DE102007013047A1 - Enzymatic treatment of polymer raw substrate involves treating polymer raw substrate with enzyme system, in order to release defined monomers or oligomer components from polymer raw substrate - Google Patents

Abstract

Enzymatic treatment involves treating polymer rough substrate with 50%, preferably 20% of an enzyme system, to release defined monomers or oligomer components from the polymer rough substrate. The monomers or oligomer components are separated, which are produced from rest of the polymer rough substrate. The physicochemical treatment is completed in aqueous solvents, organic solvents, preferably ethanol or glycerin.

Description

Background of the invention

The production of bio-based chemical building blocks from renewable sources has received increasing attention recently due to the global limitation of fossil petrochemical deposits. The preferred raw materials for the production of such biological-based chemical products are derived from renewable plant biomass ( Kamm et al., 2006 ).

current Production process based on bio-based products mainly on substrates from the food and beverage industry Feed industry, such as oils, sugars and strengths. Most raw materials of the first generation have a well-defined chemical composition and a low structural Complexity. In addition, these can Substrates in relatively high purity with only small amounts of accompanying Contaminants are obtained. Although their use is both technical as well as being economically attractive, is their long-term availability not secured on a large scale, since the use of First generation raw materials for bio-based chemical Process in strong competition with the ever-growing global Demand of the food industry stands.

Alternative substrates to the above-mentioned first-generation raw materials are derived from low-cost forest and agricultural waste products which are vegetable materials which have no applications as a food source. Examples of these so-called second generation raw materials are plant residues from agriculture, such as corn straw and wheat straw, as well as various wood-related waste products. This lignocellulosic biomass (LCB) differs from the first generation biofuels in its complex chemical and structural composition. The main constituents of LCB are high polymer materials such as cellulose (about 35-50% by weight), hemicellulose (about 20-35% by weight) and lignins (about 10-25% by weight). Proteins, lipids and other compounds are minor components of most LCB raw materials ( Saha, 2005 ), but may be present in larger quantities in particular agricultural waste products, such as oil production residues. Since LCB is composed of a large number of chemically different constituents, degradation processes are technically difficult, which in turn limits the economic feasibility of current LCB-based bioprocessing.

Technical problem

Around valuable chemical substances and building blocks with economical feasible bioprocessing based on LCB raw materials It is both important to make (i) the main part of their different chemical To extract and refine constituents, as well as (ii) in sufficient quantities To produce purity. Unlike the different and high polymer units that make up LCB have the preferred Degradation products a low molecular weight. In general, you can economically valuable products from all components of LCB cellulose derived from glucose is a versatile one Starting material for the production of high quality chemical Intermediates, such as sorbitol. From hemicellulose fractions of the LCB recovered pentose sugars, such as xylose and arabinose, serve as starting material for high-quality, low-nutrient and calorie-free Sweeteners, such as xylitol and arabinitol. Proteins out LCB can become pure enantiomers of L-amino acids be hydrolyzed. Lignins can be used as a source for Serve phenol compounds that produced in petrochemical processes Can replace aromatics. The current technical Restricting the use of raw materials of the second Generation consists mainly in their complex chemical Composition.

The Most current methods using LCBs focus on the cellulose content of the substrate. If other ingredients are used, they usually become hydrolyzed in non-selective process steps, such as by a Pretreatment with sulfuric acid for the hydrolysis of all hemicelluloses in a process step. The products of this non-selective Process steps generally have a low and in some Even a negative commercial value.

The current Iogen process for the fermentative production of bioethanol involves pretreatment with dilute acid vapor at 200-250 ° C to release the hemicellulose fraction of LCB containing a mixture of different hexose and pentose sugars. In a separate enzymatic step, the insoluble cellulose fraction is hydrolyzed to hexose sugars (glucose). After liquefaction of the hemicellulose and cellulose fractions, the insoluble lignin solids are physically separated from the mash and incinerated to provide energy for further processing of the remaining LCB fractions to win. The combined cellulose and hemicellulose fractions are fermented together in a single step to produce bioethanol. The ethanol formed is subsequently recovered by distillation from the fermentation solution. Since the majority of commercially available organisms (eg, baker's yeast, Saccharomyces cerevisiae) used in the fermentation process are unable to utilize pentose sugars, these constituents become the LCB, even though they are in their pure form , have a significant economic value, where the process is discarded together with the residual waste ( Lawford and Rousseau, 2003 ).

Recently, attempts have been made to make the pentose fraction of LCBs available for fermentative conversion to bioethanol. In this improved process, the liquefied hemicellulose fraction is separated from the components of the LCB remaining after the pretreatment step. While the remaining LCB fractions are processed as described above, particular genetically engineered microorganisms (eg, specific strains of Zymomonas mobilis) are capable of Pentose (Lawford and Rousseau, 2003 ; Lynd et al., 2005 ) and hexose sugar are used in a separate fermentation step to convert the liquefied and processed hemicellulose into bioethanol. The resulting fermentation mash is then fed to the conventional processing stream for the production of bioethanol. Although the production of bioethanol from complex pentose mixtures appears to make commercial sense, the selective processing of hexoses, pentoses and lignin into high-quality products and chemical building blocks represents an attractive alternative to the utilization of LCB constituents.

A problem inherent in all presently used pretreatment methods is the simultaneous and non-selective hydrolysis and release of various chemical building blocks composing the LCB components ( Saha, 2005 ). Currently commercially used and economically viable pretreatment methods use harsh chemical or physical processes involving a combination of acid or base treatments at elevated temperatures ( Ramos et al., 2005 ). The LCB hydrolyzates obtained contain various undesired by-products formed by the chemical modification of LCB building blocks. The presence of these impurities often prevents subsequent enzymatic or catalytic processing or whole-cell fermentation of the products ( Saha, 2005 ) and therefore significantly reduces the commercial value of the product streams generated from these LCBs.

The The technical problem underlying the invention was consequently a process for the production of chemical components from renewable sources to provide plant biomass.

Especially The technical problem was a method of manufacture to provide valuable chemical building blocks from LCB, which the disadvantages and impairments of the state of Technology avoids.

Summary of the invention

To In a first aspect, the present invention provides a method for the enzymatic treatment of a polymeric raw substrate containing the following steps comprise: (a) treatment of the raw polymeric substrate with an enzyme system, to defined monomeric or oligomeric building blocks released from the polymeric raw substrate; and (b) separating the in step (a), defined monomeric or oligomeric building blocks from the residue of the polymeric raw substrate.

On this way was found to be the separation of the released monomeric or oligomeric building blocks of the polymeric raw substrate essential after a defined treatment with an enzyme system Advantages regarding the production of chemically pure high-quality basic chemicals and chemical building blocks from complex natural substrates, such as LCB, through the use of provides selective catalytic process steps. This selective catalytic process steps set chemically defined monomers or oligomeric components from the complex polymeric feedstock free, with only small amounts of other impurities in the with contained in the process step product are contained. A preferred technical solution achieves this specificity and Selectivity in the hydrolysis of LCB by the process selective enzymatic steps. The basic chemicals can as a substitute for raw materials in conventional petrochemical Process and as raw materials for novel chemical and biochemical synthesis routes are used and therefore have one high economic value.

The invention comprises a single consolidated process or a series of successive ones the process steps. At each process step, soluble monomeric or oligomeric products are prepared from the insoluble polymeric feedstock by successive addition of a specific enzyme system, followed by separation of the soluble monomeric or oligomeric products from the insoluble polymeric feedstocks.

To In a preferred aspect, the defined monomer (s) are (are) or polymeric building block (s) derived from the polymeric raw substrate in step (a), a specific "product" from the left column of Table 1 below. In other words from the polymeric raw substrate in each treatment step (a) under Use of an enzyme system or a combination of enzyme systems, that produce the same product, just one specific one monomeric or oligomeric building block released. Examples of such Combinations of enzymes are listed in Table 1.

Further Preferred aspects and embodiments are below described in detail.

definitions

Of the The term "polymeric raw substrate" means complex mixtures of various polymeric substrates such as carbohydrates, polymeric lipids, polypeptides, Polynucleotides and polymeric phenylpropanoids in varying weight ratios, usually derived from plant material. polymers Raw substrates include, but are not limited to Waste products from forestry and agriculture and food manufacturing industry as well as municipal waste. These the main polymers are cellulose and lignin, the polymeric raw substrates are called "lignocellulosic Raw substrates "or" lignocellulosic biomass "or" LCB ". Comprising lignocellulosic raw substrates from agriculture, but are not limited to wheat straw, corn straw, Stable manure of ruminants, sugar cane press cake, sugar beet pulp and herbaceous materials, such as Common Grass, Sericea Lespedeza Serala and sudangras. Forestry waste products, but are not limited to tree bark, wood chips and wood waste. Lignocellulosic raw substrates from the food industry include, but are not limited to, fruit pulp, agave residues, Coffee residues and oil mill waste, like rapeseed press cake and mill wastewater. Raw substrates from the pulp and paper industry include but not limited to pulp and paper mill wastewater. Raw substrates from municipal waste include, but are not limited to paper waste, vegetable residues and fruit residues.

