase.When the 156h xylanase activity was the highest, cellulase activity had dropped to a low-down level (0.52U/mL), and the green sugared sporangium extracellular enzyme of sampling this moment is fit to the application requiring of pulping bleaching the most.
2, the preparation of green sugared sporangium mother liquor and crude enzyme liquid
2.1 the preparation of mother liquor and crude extract
With an amount of mycelia of picking under aseptic condition of the green sugared sporangium after the activation culture, be connected in the bottled 50mL liquid Sang Tasi substratum of 150mL triangle, shaking culture 3 days is as mother liquor under 45 ℃, 120rpm condition.The mother liquor for preparing is inoculated in to induce produces in the enzyme substratum, inoculum size is 2% (1mL/50mL).Shaking culture under 45 ℃, 120rpm.Get cultured bacterium liquid at 4 ℃, centrifugal 8min under the 10000g collects supernatant, and promptly the crude extract of enzyme can be preserved standby down in-20 ℃.
2.2 concentrating and purification of crude enzyme liquid
2.2.1 concentrating of crude enzyme liquid
The crude enzyme liquid of centrifugal back gained is packed in the dialysis tubing, place 70% glycerine, the 4～6h that dialyses, the crude enzyme liquid after obtaining concentrating.
2.2.2 cation-exchange chromatography
Adopt CM Sephadex C-50 medium in return, the pH6.0 phosphoric acid buffer is as the experiment damping fluid.The abundant mixing absorption of medium that swelling equilibrium is good and enzyme liquid back dress post (2 * 40cm chromatography column).Use 0.1,0.5 respectively, the NaCl solution of 1.0mol carries out wash-out, collects elutriant with test tube, and 2mL/ pipe, and utilize UV spectrophotometer measuring albumen elution peak is measured xylanase activity simultaneously, obtains the zytase liquid behind the purifying.Concentrate with purification process in zytase to detect data as shown in table 1.
Table 1 zytase purifying data
2.3DNS method is measured zytase and cellulase activity
2.3.1 wood sugar, the formulation of glucose typical curve
Prepare wood sugar and the glucose solution of 1mg/ml respectively with the phosphoric acid buffer of pH7.0.Take out the scale test tube of 10 25mL, suck 0,0.2,0.4,0.6,0.8,1.0,1.2,1.4,1.6 respectively, 1.8ml wood sugar (glucose) solution.Add phosphoric acid buffer, the liquor capacity that makes every test tube is 3ml.Every test tube adds 2mlDNS solution, reacts 5min in boiling water, is cooled to room temperature after the reaction rapidly.In test tube, add 20ml water, measure solution absorbency, drawing standard curve at 540nm.
2.3.2 the mensuration of enzymic activity
DNS method (Miller etc., 1959) is adopted in the determination of activity of zytase and cellulase.With 1% xylan solution of phosphoric acid buffer (pH7.0) preparation and 1%CMC solution respectively as the substrate of zytase and cellulase.In 25mL scale test tube, add the 1.0mL substrate, 0.2mL enzyme liquid and 1.8mL phosphoric acid buffer, water-bath 20min under 60 C adds 2mLDNS reagent, boils 5min in boiling water, adds water to 25mL after the cooling, measures its absorbancy behind the mixing under the 540nm wavelength.Try to achieve the amount of wood sugar in the test fluid or glucose according to calibration curve method, try to achieve enzymic activity by following formula: H=1000DC/ (tMV); In the above-mentioned formula, H enzyme (U/mL) alive; D enzyme liquid extension rate; Wood sugar in the C test fluid (glucose) content (mg/mL); The t reaction times (min); M wood sugar (glucose) molecular weight; V enzyme liquid amasss (mL); 1 enzyme unit definition alive is the required enzyme amount of per minute catalysis 1mg substrate.