Of the Term "polymeric substrate" refers to substances that either from a certain monomeric constituent or a limited one Selection of certain monomeric components exist, the Monomers in a linear or partially branched molecular structure covalently linked together. The insoluble fraction of the lignocellulosic raw substrate contains significant Amounts of such polymeric substrates as cellulose, xylan, mannan and galactan. In addition, it contains polymers Substrates such as lignin, arabinoxylan, glucoronoxylan, glucomannan and xyloglucan.

Of the Term "enzyme systems" refers to proteinaceous entities, the polymeric or oligomeric substrates catalytically into smaller oligomers or disassemble monomeric components (building blocks). In addition to using a single enzyme for production monomeric or oligomeric products from a polymeric substrate The term enzyme system also includes mixtures that more than include an enzyme and a specific monomeric or oligomeric Product characterized by synergistic or parallel activity produce a polymeric substrate. The terms "enzyme system" and "Enzyme mixtures" are therefore interchangeable here and both may be one or more enzyme (s) or enzyme activity (s) include.

Of the Term "monomeric or oligomeric building blocks" refers to monomers or oligomeric products produced using an enzyme system be released from the polymeric raw substrate. The term "oligomer" includes compounds having two or more monomeric units.

Of the The term "enzymatic activity" of an enzyme refers on the catalytic activity of the enzyme at appropriate Conditions in which the enzyme acts as a protein catalyst and certain polymeric or artificial substrates in certain converts oligomeric or monomeric products.

Of the Term "contaminating enzymatic activity" enzymatic activities of an enzyme system used, which results in oligomeric or monomeric products other than the lead to desired oligomeric or monomeric product (s), which after the intended (main or first) enzymatic activity of the enzyme system can be generated.

Of the Term "artificial substrate" refers to a substance usually of low molecular weight, which after the Contact of the artificial substrate with an enzyme system a measurable change in physicochemical properties of the artificial substrate that is on the Activity of a selected enzyme system attributed can be. These physicochemical properties and the artificial Substrates that undergo these changes of physicochemical Properties after contact with an enzyme system are those skilled in the art and include, but are not limited on changes in spectrophotometrically measurable absorption or fluorescence emission, changes in the chromatographic Mobility caused by liquid or gas chromatographs can be determined and changes in molecular weight, which can be determined by mass spectroscopy.

suitable artificial substrates for enzymes and enzyme systems and measurable reaction products are in the table below 2 listed.

Of the The term "release" means the transformation of an insoluble one polymeric substrate into a soluble monomeric or oligomeric Product through a physical, chemical or catalytic table Processes such as hydrolysis, oxidative or reductive Depolymerization, etc.

Detailed description of the invention

General

The The invention relates to a single consolidated process or a Series of sequential process steps for the preparation of basic chemicals or chemical building blocks from polymeric raw substrates like LCB. At each process step become soluble monomers or oligomeric products (building blocks) from the insoluble polymeric raw substrate formed by the polymeric raw substrate with a specific enzyme system is brought together, preferably is essentially free of enzymatic activities, other than the intended reaction products (monomers or polymer building blocks), followed by the separation of the (the) soluble monomeric or oligomeric building block (s) of the insoluble polymeric raw substrate.

method for the separation of soluble and insoluble components are known in the art and include process steps such as sedimentation, Decantation, filtration, microfiltration, ultrafiltration, centrifugation, Evaporating volatile products and extraction with organic Solvents. According to a preferred embodiment For example, the physicochemical treatment step involves a treatment with aqueous solvents, organic solvents or any combination or mixture thereof, preferably with Ethanol or glycerin.

To A preferred embodiment of the invention is the enzymatic treatment carried out in an aqueous medium, and the defined monomeric or oligomeric building blocks consisting of are released to the polymeric raw substrate are in the aqueous-grained Soluble medium and the separation according to the above step (b), the solid / liquid separation of soluble building blocks in the aqueous medium of the insoluble polymeric raw substrate.

relevant Examples of polymeric substrates, the main components of polymeric Represent raw materials, their monomeric or oligomeric degradation products (Building blocks) and enzyme systems used in the production of essentially Pure products from any polymeric substrate that are in a polymeric Raw substrate is included, are useful, are in the following Table 1 given.

at be the inventive steps chemical building blocks of polymeric substrates, such as hemicellulose, Cellulose, lignins, glucans, proteins and lipids, by bringing together of the polymeric substrate with specific enzyme systems. The enzymatic degradation of the individual components of the substrate to essentially pure, soluble products on the application of substrate-specific enzyme preparations, in a preferred embodiment substantially are free from activities that have other ingredients than release the intended products at the respective process step.

Determination of composition of the polymeric raw substrate

The molecular composition of the raw substrate to be used in the methods described herein may be determined by methods known to those skilled in the art. For example, can the composition of lignocellulosic material can be determined by a combination of pyrolysis, gas chromatography and mass spectroscopy. An overview of the methods that describe possible analytical procedures for determining LCB constituents can be found at:
http://www1.eere.energy.gov/biomass/analytical procedures.html # samples ,

To In one embodiment, standard methods, comprising several physical and enzymatic reaction steps, be used to base the constituents of the substrate to empirically quantify the reaction products obtained.

For conventional substrates, there is a database that lists the mass distribution for most common resource classes, at:
http://www1.eere.energy.gov/biomass/feedstock databases.html ,

In a representation for analyzing the components of the substrate must first partially hydrolyzed the polymeric raw substrate be done before any further analysis can.

In front the hydrolysis of the substrate, the sample of the substrate (1 g) in introduced a crucible and at 50 ° C to constant weight dried. The dry weight is noted down to three decimal places to obtain the oven dry weight (ODW) of the sample.

For the hydrolysis process is 1 g of the finely ground substrate (2 μm) placed in a pressure tube and 3 ml of 72% sulfuric acid (v / v) added. The pressure tube is placed in a water bath brought at 30 ° C and incubated for 1 h. With the help of a stir bar The sample is stirred every 5 to 10 minutes without the sample is taken out of the bath. Stirring is essential for a uniform acid-particle distribution to ensure. The acid is diluted to 4%, Add 84 ml of double distilled water using a Burette are added and the sample is mixed, by inverting the tubes several times to phase separate to prevent.

Around the acid-insoluble lignin fraction of the substrate to determine the autoclave hydrolysis solution through vacuum filtered a previously weighed filter crucible.

The Filtrate is collected in a filter bottle and a 50 ml aliquot is transferred to a sample storage bottle. This sample is used in the following procedure to obtain the To determine lignin and carbohydrate content. The determination of the Acid-insoluble lignin must be within six hours be carried out after the hydrolysis.

to Determination of the acid-insoluble lignin is double distilled water was added to make up any remaining insoluble Add substances from the pressure tube quantitatively into the filter crucible to convict. The insoluble substances need to use at least 50 ml of double distilled water be rinsed, and then the crucible and the acid-insoluble residues 4 hours at 105 ° C or dried to constant weight become. After incubation, the samples are removed from the oven and cooled in a desiccator. The weight "w2" of the Tiegel and dried residue to 0.1 mg, before crucibles and residues Be placed in a muffle furnace at 575 ° C for 24 hours.

Of the Crucible is carefully removed from the oven, transferred directly to a desiccator and a certain time chilled, that of the initial one Cooling time of the crucible corresponds. The crucible and the ashes are weighed (weight "w3") and put back into the oven until a constant weight is reached. The one with the remaining ashes ("w3") corrected weight "w2" corresponds to the weight of the acid-insoluble Lignins, which is contained in the raw substrate.