2.3.3 the preparation of related solution
Phosphoric acid buffer (pH7): first liquid 39.0ml, second liquid 61.0ml, mixing.First liquid is 0.2mol/LNaH2PO4.H20 (27.6g/1000ml); Second liquid is 0.2mol/LNa2HPO4.2H20 (35.61g/1000ml).DNS reagent (3-5 edlefsen's reagent): Seignette salt 182.0g is dissolved in 500ml distilled water, heating (being no more than 50 C), in hot solution, add 3 successively, 5-dinitrosalicylic acid 6.3g, sodium hydroxide 21.0g, phenol 5.0g, anhydrous sodium sulphate 5.0g, stir to molten, the cooling back is settled to 1000ml with distilled water, is stored in the brown bottle, room temperature preservation was used after 7-10 days.
2.4 the alkali resistance analysis of zytase
Respectively with the wide region pH value damping fluid of pH3.0～9.0 as treatment condition, measure xylanase activity, explore its optimal reaction pH value.
2.5 the alkali resistance of the zytase of zytase is analyzed stability analysis
Adopt orthogonal design, the crude enzyme liquid that extracts is handled the different periods down at 50～90 ℃, per 5 ℃ is a gradient.The period of each Temperature Treatment is 0.5～3h, and every 0.5h is a gradient, measures xylanase activity then, is that residual enzyme is lived with it with respect to the active per-cent of undressed enzyme liquid, analyzes the thermostability of this enzyme.
2.6 result
2.6.1 the alkali resistance analysis of zytase
The enzyme that green sugared sporangium excretory zytase is showed under pH3.0～9.0 environment is lived, and the result as shown in figure 10.The activity change that zytase is showed under Conditioning between pH3.0～5.0 is little and enzyme is alive not high, is 10.80U/mL～19.73U/mL.The highest enzyme of reaction performance is lived when pH7.0, and with preceding compare remarkable increase, reach 47.35U/mL, when pH8.0, also keep higher enzyme running water flat (44.51U/mL), but under the alkaline environment of pH8.0～9.0, react, the xylanase activity that is showed with preceding compare rapid reduction, reaction enzymes is only lived to only being 22.50U/mL under the pH9.0 condition.The research work done of experimental result and forefathers as can be seen thus, compare with fungi and some bacterium excretory acidic xylanase, green sugared sporangium excretory zytase has alkali resistance preferably, and this enzymatic property meets the alkaline environment needs of pulping bleaching.
2.6.2 the thermotolerance analysis of zytase
By orthogonal design, after the processing of green sugared sporangium excretory zytase through the differing temps different time sections, the result is as shown in table 2.Under 50～65 ℃ of conditions, the length in treatment time is little to the influence of xylanase activity, compares with untreated control enzyme work, remains on more than 90% of initial enzyme work substantially.Under 70 ℃～75 ℃ conditions, enzyme work slightly reduces with the growth of the time of processing, remains on about 80% of initial enzyme work substantially.Under 75 ℃～85 ℃, the treatment time is shorter than 2.0h, and residual enzyme work is more than 70%; Treatment time, residual enzyme work was more than 65% of initial enzyme work when being 2.0～3.0h.Under 90 ℃, the treatment time is shorter than 1.5h, and enzyme is lived and kept more than 70%, and when handling 1.5～3.0h, residual enzyme work is more than 60%.Thus the result as can be known, under 60 ℃～90 ℃ hot environment (this temperature range is the temperature range of actual pulping bleaching process environments), green sugared sporangium excretory zytase also keeps greater activity within a certain period of time, has thermostability preferably.And compare with streptomyces albus excretory zytase, the thermotolerance under the high temperature is better.
The thermotolerance analysis (%) of table 2 zytase
3. the outer prozyme of green sugared monospore mycetocyte floats the back pulp property and detects
3.1 pulp preparation
Speed is given birth to Triploid of Populus Tomentosa and is originated from the Linfen Prefecture, Shanxi Province, the age of tree 5 years.The section back adopts kraft cooking to obtain paper pulp by laboratory 15L electric heating revolving boilers.The pulping process condition sees Table 3-1, gained Triploid of Populus Tomentosa sulfate pulp will expect that performance is shown in table 3-2 (Yu Weidong, among the Chen Hao, Yu Huisheng, 1998).