ODW

= Ofen-Trockengewicht der Probe des Rohsubstrats

UV_abs

= mittlere UV-vis-Absorption der Probe bei 320 nm

Volumen

_Filtrat = Volumen des filtrierten Hydrolysats

e

= Extinktionskoeffizient des Hydrolysats der Biomasse bei maximaler Absorption der Probe (zahlreiche Werte der Extinktionskoeffizienten für eine große Anzahl von polymeren Rohsubstraten können gefunden werden unter http://wwwl.eere.energy.gov/biomass/analytical procedures.html#samples )

In contrast to the elaborate measurements required to obtain the amount of acid-insoluble lignin, the amount of acid-soluble lignin can be easily determined spectrophotometrically. First, a blank reading is taken with 4% sulfuric acid (v / v) on any spectrophotometer. Using the initial hydrolyzate, the absorbance at 320 nm and the maximum absorbance of the filtered hydrolyzate, which is usually about 198 nm, are measured. If necessary, dilute the sample so that the absorption falls within the range of 0.7-1.0 A units. The absorbance of the sample to 3 decimal places is used to calculate the amount of acid soluble lignin present in the sample according to the following equation: % ASL = (UV Section ·Volume filtrate · Dilution) / (s · ODW sample ) X 100

ODW

= Oven dry weight of the sample of the raw substrate

UV _abs

= average UV-vis absorption of the sample at 320 nm

volume

_Filtrate = volume of filtered hydrolyzate

e

= Extinction coefficient of the hydrolyzate of the biomass with maximum absorption of the sample (numerous values of the extinction coefficients for a large number of polymeric raw substrates can be found under http://wwwl.eere.energy.gov/biomass/analytical procedures.html # samples )

The Liquid hydrolyzate of the sample can also be used around the structuring carbohydrates that are in the hemicellulose fraction of the substrate using a method to be determined on an HPLC basis.

These Determination first requires a calibration mixture for each D-cellobiose, glucose, xylose, galactose, arabinose and Mannose. The concentrations produced for each sugar standard should be in the range 0.1-4 mg / ml. For Each set of calibration standards should be an independent one Calibration verification standard (calibration verification standard, CVS) which are in the middle of the validated area the calibration curve falls (eg 2.5 mg / ml). The CVS should be in the HPLC after each calibration set and in regular Intervals analyzed during the analysis sequence be grouped together. The CVS will be used the quality and stability of the Verify calibration curves of each run.

20 ml in the initial steps after hydrolysis the hydrolysis liquid obtained in the sample are placed in a Transferred 50 ml Erlenmeyer flask. It becomes calcium carbonate added to neutralize the sample to a pH of 5-6, and after settling the solution, the supernatant to be decanted. After settling, the solution has a approximately neutral pH.

The Sugar calibration standards (CVS) and the samples can now on a Shodex sugar SP0810- (Phenomenex) or an AMinex HPX-87P column (BioRad) equipped with the appropriate Protective column is equipped, analyzed by HPLC.

The Sample injection volume should vary depending on the Concentration and detection limits between 10 and 50 μl lie. The samples are washed with double distilled water at a Flow rate of 0.6 ml / min and a column temperature eluted from 80 ° C. The sample elution is best with the measurement of the refractive index are monitored.

The Chromatograms should be integrated before analysis, and the individual sugar contents should be determined by reference to the appropriate Standard curves determined for each saccharide component become.

enzyme systems

at The embodiments described herein become specific the enzyme mixtures used to pure monomeric product streams (defined monomeric or polymeric building block (s)) from different to obtain polymeric substrate classes.

at a preferred embodiment of the invention may have enzyme systems that are specific to a polymeric Starting material to be applied, not more than 50% other enzymatic Include activities that are different from the preferred ones Lead product from the specific polymeric substrate.

at a more preferred invention Embodiment allowed enzyme systems to be used in a polymeric substrate used, not more than (or less than) 20%, preferably not more than (or less than) 10%, in particular preferably not more than (or less than) 5%, and more particularly preferably not more than (or less than) 2%, and most preferred not more than (or less than) 1% other enzyme activities contain, which is different from the preferred product lead the particular polymeric substrate.

The percentage of other enzymatic activities can be routinely determined using standard methods for determining the particular enzyme activity. The in the enzyme system Therefore, at least 50%, preferably at least 80%, particularly preferably at least 90%, particularly preferably at least 95%, particularly preferably at least 98%, of the essential enzymatic "main" activity which leads to the release of the desired monomeric or polymeric building block from the polymeric substrate. , particularly preferably at least 99% of all enzyme activities present in the enzyme system.

The Enzyme activities may be known to those skilled in the art Standard procedures and as described herein.

To In a preferred embodiment, the foregoing Percentages are easily determined by the polymer Substrate with the enzyme system according to step (a), above, and the released monomers or oligomeric building blocks after separation from the polymeric substrate according to step (b). If so the released monomeric or oligomeric building blocks more than 50 Mole percent of the particular building block desired (based on the total solids content), it is believed that the others enzymatic activities in the enzyme system less than 50% be. When equally the released monomers or oligomeric building blocks more than 80, 90, 95, 98 or 99 mol% of the particular desired building block, it is assumed that the other enzymatic activities in the enzyme system less than 20, 10, 5, 2 and 1%, respectively. Accordingly, it is assumed that the enzymatic "main" activity occurring in the enzyme system is present and to release the desired monomer or oligomeric building blocks from the polymeric raw substrate, in this case at least 50%, preferably at least 80%, especially preferably at least 90%, more preferably at least 95%, especially preferably at least 98%, more preferably at least 99% of all enzymatic activities present in the enzyme system is. According to another embodiment is used in the above determination of the percentage of enzymatic Activity (s) "Mol%" replaced by "wt%".

According to one Embodiment may be the enzymatic activities be determined by the conversion rate of a selected polymeric substrate as defined above. According to one Alternative embodiments may be made in the Enzyme system determined enzymatic activities present be used by using artificial substrates. ever after feasibility and reliability of the The determination method used can either be the conversion of a substrate itself or the formation of one by the enzymatic catalyzed reaction of certain product obtained. In special cases, catalytic intermediates of the enzyme itself (eg, oxidoreductases) measured spectrophotometrically become.

The determination of enzymatic activities in prefabricated enzyme mixtures is known to the person skilled in the art. An overview of suitable test procedures for certain enzymatic activities is listed under:
http://www.sigmaaldrich.com/Area of Interest / Biochemicals / Enzyme Explorer / Key Resources / Assay Library / Assays by Enzyme Name.html

Examples specific test method for the determination of enzymatic activities of certain classes of enzymes are listed below.

In a particular case, both the endo and exocellulase activity can be measured in 0.5 ml of a 50 mM MES reaction buffer (pH 6) containing 10 mM CaCl ₂ , 4 mM p-nitrophenyl-beta-D-cellobioside and 200 ul of a diluted enzyme solution. The reaction solution should be 30 min. be incubated at 50 ° C. Glycine buffer (100 mM, pH 4) is added to stop the reaction. The enzymatic activity can then be determined by measuring the amount of liberated p-nitrophenol spectrophotometrically at 430 nm. The absorbance values for p-nitrophenol are converted to micromoles of nitrophenol using a standard curve that relates micromoles of nitrophenol for absorption. One unit of cellulase activity is the amount of enzyme required to release 1 μmol nitrophenol / min under the assay conditions ( Wood, TM and Bhat, K., 1988 ).

In another particular case, the arabinofuranosidase activity can be determined in 1 ml of 50 mM sodium phosphate (pH 7) containing 4-100 μM 4-nitrophenyl-alpha-L-arabinofuranoside (PNP-Araf) and diluted enzyme solution (4 nM-8 uM) , The reaction solution is incubated at 37 ° C and the amount of liberated 4-nitrophenol measured at 400 nm. The enzymatic activity can be calculated using the extinction coefficient of 4-nitrophenol (10500 M ^-1 cm ^-1 ) at 400 nm ( Taylor et al., 2006 ).

In another particular case, xylosidase activity using o-nitrophe nol substituted beta-D-xylopyranoside (ONP-β-D-xylopyranoside) as a substrate ( Chen et al., 1986 ). A stock solution of the substrate (10 mM) in 100 mM citrate buffer (pH 5) is prepared. The diluted enzyme solution to be tested is prepared in bidistilled water. The reaction solution contains equal molar amounts of substrate and enzyme solution in 200 mM borate buffer at 25 ° C and pH 9.8. To determine the enzymatic activity, the release of the o-nitrophenol is recorded spectrophotometrically at a wavelength of 410 nm. The enzymatic activity in units / mg enzyme is directly proportional to the amount of o-nitrophenol released and can be calculated as described for the determination of cellulase activity.

In another particular case, laccase activity is determined using the Guiacol oxidation assay. A stock solution of 10 mM guiacol fresh in 50 mM citrate buffer (pH 4.3, 40 ° C) is prepared. The reaction is carried out in 2 ml of 50 mM citrate buffer using 10 μl of diluted enzyme solution (1-3 nM) and various amounts of substrate (5-20 μl). The rate of guiacol oxidation is measured spectrophotometrically at 465 nm. The rate of guiacol oxidation can be determined using the extinction coefficient of 5200 M ^-1 cm ^-1 for guiacol oxidation products ( Smirnov et al. 2001 ).

In another particular case, the manganese peroxidase (MnP) activity in 50 mM tartrate buffer (pH 3, 25 ° C) can be measured by adding 100 μM H ₂ O _{2 to} a reaction mixture containing 0.5 μM / ml MnP and 5-200 μM Mn ^{2 +} , is added. The formation of Mn ³⁺ is recorded spectrophotometrically at 238 nm ( Kmaitisuji et al., 2005 ).

Around determine whether a particular enzyme system (as listed in Table 1), which is useful for producing a desired product is unwanted activity of other enzyme systems (as listed in Table 1) containing the different, unwanted product from the same or a different polymeric substrate, can be a test be carried out for this activity by a specific polymeric substrate or artificial Substrate as described above, and one of the above described method is used.

Becomes For example, it is suspected that a particular enzyme system, which, as listed in Table 1, contains cellulases, unwanted xylosidase activity (as in Table 1 listed), the primary cellulase activity can be determined by an artificial cellulosic substrate is used and the amount on glucose, after adding a certain amount of the enzyme system is released per unit time is determined. Is the cellulase activity The composition determines the xylosidase activity The same enzyme preparation can be determined by the enzyme system contacted with an artificial xylan substrate and then the amount of xylose produced by a certain amount of the enzyme per Time unit is released, is measured. By measuring the relative Conversion rates in a given time unit with a certain amount of enzyme with substrates for both cellulase and xylosidase can do the specific activities for each substrate class can be calculated.

suitable Substrates for the determination of all listed in Table 1 enzymatic activities are listed in Table 2.

at an alternative embodiment are used to determine whether an enzyme system is an independent contaminant enzymatic Activity contains the components of the preparation with methods such as electrophoresis or chromatography, separated. The individual components of the preparation can by using specific methods, such as color reactions or detection of components by absorption or others, the Known to those skilled in the art. The relative percentage Distribution of proteinaceous ingredients can be done using quantitative methods, such as densitometry or any other equivalent methods known to those skilled in the art be determined with suitable mathematical calculations.

Determination of the order

Optionally, methods of pretreating the polymeric substrate, such as hot water extraction, low temperature steam explosion, acidic steam explosion, ammonia steam explosion, or ultrasound, can be used to physically alter polymeric substrates to increase the surface accessibility of vegetable fibers and increase the crystallinity of the polymer Cellulose fraction is reduced ( Puls et al., 1985 ; Ramos et al., 2005 ; Kinley et al., 2005 ). Preferably, the above-mentioned methods change the physical properties of the polymeric raw substrate such that the substrate is suitable for subsequent enzymatic better open-minded, but either limited amounts or no chemical building blocks are released.

Optional can be a pre-treatment, such as hot water extraction, low-temperature steam explosion, acid steam explosion, ammonia steam explosion or ultrasound used to be soluble in the polymeric raw substrate Remove substances before the polymeric raw substrate with an enzyme system is contacted to contamination of the products of the enzymatic Process step by soluble in the raw substrate contained To avoid substances.

Become two or more process steps applied sequentially depends the order of these steps and therefore the order the addition of the enzyme mixtures as described in Table 1 of the specific composition of the polymeric raw substrate used from. The order of process steps and enzyme systems becomes chosen so that the amount and cost of catalytic enzymes and the contact time with the substrate, which releases the desired Products leads, minimized. Additionally selected Steps in the process should include both purity and Yield of the monomers released from the polymeric substrate or Optimize oligomeric reaction products. The order of the process steps is therefore dependent on the starting material and the product.

The specific order of enzymatic treatments used for Degradation of a particular substrate into its components necessary is, can be determined empirically, even if the composition of the substrate is unknown. It is therefore possible that polymeric raw substrate in separate treatment steps with a Number of enzyme mixtures of different products, as shown in Table 1 listed, to disassemble. After each of the treatment steps is the purity and composition of the product stream obtained by using analytical methods known to those skilled in the art, including but not limited to spectroscopic and chromatographic procedures, as described above in the analysis the composition of the substrate and the determination of the enzymatic Activity were determined.

Based From the results of these measurements it is possible to do that kinetically preferred enzyme mixture to select as first treatment step of the starting material leads to the purest product flow. After repeated washing of the insoluble residues, which remain after this particular enzymatic treatment, The residues are mixed with another enzyme mixture treated, except with the enzyme mixture, which for the first treatment was selected. With all these enzymatic treatments is the composition of the obtained Product determined by the methods described above. The Enzyme mixture, which leads to the purest product flow, is as a second treatment step for the particular substrate selected. The remaining after the second process step insoluble material is again washed thoroughly and again tested with all remaining enzyme systems as above described. This method of analytical determination and selection the purest product streams, with each of the remaining Enzyme mixtures are obtained from Table 1 is repeated until the Starting material is either completely degraded or the insoluble residues of the starting material, which remain after repeated enzymatic treatments, in Comparison to the costs of further treatment options no longer gain much economic profit.

When Another possibility is the composition of the substrate determined by the analytical methods mentioned above, before the order of enzymatic treatment options is selected. According to a preferred embodiment The invention relates to the enzyme systems to be used and the order Their use is therefore determined by first the polymeric Raw substrate is analyzed.

• (a) zuerst in getrennten enzymatischen Behandlungen mit jedem einer Vielzahl von Enzymsystemen(-gemischen), wie sie in Tabelle 1 aufgeführt sind, behandelt wird;
• (b) für jede enzymatische Behandlung die definierten monomeren oder oligomeren Bausteine, die aus dem polymeren Rohsubstrat freigesetzt werden, auf Reinheit der definierten monomeren oder polymeren Bausteine untersucht werden, vorzugsweise nach der Abtrennung der (löslichen) definierten monomeren oder oligomeren Bausteine von dem (unlöslichen) polymeren Rohsubstrat;
• (c) das Enzymsystem, das zur höchsten Reinheit führt, für den ersten enzymatischen Behandlungsschritt gemäß Schritt a) von Anspruch 1 ausgewählt wird;
• (d) wahlweise die Reihenfolge der Schritte a. bis c. mit dem Rückstand des polymeren Rohsubstrats wiederholt wird, um das Enzymsystem für den folgenden enzymatischen Behandlungsschritt zu bestimmen.

A preferred embodiment of the invention is directed to a method, in particular for determining the enzyme systems to be used and their sequence, wherein the polymeric raw substrate

• (a) first treated in separate enzymatic treatments with each of a plurality of enzyme systems (mixtures) as listed in Table 1;
• (B) for each enzymatic treatment, the defined monomeric or oligomeric building blocks released from the polymeric raw substrate are tested for purity of the defined monomeric or polymeric building blocks, preferably after separation of the (soluble) defined monomeric or oligomeric building blocks from the (insoluble ) polymeric raw substrate;
• (c) selecting the enzyme system which gives the highest purity for the first enzymatic treatment step according to step a) of claim 1;
• (d) optionally the order of steps a. to c. is repeated with the residue of the polymeric raw substrate to determine the enzyme system for the subsequent enzymatic treatment step.

To an alternative embodiment will be selected Enzyme mixtures as listed in Table 1 are used, which attack the component of the substrate which has the largest Has a mass fraction of the composition of the substrate. After enzymatic treatment, the purity of the product stream obtained, as above in the analytical determination of the constituents of the substrate described. After a preferred Embodiment, it is desirable that the purity is so high that more than 75% by weight, preferably more than 90% by weight, more preferably more than 95% by weight, more preferably more as 99% by weight of the total solids content (preferably the soluble fraction) consist of the defined monomeric or oligomeric building blocks.

If the enzymatic degradation of the constituent of the substrate with the highest Mass fraction to a pure product stream (as defined above) This treatment may be the first step for the processing of the substrate can be applied. By washing the obtained insoluble pulp and subsequent Separation of the particles from the supernatant will be the next Prepared process steps. The after the first treatment of the Substrate remaining residues are with specific enzyme systems, such as those listed in Table 1 Enzyme systems treated which attack the constituent of the substrate, the second largest mass fraction of the composition of the substrate. The product stream must again with The analytical methods mentioned are analyzed for product purity to determine before the selected enzymatic treatment selected for the second enzymatic treatment of the substrate can be. The resulting insoluble residues from the second enzymatic treatment are then washed and processed as described above. Through repeated treatments with the specific enzyme systems listed in Table 1, which always attack the component of the substrate which is the largest Mass fraction of the remaining insoluble residue of the substrate obtained by the preceding enzymatic treatment cycles is obtained, the substrate can gradually into the units of his Components are degraded.