Table 3-1 pulping process condition
Table 3-2 size performance
3.2 laboratory apparatus
Table 3-3 tests used instrument
3.3 experimental technique
3.3.1 compound extracellular enzyme bleaching
According to the buffered soln amount of the required adding of the dense calculating of bleached pulp, with buffered soln with do not float paper pulp and in polyethylene bag, leave standstill 30min behind the uniform mixing, make the pH value of paper pulp actual pH near the bleaching design.Add Sav in the paper pulp and mix, H202 (setting up the catalytic environment of a suitable lignin peroxidase the reaction) (Lin Lu that adds about 0.1mmol/L simultaneously, Zhan Huaiyu, 2002), place the water bath with thermostatic control that configures temperature to react, every 15min the polyethylene bag taking-up is rubbed once in the process.To the specified time, paper pulp is taken out, carefully clean residual enzyme liquid, copy to get at pattern and copy unified quantitative lodicule on the device, use for the experiment of slurry analytical test behind the natural air drying with distilled water.
3.3.2 subsequent chemistry bleaching
The required chemistry of bleaching is floated agent to add according in the dense distilled water that draws consumption of slurry, after mixing, pour into together to be equipped with and wait to float in the polyethylene bag of paper pulp, rub mixing, place the water bath with thermostatic control that configures temperature to react, every 15min the polyethylene bag taking-up is rubbed once in the process.To the specified time, paper pulp is taken out,, grind defibrination after the design beating degree, copy to get at pattern and copy unified quantitative lodicule on the device, test use for the slurry analytical test behind the natural air drying through PFI with the carefully clean remaining bleaching liquor of distilled water.
3.4 slurry analyzing and testing
3.4.1 whiteness
Paper pulp is copied to get at ZQJ1-B type pattern and is copied sheet on the device, treat air-dry after, show on the whiteness instrument in YQ-Z-48B numeral according to iso standard and to measure.
3.4.2 ageing whiteness
Adopt the oven ageing method, the lodicule of measuring whiteness is placed 105 ℃ constant temperature oven, dry by the fire 3h continuously and wear out, take out cooling, show on the whiteness instrument in the YQ-Z-48B numeral and measure.
3.4.3 kappa value
Adopt standard kappa value method to measure according to GB/T1546-1989.
3.4.4 viscosity
Adopt the cupri ethylene diamine viscosimetry to measure according to GB/T1548-1989.
3.5 experimental result
In preliminary experiment, fix other four major influence factors (pH value, temperature, time and slurry are dense), the consumption of setting Sav is respectively a 1IU/g (oven dry stock, 10IU/g, 50IU/g, 100IU/g, 200IU/g down together),, float the whiteness, kappa value of back paper pulp and viscosity and change and see Table 3-4 and Figure 11, Figure 12 with under the condition Triploid of Populus Tomentosa sulfate pulp being carried out the bio-bleaching analysis.
Table 3-4 analyzes whiteness, kappa value and the viscosity result of variations of floating back paper pulp
From above-mentioned experiment as can be seen, paper pulp kappa value after the Sav bleaching descends, and pulp brightness raises, and viscosity also slightly promotes.Can see that from Figure 11 and Figure 12 along with the increase of Sav consumption: the whiteness of paper pulp ascendant trend when enzyme dosage is lower than 50IU/g is obvious; Enzyme dosage is after 50IU/g, and whiteness no longer includes tangible lifting, and this point is reflected in the variation of pulp viscosity also identical trend.Meanwhile, the kappa value that floats back paper pulp is before enzyme dosage 50IU/g, and it is comparatively remarkable to descend, and delignification's effect is obvious; After 50IU/g, just descend relatively steadily, there is not delignification's effect basically.The consumption that the Sav bleached pulp is described should be below 50IU/g, if surpass this consumption, unnecessary extracellular enzyme might be in bleaching action inactivation, do not have the effect of bleached pulp.
4. the best enzyme dosage of bleached pulp
4.1 experimental technique is with 3.3
4.2 experimental result
The conclusion that draws according to experiment, the consumption of Sav bleached pulp is limited in below the 50IU/g, setting enzyme dosage respectively is 10IU/g (oven dry stock, 20IU/g, 30IU/g, 40IU/g, 50IU/g down together),, with bio-bleaching Triploid of Populus Tomentosa sulfate pulp under the condition, whiteness, kappa value and viscosity that back paper pulp is floated in analysis change, and find out the best enzyme dosage of Sav bleaching Triploid of Populus Tomentosa sulfate pulp, and the result is as table 4-1 and Figure 13, shown in Figure 14.
Table 4-1 enzyme dosage is to the influence of bleaching
Annotate: other conditions of bleaching: pH value 7,80 ℃ of temperature, time 120min, starch dense 10%, H
2O
20.1mmol/L.
From Figure 13 and Figure 14 as can be seen, after the Sav bleaching, the whiteness of paper pulp and viscosity all have rising, and kappa value descends.Along with the Sav consumption increases from 10IU/g to 50IU/g: float the back pulp brightness before enzyme dosage reaches 30IU/g the time, rises apparent in viewly, rise to 35.8%ISO, whiteness lifting 3.2%ISO from the 32.6%ISO of magma; Pulp viscosity rises to 1194mL/g from the 1170mL/g of magma, rises 2.1%, and trend is also apparent in view; The paper pulp kappa value has obvious decline in this interval, drops to 11.7 from 13 of magma, descends 10%, illustrate that Sav has certain delignification and acts on, and reason may be woodenly in wherein the lignin peroxidase centering paper pulp to have a degraded.When enzyme dosage when 30IU/g is between 50IU/g, the whiteness and the viscosity of floating back paper pulp all no longer include tangible rising, have only improved 0.1%ISO respectively and not do not change; Kappa value also remains unchanged substantially, has not had delignification's effect.The consumption that Sav is described surpasses after the 30IU/g, paper pulp is not had again tangible raising whiteness and delignification's effect, increase enzyme dosage again association with pulp bleaching is not had bigger improvement, this may be because unnecessary Sav inactivation in pulp bleaching process, does not have the cause of bleached pulp effect.In addition, in the process of zytase bleached pulp, the low-molecular-weight xylan of part in the preferential degraded of the zytase meeting paper pulp, therefore, the pulp viscosity after the zytase bleaching has lifting (69 Kantelinen et al, 1993 to a certain degree; Suurnakki et al, 1994), Sav is when 30IU/g in this experiment, and the viscosity of floating back paper pulp has risen 2.1% than magma, and this has also verified the viewpoint of Kantelinen and Suurnakki simultaneously.So, from using this principle of bleaching effect that makes paper pulp acquisition the best under the minimum situation of enzyme amount, comprehensive above-mentioned experiment conclusion can draw, and selecting 30IU/g (with zytase work) for use is relatively more suitable as the best enzyme dosage of Sav bleaching Triploid of Populus Tomentosa sulfate pulp.
Technique scheme has only embodied the optimal technical scheme of technical solution of the present invention, those skilled in the art to some part wherein some changes that may make all embodied principle of the present invention, belong within protection scope of the present invention.
Claims (9)
1. the preparation method of zytase or lignin peroxidase is characterized in that, this preparation method may further comprise the steps,
(1) the sugared sporangium strain activation and culture of green is made mother liquor;
(2) mother liquor that (1) step is made is inoculated in to contain and induces inducing in the product enzyme Sang Tasi substratum of substrate to cultivate;
(3) stillness of night is collected in the centrifugal back of bacterium liquid that (2) step is made, and promptly gets the crude extract of enzyme;
(4) will carry out chromatography after (3) step crude extract dialysis and purifying obtains zytase or lignin peroxidase liquid.
2. the preparation method of zytase according to claim 1 or lignin peroxidase, it is characterized in that, mother liquor preparation method in the step (1) is the green sugared sporangium bacterial classification that 4 ℃ are preserved down, an amount of mycelia of picking or spore under aseptic condition, be inoculated on the Sang Tasi solid culture base plane, cultivate 72h down in 45 ℃, then the green sugared sporangium after above-mentioned activation culture, the an amount of mycelia of picking under aseptic condition, be connected in the bottled 50mL liquid Sang Tasi substratum of 150mL triangle, at 45 ℃, shaking culture 3 days is as mother liquor under the 120rpm condition.