• (a) zunächst in getrennten enzymatischen Behandlungen mit jedem einer Vielzahl von Enzymsystemen(-gemischen), wie sie in Tabelle 1 aufgeführt sind, behandelt wird, um die monomeren oder oligomeren Bausteine zu bestimmen, die den größten Massenanteil an der Zusammensetzung des polymeren Rohsubstrats darstellen;
• (b) die definierten monomeren oder oligomeren Bausteine, die den größten Massenanteil an dem polymeren Rohsubstrat darstellen, auf Reinheit untersucht werden, vorzugsweise nach der Trennung von dem polymeren Rohsubstrat;
• (c) falls die bestimmte Reinheit ergibt, dass mehr als 75 Gew.-%, vorzugsweise mehr als 90 Gew.-%, besonders bevorzugt mehr als 95 Gew.-%, besonders bevorzugt mehr als 99 Gew.-% des Gesamt-Feststoffgehalts aus den definierten monomeren oder oligomeren Bausteinen besteht, das betreffende Enzymsystem für den ersten enzymatischen Behandlungsschritt gemäß Schritt a) von Anspruch 1 ausgewählt wird;
• (d) falls die nach Schritt b) bestimmte Reinheit niedriger als die nach Schritt c) erforderliche Reinheit ist, die definierten monomeren oder oligomeren Bausteine, die den nächstgrößten Massenanteil an dem polymeren Rohsubstrat darstellen, auf Reinheit untersucht werden, vorzugsweise nach der Abtrennung von dem (unlöslichen) polymeren Rohsubstrat, bis eine Reinheit die Forderungen nach Schritt c) erfüllt und das betreffende Enzymsystem für den ersten enzymatischen Behandlungsschritt gemäß Schritt a) von Anspruch 1 ausgewählt wird;
• (e) wahlweise die Reihenfolge der Schritte (a) bis (d) mit dem Rückstand des polymeren Rohsubstrats wiederholt wird, um das Enzymsystem zu bestimmen, das bei dem anschließenden enzymatischen Behandlungsschritt eingesetzt wird.

Another preferred aspect of the invention relates to a method, in particular for determining the enzyme systems to be used and their sequence, wherein the polymeric raw substrate

• (a) first treated in separate enzymatic treatments with each of a plurality of enzyme systems (mixtures) as listed in Table 1 to determine the monomeric or oligomeric building blocks which represent the largest mass fraction of the composition of the polymeric raw substrate ;
• (b) the defined monomeric or oligomeric building blocks representing the largest mass fraction of the polymeric raw substrate are examined for purity, preferably after separation from the polymeric raw substrate;
• (c) if the determined purity is greater than 75% by weight, preferably greater than 90% by weight, more preferably greater than 95% by weight, most preferably greater than 99% by weight of the total solids content consists of the defined monomeric or oligomeric building blocks, the enzyme system in question for the first enzymatic treatment step according to step a) of claim 1 is selected;
• (d) if the purity determined after step b) is lower than the purity required after step c), the defined monomeric or oligomeric building blocks constituting the next largest mass fraction of the polymeric raw substrate are examined for purity, preferably after separation from the latter (insoluble) polymeric raw substrate until a purity meets the requirements of step c) and the enzyme system in question is selected for the first enzymatic treatment step according to step a) of claim 1;
• (e) optionally repeating the sequence of steps (a) through (d) with the backbone of the bulk polymeric substrate to determine the enzyme system used in the subsequent enzymatic treatment step.

• (a) zunächst in getrennten enzymatischen Behandlungen mit jedem einer Vielzahl von Enzymsystemen(-gemischen), wie sie in Tabelle 1 aufgeführt sind, behandelt wird, um die enzymatische Behandlung zu bestimmen, welche zum höchsten Ertrag an monomeren oder oligomeren Bausteinen führt, welche in dem polymeren Rohsubstrat enthalten sind;
• (b) die enzymatischen Behandlungen ausgewählt werden, die zu einem definierten monomeren oder oligomeren Produkt mit einer Reinheit von mehr als 75 Gew.-%, vorzugsweise mehr als 90 Gew.-%, besonders bevorzugt mehr als 95 Gew.-%, besonders bevorzugt mehr als 99 Gew.-% der Gesamt-Feststoffe führen (vorzugsweise nach der Abtrennung von dem unlöslichen polymeren Rohsubstrat);
• (c) unter den verbleibenden enzymatischen Behandlungen die Behandlung mit dem höchsten Ertrag an monomeren oder oligomeren Bausteinen bestimmt wird;
• (d) wahlweise die Reihenfolge der Schritte (a) bis (c) mit dem Rückstand des polymeren Rohsubstrats wiederholt wird, um das Enzymsystem zu bestimmen, das bei dem anschließenden enzymatischen Behandlungsschritt eingesetzt wird.

Another preferred aspect of the invention relates to a method, in particular for determining the enzyme systems to be used and their sequence, wherein the polymeric raw substrate

• (a) first treated in separate enzymatic treatments with each of a variety of enzyme systems (mixtures) as listed in Table 1 to determine the enzymatic treatment which results in the highest yield of monomeric or oligomeric building blocks found in the polymeric raw substrate are included;
• (B) the enzymatic treatments which are selected to a defined monomeric or oligomeric product having a purity of more than 75 wt .-%, preferably more than 90 wt .-%, particularly preferably more than 95 wt .-%, particularly preferably more than 99% by weight of the total solids (preferably after separation from the insoluble polymeric feedstock);
• (c) determining, among the remaining enzymatic treatments, the treatment with the highest yield of monomeric or oligomeric building blocks;
• (d) optionally repeating the sequence of steps (a) through (c) with the backbone of the polymeric raw substrate to determine the enzyme system used in the subsequent enzymatic treatment step.

As mentioned above should, according to a preferred aspect The invention, the enzyme systems used only a small amount to have contaminating enzymatic activities, the other than the intended monomeric or oligomeric building blocks release from the polymeric raw substrate, or completely be free from it.

To a preferred embodiment contains the used in a particular enzymatic treatment step Enzyme system not more than 50%, preferably not more than 20%, more preferably not more than 10%, more preferably not more than 5%, more preferably not more than 2%, more preferably not more than 1% of contaminating (other) enzymatic activities, not in a previous enzymatic treatment step were used when using a different enzyme system, or for the release of other defined monomers or oligomeric building blocks that are not in preceding enzymatic Treatment steps have been released, lead, or according to a further embodiment, only for release of products from polymeric substrates that originally not substantially present in the polymeric starting material are. The percentage of other or contaminating enzymatic Activities in the enzyme system can be explained as above be calculated. According to a preferred embodiment "essentially non-present" means less than 20% by weight, preferably less than 10% by weight, preferably less than 5% by weight, preferably less than 2% by weight, preferably less than 1% by weight of the entire polymeric substrate.

To an advantageous and preferred embodiment of Invention contains that at a certain enzymatic Treatment step used enzyme system as contaminating enzymatic activities one or more such enzymatic activities, such as in a previous enzymatic treatment step Using a different enzyme system were used, or, in another embodiment, enzyme systems, the only for the release of other monomeric or oligomeric building blocks can result from polymeric substrates that originally are substantially absent in the raw polymeric substrate. In other words, it was found that when a particular monomeric or oligomeric building block from the polymeric raw substrate previously in released from a previous enzymatic treatment step was (or not present in the polymeric raw substrate from the beginning it was not essential that in a subsequent The step used enzyme system substantially free of those Enzymatic activity is the main activity used in one of the preceding enzymatic treatment steps has been. An advantage of this embodiment is that less pure and therefore less expensive enzyme systems for the second and subsequent enzymatic treatment steps can be used.

In In a specific case of such a process, the enzymatic Conversion of xylan into xylose in a process step after enzymatic processing of cellulose followed by Removal of the product glucose in a previous process step carried out. Accordingly, the enzyme mixtures in Table 1, which serve to process xylan into xylose, too Contain contaminating enzymatic activities that specific for the processing of cellulose.

additionally can according to another preferred embodiment of the invention Enzyme systems used to process a particular Component of the polymeric raw substrate can be used contaminating contain (other) enzymatic activities, if these act on certain reaction intermediates that in earlier enzymatic reactions were obtained, and when the final products are identical. In other words, another preferred Embodiment of the invention A method in which the enzyme system, used in a particular enzymatic treatment step becomes, a main enzymatic activity (as above described) and at least one additional enzymatic Has activity leading to the same defined monomers or oligomeric building blocks from the polymer raw substrate such as leading enzymatic activity of the enzyme system, in particular starting from a different polymeric substrate, which is present in the polymeric raw substrate.