3. the preparation method of zytase according to claim 1 and 2 or lignin peroxidase, it is characterized in that, used Sang Tasi substratum (STS) consist of soy peptone 10.0g, yeast powder 2.0g, enzymic hydrolysis casein 2.0g, sodium-chlor 6.0g, glucose 10.0g, distilled water 1000.0mL.
4. the preparation method of zytase according to claim 1 or lignin peroxidase, it is characterized in that being used to prepare the used substrate of inducing of zytase in the step (2) is one of the following: cotton yarn, xylan, xylan and cotton yarn mixture, poplar powder, poplar powder and cotton yarn mixture, pine powder or pine powder and cotton yarn mixture.
5. the preparation method of zytase according to claim 1 or lignin peroxidase, it is characterized in that being used to prepare the used substrate of inducing of lignin peroxidase in the step (2) is one of the following: Stem or leaf of Shrub Lespedeza, wheat straw, Cortex Populi Tomentosae, pine powder, Straw Pulp, rugose wood element, phenylcarbinol, veratryl alcohol, two or four chlorophenesic acid or guaiacin.
6. the preparation method of zytase according to claim 1 or lignin peroxidase is characterized in that, in the step (2), mother liquor is 2% (1mL/50mL) in the inoculum size that inoculation contains when inducing inducing of substrate to produce enzyme Sang Tasi substratum.
7. the preparation method of zytase according to claim 1 or lignin peroxidase, it is characterized in that, in the step (3), adopt CM Sephadex C-50 medium in return, the pH6.0 phosphoric acid buffer carries out cation-exchange chromatography as the experiment damping fluid to crude extract.
8. the preparation method of zytase according to claim 1 or lignin peroxidase is characterized in that, also the mother liquor that makes of (1) step can be inoculated in to contain to induce to carry out fermentation culture behind the substrate and make lignin peroxidase.
9. the preparation method of zytase according to claim 1 or lignin peroxidase is characterized in that, utilizes the zytase of method for preparing or lignin peroxidase can be used for industrial bleaching field.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010156197 CN101857859A (en) | 2010-04-27 | 2010-04-27 | Preparation method of xylanase or lignin peroxidase and application thereof to industrial bleaching field |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010156197 CN101857859A (en) | 2010-04-27 | 2010-04-27 | Preparation method of xylanase or lignin peroxidase and application thereof to industrial bleaching field |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101857859A true CN101857859A (en) | 2010-10-13 |
Family
ID=42944019
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010156197 Pending CN101857859A (en) | 2010-04-27 | 2010-04-27 | Preparation method of xylanase or lignin peroxidase and application thereof to industrial bleaching field |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101857859A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013127069A1 (en) * | 2012-02-29 | 2013-09-06 | Danisco Us Inc. | Thermostable xylanase from thermobifida fusca and methods of use thereof |
| CN104807764A (en) * | 2015-04-21 | 2015-07-29 | 华南理工大学 | Alkaline xylanase activity determination method |
| CN106318993A (en) * | 2016-09-02 | 2017-01-11 | 伊春林业科学院 | Technology for increasing xylanase yield of polysaccharide production using auricularia auricula |
| CN111172126A (en) * | 2020-01-16 | 2020-05-19 | 黑龙江八一农垦大学 | Method for increasing yield of trametes hirsuta lignin peroxidase |
| CN113668281A (en) * | 2021-06-30 | 2021-11-19 | 湖北骏马纸业有限公司 | High-whiteness coated white cardboard and preparation method thereof |
-
2010
- 2010-04-27 CN CN 201010156197 patent/CN101857859A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 《生命科学研究》 20050331 谢响明 绿色糖单孢菌产木聚糖酶规律及其耐碱耐热性的初步研究 第9卷, 第1期 2 * |