In one particular case, the crude polymeric substrate contains both cellulose and starch (amylose) and the target product in the process step concerned is glucose. Here, the contamination of an enzyme mixture listed in Table 1 for the conversion of cellulose (cellulase activity) with accompanying side activities for the conversion of starch (amylase activity) can be tolerated. The enzyme system must therefore not have any other enzyme activities above a certain ratio, except amylase activities included. Here, the starting materials may differ for the different enzymatic reactions, but the product is glucose in both cases.

In In the preferred case, the enzyme mixtures used must be essentially free of specific enzyme activities be that cause contamination of the product stream obtained could.

In In another specific case, glucose is the target product, the polymeric raw substrate contains cellulose as a polymeric substrate (eg wheat straw) and it becomes an enzyme mixture of cellulases used. Useful enzyme mixtures are listed in Table 1. Such cellulase mixtures may be used in conjunction with β-glycosidases, Glucohydrolasen and alpha or beta-amylases are used, as listed in Table 1 to the cellulose fraction of the polymeric Convert crude substrates into monomeric glucose units. To one To obtain pure product flow of glucose, that of the cellulose fraction added enzyme mixture free of any hemicellulase activity which includes, but is not limited to, enzyme activities arabinofuranosidase, arabinase, galactosidase, mannanase, mannanosidase, Xylanase and xylosidase. In this enzymatic process is the polymeric cellulose is converted into soluble glucose units, which physically from the insoluble starting material can be separated as described above. The remaining one Insoluble material can be further processed to to produce various pentose sugars from the hemicellulose fraction.

In In another specific case, arabinose and xylose are the target products, the polymeric raw substrate contains polymeric substrates, u. a. heterogeneous hemicellulose polymers such as arabinoxylan, and it becomes Enzyme mixture, which in Table 1 for conversion and mobilization of branched arabinose units, first used. Subsequently, the soluble Arabinose units from the remaining insoluble substrate separated before a second enzyme mixture, which in Table 1 to Mobilization of xylose units contained in xylan polymers is used. The soluble xylose units then become also separated from the remaining insoluble substrate.

Enzymatic process steps can be combined with physico-chemical process steps. Such physico-chemical process steps may be either nonselective to extract low commercial value ingredients which would otherwise contaminate high quality product streams from the process. In one embodiment, therefore, one or more physicochemical process steps are performed to extract or otherwise remove defined constituents. Alternatively, selective physico-chemical process steps can be used, which provide a comparable selectivity to enzymatic process steps in the digestion of individual chemical constituents of the polymer raw substrate. According to one embodiment, therefore, one or more physico-chemical process steps are used to increase the selectivity of subsequent enzymatic treatment steps. Examples of such process steps are the digestion of the lignin fraction of LCB by organic solvents, such as ethanol or glycerol ( Itoh et al., 2003 ; Demirbas, A., 1998 ). These individual process steps and conditions are known to the person skilled in the art. In an additional alternative, the nonselective physico-chemical process steps described are applied to insoluble residues which have been freed of impurities in previous selective process steps. An example of such a process step is the complete hydrolysis of the protein fraction by acid treatment after selective digestion of the hemicellulose, cellulose and lignin fractions of LCB by the above selective process steps.

Another specific example of an embodiment of the invention is shown in FIG 1 shown. 1 therefore, shows a flow chart for the stepwise enzymatic processing of LCB.

At the in 1 The illustration of the processing of a substrate with high content of cellulose, hemicellulose and lignin and with small amounts of proteins and lipids, such as wheat straw, maize straw or softwood, is the digestion of the various pentose constituents contained in the hemicellulose fraction by stepwise treatment with xylanase Achieved, arabinase and mannase enzymes which specifically release xylose, arabinose or mannose sugar. Suitable enzymes and process conditions to provide optimal process conditions for the enzymes are known to those skilled in the art. The soluble fraction is removed after each step and the insoluble constituents are treated with the subsequent enzyme. Following processing of the pentose fraction, a similar process step is followed to convert cellulose to glucose by using a mixture of exo- and endocellulases, optionally in combination with cellobiase or β-glucosidase, to liberate glucose and cellobiose from the cellulosic fraction of the LCB substrate , These soluble reaction products are removed from the reaction mixture with the supernatant. Similarly, in the insoluble Phase remaining lignin fraction into its various phenolic building blocks using specific enzyme systems, such as by laccases and lignin peroxidases. These phenolic and oligophenolic compounds are then extracted from the reaction mixture with the supernatant or by solvent extraction. The conditions for carrying out this process step are known to the person skilled in the art. Each of the individual products resulting from the enzymatic conversion of hemicellulose, cellulose or lignin or other LCB components can be isolated after each successive enzyme application (see 1 ). The resulting, essentially pure chemical building blocks of phenolic substances, pentose and hexose sugars can then be further processed into high-quality commercial products (see 1 ). In general, in one embodiment, the defined monomeric or oligomeric building blocks released from the polymeric feedstock are purified and optionally further processed.

In another illustration will be agricultural residues, which are rich in proteins and lipids, such as residues from the production of rapeseed, sunflower or olive oils, subsequently with the above hemicellulases and cellulases and optionally contacting pectinases, followed by contacting the residues of these process steps with a non-specific protease. Such non-specific proteases are the Skilled in the art and can be scaled up getting produced. The protease treatment dissolves amino acids and peptides from the complex starting material. These amino acids and Peptides can then be used as a mixture or by methods known in the art into individual substances be separated.

In In one case, the target products are arabinose, xylose, glucose, oligophenylpropanoids, Monolignole and / or monophenolic substances, and these products be by sequential enzymatic conversion of the polymeric Raw substrates made of wheat straw into its components.

In the first step of such a stepwise process, finely ground wheat straw (1 kg weight, 0.2 μm) having a moisture content of about 5% by weight is placed in a steel container. At least 2 liters of water are added and mixed with the substrate. The resulting slurry is soaked for 4 hours at room temperature. Excess liquid is removed to about 200 ml of the solution. The container is sealed and heated at 10 bar pressure for 1 h at 121 ° C in a conventional autoclave system for sterilization ( Puls et al., 1985 ; Harms, 1989 . Foody et al., 1998 ). The pressure in the container is drained quickly and the contents allowed to cool to room temperature. Under these conditions, the degree of polymerization of the constituents of the substrate decreases, but only a small fraction of its constituents is released in soluble form. The resulting liquid phase (containing salts and small amounts of various soluble constituents) and the insoluble phase (containing insoluble polymeric substrates such as cellulose, hemicellulose and lignin) is separated by filtration, sieving or centrifugation, and the insoluble phase is further processed.

In the next step, a mixture of alpha-L-arabinofuranosidases is used to liberate the arabinose components contained in the insoluble hemicellulose fraction. All of the reactions described below are performed for 24 hours at a minimum of 40 ° C in 50 mM phosphate buffer of pH 5-7. 0.08-1 g of GH51 alpha-L-arabinofuranosidase from Clostridium thermocellum / kg of starting material is added to hydrolyze the alpha-1,2 / 1,3-arabinofuranosyl groups of arabinan and xylan ( Taylor et al., 2006 ). Subsequently, 0.08-1 g of GH43 alpha-L-arabinofuranosidase from Humicola insolens / kg stock is added to hydrolyze the alpha-1,5-arabinofuranosyl units ( Sorensen et al., 2006 ). Due to the specificity of the enzyme mixture, the resulting liquid phase contains mainly arabinose. The liberated and soluble arabinose units are separated from the insoluble pulp by filtration or centrifugation. The remaining insoluble fraction is retained for further processing.

in the The next step is a mixture of xylanases and xylosidases used to make the insoluble xylan components soluble Implement xylose units. All these reactions are over 24 h at a minimum of 40 ° C in 50 mM phosphate buffer with a pH of 5-7. 0.08-1 g endo-1,4-beta-xylanase (GH 10 or 11) from Humicola insolens / kg Starting material and 0.08-1 g of beta-xylosidase (GH 3) Trichoderma reesei / kg of starting material are added to xylo-oligosaccharides or xylanose units release. Due to the specificity the enzyme mixture contains the resulting liquid Phase mainly xylose. The soluble xylose is separated from the insoluble pulp by filtration or centrifugation.

In the next step, the insoluble constituents with an optimized enzyme mixture, ent holding endo and exocellulases, to mobilize the hexose sugars remaining in the hemicellulose and cellulose fraction. All reactions are performed for 16 h at a minimum of 50 ° C in 50 mM sodium acetate buffer (pH 5-6). Mixtures of each of 0.005-1 g of 1,4-beta-endoglucanases (Cel5A, Cel7B, Cel12A, Cel61A) and 1,4-beta-cellobiohydrolases (Cel7A, Cel6A) from Trichoderma reesei / kg of insoluble starting material are added to increase the hexose sugars mobilize ( Irwin et al., 1993 ; Kim et al., 1998 ).

The efficiency and kinetics of conversion of cellulose to monomeric sugar units can be optionally achieved by adding 0.0005-0.01 g of cellobiose dehydrogenase from Phanerocaete chrysosporium in combination with 0.05-1 g of ferrocyanide and 0.0005-0.1 g of β Glycosidase (10 wt. Enzyme mixture) from Aspergillus niger / kg starting material ( Igarashi et al., 1998 ; Rosgaard et al., 2006 ). Due to the specificity of the enzyme mixture, the resulting liquid phase contains mainly hexoses, predominantly glucose. These are further separated by filtration or centrifugation from the fine particulate residues of the remaining substrate.

In the next step, the insoluble lignin remaining after previous enzyme treatments is broken down into its components by sequential contact with lignin peroxidase and laccase enzyme systems. The reactions with lignin peroxidase are carried out in a minimum of 50 mM sodium tartrate (pH 3.5) at a maximum temperature of 32 ° C. High polymer insoluble lignins are prepared by contacting with in each case 0.5-1 g lignin peroxidase (LIP) from Phanerocaete chrysosporium / kg starting material ( Ward et al., 2003 ) oxidatively degraded. To investigate the catalytic inactivation of LIP ( Formation of component III; Wariishi and Gold, 1990 ) by an excess of H ₂ O _{2 present} in the reaction mixture, an H ₂ O ₂ -generating soluble enzymatic system is used to provide a controlled and continuous environment of H ₂ O ₂ formation. It can produce H ₂ O ₂ produced by glyoxaloxidase (GLOX), a natural adjuvant enzyme that works synergistically with lignin peroxidases ( Kersten, 1990 ) are used. The reaction of GLOX requires the same profile of pH, ionic strength and temperature as described for LIP. The production of H ₂ O ₂ is induced by the addition of 0.05-1 g GLOX and 0.01-1 g of the GLOX substrate methylglyoxal / kg stock. To induce and enhance the oxidative degradation of polymeric lignin by LIP, verartyl alcohol (3,4-dimethoxybenzyl alcohol) is added as a redox mediator until completion ( Ferapontova et al., 2006 ). More efficient degradation of insoluble lignin can be achieved by adding 2 g of verartyl alcohol / kg of substrate ( Barr et al., 1993 ) can be achieved. The main reaction products of the LIP-catalyzed oxidative degradation of insoluble lignin are oligophenylpropanoids, while monolignols are only minor constituents of the product mixture.

In order to increase the amount of monophenolic substances in the reaction mixture, the product mixture obtained with LIP is further reacted with laccase ( d'Acunzo et al., 2002 ). The reaction is carried out for 6 hours at a minimum of 40 ° C in 100 mM phosphate buffer with a pH of 5-6. 0.0004 g of Trametes versicolor laccase and 0.0005 g of 2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO) are added as reducing agent / kg of substrate ( Arias et al., 2003 ) to oxidize oligophenols to monophenolic lignin units. Due to the specificity of the enzyme mixture, the resulting liquid phase contains mainly monophenolic lignin units. The resulting product mixture is separated from the remaining pulp by membrane filtration and simple centrifugation.

literature

• Hamsen, G. et al. (1989) Process for the treatment of biomass with steam reactor. EP 0 187 422 A2
• Chef, W.P., Matsuo, M., Yasui, T. (1986) Agric. Biol. Che ,. 50, pp. 1183-1194
• Arias, M.E., Arenas, M., Rodriguez, J., Solviveri, J., Ball, R.S., Hernandez, M. (2003) Kraft pulp biobleaching and mediated oxidation of a non-phenolic substrate by laccase from Streptomyces cyaneus CECT 3335. Appl. Envir. Microbiol. 69, pp. 1953-1958
• D'Acunzo, F. Galli, C., Masci, B. (2002) Oxidation of phenols by laccase and laccase mediator systems. Solubility and steric issues. Eur. J. Biochem. 269, pp. 5330-5335
• Barr, D., Sha, M.M., Aust, S.D. (1993) Veratryl alcohol-dependent production of molecular oxygen by lignin peroxidase. J. Biol. Chem. 268, pp. 241-244
• Currie, H.A., Perry, C.C. (2006) Resolution of complex Monosaccharide mixtures from plant cell wall isolates by high pH anion exchange chromatography. J. Chromatography. 1128 (1-2), pp. 90-96
• Demirbas, A. (1998) Aqueous glycerol delignification of wood chips and ground wood. Bioresource Technol. 63 (2), pp. 179-185
• Irwin, D.C., Spezio, M., Walker, L.P., Wilson, D. B. (1993) Biotech. Bioengineer. 42, pp. 1002-1013.
• Itoh, H., Wada, M., Honda, Y., Kuwahara, M., Watanabe, T. (2003) Bioorganosolve pretreatments for simultaneous saccharification and fermentation of beech wood by ethanolysis and white red fungi. J. Biotechnol. 103, pp. 273-280
• Igarashi, K., Samejima, M. Eriksson, K.-L. (1998) Cellobiose dehydrogenase enhances Phanerocaete chrysosporium cellobiohydro lase I activity by relieving product inhibition. EUR. J. Biochem. 253, pp. 101-106
• Ferapontova, E.E., Castillo, J., Gorton, L. (2006) Bioelectrocatalytic properties of lignin peroxidase from Phanerocaete chrysosporium in reactions with phenols, catechols and ligninmodel compounds. Biochem. Biophys. Acta 1760 (9): pp. 1343-54
• Foody, B. et al. (1998) Pretreatment process for the conversion of cellulose to fuel ethanol. US 6,090,595
• Kamm, B., Gruber, P.R., Kamm, M. (2006) Industrial processes and products
• Status quo and future direction. Biorefineries 1, pp. 1-39 Kamitsuiji, H., Watanabe, T., Honda, Y., Kuwahara, M. (2005) Direct oxidation of polymeric substrates by multifunctional manganese peroxidase isoenzymes from Pleurotus ostreatus without redox mediators. Biochem. J. 386, pp. 387-393.
• Kaschemekat, J. Klose, M. (1985) Separation of components a liquid mixture. DE 3410155 C1
• Kersten, P.J. (1990) Glyoxal oxidase of Phanerocaete chrysosporium: its characterization and activation by lignin peroxidase Proc. Natl. Acad. Sci. 87, pp. 2936-2940
• Kim, E., Irwin, D.C., Walker, L.O., Wilson, D.B. (1998) Factorial optimization of a six-cellulase mixture. Biotech. Bioengineer. 58 (5), pp. 494-501
• Kinley, M.T., Krohn, B. Biomass conversion to alcohol using ultrasonic energy. US pat. 200570136520 A1
• Lawford, H.G., Rousseau, J.D. (2003) Cellulosic fuel ethanol. Appl. Biochem and Biotechnol. 105, pp. 457-469
• Lawford, H.G., Rousseau, J.D. (2003) Cellulosic fuel ethanol. Alternative fermentation process designs with wild-type and re combinant Zymomonas mobilis. Appl. Biochem. Biotechnol. 105 pp. 457-469
• Lynd, L.R., van Zyl, W.H. v., McBride, J.E., Laser, M. (2005) Consolidated bioprocessing of cellulosic biomass: an update. Curr. Opin. Biotechnol. 16, pp. 577-583
• Mammela, P. (2001) Phenolics in selected European hardwood species by liquid chromatography-electrospray ionization mass spectrometry. Analyst 126 (9), pp. 1535-1538
• Palla, G. (1981) C18 reversed-phase liquid chromatography determination of invert sugar, sucrose and raffinose. Anal. Chem. 53, pp. 1966-1967
• Puls, J., Poutanen, K., Körner, H.-U., Viikari, L. (1985) Biotechnological utilization of wood carbohydrates after steaming pretreatment. Appl. Microbiol. Biotechnol. 22, pp. 416-423
• Ramos, L.P., Silva, T.A., Martins, L.F., Satyanarayana, K.G. (2005) Conversion of lignocellulosics to fules, chemicals and environmentally-friendly materials. Metals and Processes 117, pp. 299-318
• Rosgaard, L., Peterson, S., Cherry, J.R., Harris, P., Meyer, A.S. (2006) Efficiency of new fungal cellulose systems in boosting enzymatic degradation of barley straw lignocellulose. Biotechnol. Prog. 22 (2), pp. 493-498
• Saha, B.C., Enzymes as biocatalysts for conversion of lignocellulosic biomass to fermentable sugars (2005) in Handbook of industrial biocatalysis, ed. Ching T. Hou, CRC Press, Chapter 24, pp. 1-12
• Smirnov, S.A., Korovela, O.V., Gavrilova, V.P., Belova, A. B., Klyachko, N.L. (2001) Laccases for basidomyces: Physicochemical characteristics and substrate specificity towards methoxyphenolic compounds. Biochem. (Moscow) 66 (7), pp. 774-779
• Sorensen, H., Pederson, S., Viksoe-Nielsen, A. et al. (2006) Hydrolysis of arabinoxylan. WO 2006/114095 A1
• Taylor, E. Smith, N., Turkenburg, J. et al. (2006) Structural insights into the ligand specificity of a thermostable family 51 arabinofuranosidase, Araf51, from Clostridium thermocellum. Biochem. J. 395, pp. 31-37
• Ward, G., Hadar, Y., Bilkis, I., Dosoretz, C. (2003) Mechanistic features of lignin peroxidase-catalysed oxidation of substituted phenols and 1,2-dimethoxyarenes. J. Biol. Chem. 278, pp. 39726-39734
• Wariishi, H., Gold, M.H. (1990) Lignin Peroxidase Compound III. Mechanism of formation and decomposition. J. Biol. Chem. 265, pp. 2070-2077
• Wood, T.M. and Baht, K.M., Methods for Measuring Cellulose activities. Methods in Enzymology. 160, pp. 87-112