| 《生物技术通报》 20090930 杨暖等 绿色糖单孢菌2木素过氧化物酶的发酵工艺研究 , 第9期 2 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013127069A1 (en) * | 2012-02-29 | 2013-09-06 | Danisco Us Inc. | Thermostable xylanase from thermobifida fusca and methods of use thereof |
| CN104807764A (en) * | 2015-04-21 | 2015-07-29 | 华南理工大学 | Alkaline xylanase activity determination method |
| CN106318993A (en) * | 2016-09-02 | 2017-01-11 | 伊春林业科学院 | Technology for increasing xylanase yield of polysaccharide production using auricularia auricula |
| CN111172126A (en) * | 2020-01-16 | 2020-05-19 | 黑龙江八一农垦大学 | Method for increasing yield of trametes hirsuta lignin peroxidase |
| CN113668281A (en) * | 2021-06-30 | 2021-11-19 | 湖北骏马纸业有限公司 | High-whiteness coated white cardboard and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yang et al. | High-level of xylanase production by the thermophilic Paecilomyces themophila J18 on wheat straw in solid-state fermentation | |
| Kuhad et al. | Lignocellulose biotechnology: current and future prospects | |
| Song et al. | Production and characterization of cellulases and xylanases of Cellulosimicrobium cellulans grown in pretreated and extracted bagasse and minimal nutrient medium M9 | |
| Saritha et al. | Pretreatment of paddy straw with Trametes hirsuta for improved enzymatic saccharification | |
| Irfan et al. | Effect of various pretreatment conditions on enzymatic saccharification. | |
| CN101460614A (en) | Talaromyces emersonii enzyme systems | |
| Khalil et al. | Production of cellulase by Pleurotus ostreatus and Pleurotus sajor-caju in solid state fermentation of lignocellulosic biomass | |
| Asgher et al. | Optimization of lignocellulolytic enzyme production by Pleurotus eryngii WC 888 utilizing agro-industrial residues and bio-ethanol production | |
| Coconi Linares et al. | Enhanced delignification of lignocellulosic biomass by recombinant fungus Phanerochaete chrysosporium overexpressing laccases and peroxidases | |
| Sermanni et al. | The production of exo-enzymes by Lentinus edodes and Pleurotus ostreatus and their use for upgrading corn straw | |
| Han et al. | Solid-state fermentation on poplar sawdust and corncob wastes for lignocellulolytic enzymes by different Pleurotus ostreatus strains | |
| Bhardwaj et al. | Role of enzymes in deconstruction of waste biomass for sustainable generation of value-added products | |
| CN105647813B (en) | One plant of Trichoderma viride and its application | |
| CN101857859A (en) | Preparation method of xylanase or lignin peroxidase and application thereof to industrial bleaching field | |
| JP4793781B2 (en) | White-rot fungi having lignocellulose-degrading action and use thereof | |
| Peláez et al. | Co-culturing of micro-and macro-fungi for producing highly active enzyme cocktail for producing biofuels | |
| AU2013279186A1 (en) | Method for producing an enzyme cocktail using the liquid residue from a method for biochemically converting lignocellulosic materials | |
| CN108342332A (en) | The method for producing enzymatic mixture using the solid residue of the biochemical conversion method from lignocellulosic material | |
| CN109055459A (en) | Full bacterium method for saccharifying for lignocellulosic | |
| Cohen et al. | The roles of fungi in agricultural waste conversion | |
| Sharma et al. | An evaluation study of different white rot fungi for degradation of pine needles under solid state fermentation | |
| Asiegbu et al. | Inhibition of cellulose saccharification and glycolignin-attacking enzymes of five lignocellulose-degrading fungi by ferulic acid | |
| CN109161535A (en) | A kind of fermentation medium and its application for improving white-rot fungi and producing lignin-degrading enzymes enzyme activity | |
| Benaddou et al. | Optimizing fungal treatment of lignocellulosic agro‐industrial by‐products to enhance their nutritional value | |
| Fatima | Recent Insight into production of cellulase by fungi and its industrial applications |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20101013 |