Table 1 Enzyme Systems product Polymeric substrate enzyme activity Number of the enzyme mixture 1-acylglycerophosphocholine phosphatidylcholine phospholipase 1 1,5-anhydro-D-fructose + D-glucose Alpha-glucan Exo-alpha-1,4-D-glucan lyase 1 Alcohol + acetate xyloglucan acetyl 1 rhamnogalacturonan amino acids proteins protease 1 arabinose arabinan arabinofuranosidase 2, 5 arabinoxylan Endo-alpha-1,3-L-arabinanase 1, 3, 4 xyloglucan Endo-alpha-1,5-L-arabinanase 1 Exo-alpha-1,3-L-arabinanase 1, 2, 3 Exo-alpha-1,5-L-arabinanase 1, 2, 5 choline Essigsäureester acetylcholinesterase 1, 2 choline cholinesterase 1, 3 D-xylonate Xylono-1,4-lactone Xylono-1,4-lactonase 1 diacylglycerol triglyceride Triacylglycerol lipase 1 fucose xyloglucan Endo-alpha-1,2-L-fucosidase 1, 2 Exo-alpha-1,2-L-fucosidase 1, 2, 3, 4 pectin pectinase 1, 3 galactose galactan Endo-beta-1,4-D-galactosidase 1 galactomannan Exo-beta-1,4-D-galactosidase 1, 2 xyloglucan gallate digallate Acylglycerollipase 1, 3 Glycerol monoesters of long-chain fatty acids tannase 1, 2 glucose cellulose cellulase 1, 2, 3, 4, 5, 6, 7, 8 glucomannan Alpha-amylase 1, 2, 3, 4, 6 glucoronoxylan Beta-amylase 1, 2, 3, 5, 6 xyloglucan Beta-glucosidase 1, 2, 3, 4, 6 Cellobiohydrolase I 1, 3, 4, 6, 7 Cellobiohydrolase II 2, 3, 4, 6, 8 Endo-beta-1-4-D-glucanase 1, 2, 3, 4, 5, 6 Endoglucanase I 1, 2, 4, 6 Endoglucanase II 1, 2, 4, 5, 6 Endoglucanase III 1, 2, 3, 5, 6 Endoglucanase IV 1, 2, 3, 5, 6 Endoglucanase V 1, 3, 4, 6 Endoglucanase VI 1, 3, 4, 6 Endoglucanase VII 1, 3, 4, 6 Exo-beta-1,4-D-glucanase 1, 2, 3, 4, 5, 6, 7, 8 glucohydrolase 1, 2, 3, 4, 6 Strength Alpha-amylase 1, 2 Beta-amylases 1, 3 Glycerophosphocholine 2-lysophosphatidylcholine lysophospholipase 1 L-arabinonate L-arabinono-1,4-lactone L-Arabinonolactonase 1 Long-chain alcohols wax Sester Wax Sester hydrolase 1 Long-chain fatty acids Long-chain fatty acid ethyl esters Fettsäureacylethylester synthase 1 mannose galactomannan Beta-1,4-D-mannosidase 2 mannan Endo-beta-1,4-D-mannose anase 1 Exo-beta-1,4-D-mannanase 1, 2, 3 Methanol + pectate pectin pectin esterase 1, 2, 3 Pektindemethoxylase 1 Pektinmethoxylase 1, 2 Oligolignan, monolignols, phenolic compounds, oligophenylpropanoids lignin Laccase (TEMPO) 1, 2, 6 Lignin peroxidase (veratryl alcohol) + glyoxal oxidase (primary aldehyde or methylglyoxal) 1, 3, 4 Manganese peroxidase (Mn ²⁺ organic acids) 1, 2, 3, 5 oligopeptides proteins aminopeptidase 1, 2, 8 carboxypeptidase 1, 3, 8 Carboxylproteinase 1, 4 endopeptidase 1, 4, 5, 6, 7 exopeptidase 1, 2, 3, 8 metalloproteinase 1, 5 serine 1, 6 thiol proteinase 1, 7 Oligosaccharides with terminal 4-deoxy-alpha-D-galact-4-enuronosyl groups Alpha-1,4-D-galacturonan Pectate lyase (alpha-1,4-D-endopolygalacturonic acid lyase) 1 phytol chlorophyll chlorophyllase 1 ribonucleotides RNA endoribonuclease 1, 2, 3 Exoribonuclease 1, 2, 4, 5 ribonuclease 1, 3, 4, 5 sterol steryl sterol esterase 1, 2 Triterpenolesterase 1, 3 xylose arabinoxylan Endo-beta-1,3-D-xylanase 1, 3, 6 glucoronoxylan Endo-alpha-1,6-D-xylosidase 1, 2 xylan Endo-beta-1,4-D-xylanase 1, 2, 3 xyloglucan Exo-alpha-1,6-D xylanase 1, 2, 7 Exo-beta-1,3-D-xylanase 1, 3, 5, 6 Exo-beta-1,4-D-xylanase 1, 2, 3, 4 xylosidase 1, 2, 3 Exemplary enzyme combinations for the production of a specific product from a specific component of the polymeric starting material are identified individually by numbers from 1 to 8. Table 2 substratum Sigma cat. Number enzymes product cellulose C6288 Endocellulase, exocellulase, β-glycosidase glucose cellodextrines C4642 Endocellulase, exocellulase, β-glycosidase glucose β-oligosaccharides Methlylumbelliferyl M6018 Endocellulase, exocellulase, β-glycosidase glucose p-Nitrophenol-Oligosa ccharide N0145 Endocellulase, exocellulase, β-glycosidase glucose CMC C9481 endocellulase Oligoscaccharide Avicel PH-101 11365 exocellulase glucose 4-Nitrophenyl-β-D-cellobioside N5759 Glucose and pNP xylan X4252 Xylanase, xylanosidase xylose 4-Nitrophenyl-β-D-xylopyranoside N2132 cellulase Xylose and pNP Mannan from yeast M7504 mannanase Mannanoside and mannose D-galacto-D-mannan from Ceratonia siliqua 48230 Galactase and mannanase Galactose and mannose 4-nitrophenyl-α-L-arabinofuranoside N3641 Arabinofucosidase Arabinose and pNP 2,3-dimethoxybenzyl alcohol (veratryl alcohol) 38700 Lignin peroxidase (LIP) Veratrylalcohol radical cation Lignin, hydrolytic 371076 Lignin peroxidase (LIP) phenylpropanoids Lignin, organosolv 371017 Lignin peroxidase (LIP) phenylpropanoids 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) A1227 Lignin peroxidase (LIP) ABTS radical cation 2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS) A1227 laccase ABTS radical cation Manganese chloride (MnCl ₂ ) 416479 Manganese peroxidase (MnP) Mn ³⁺ ion

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• EP 0187422 A2 [0096]
• - US 6090595 [0096]
• - DE 3410155 C1 [0096]
• US 200570136520 A1 [0096]
• WO 2006/114095 A1 [0096]

Cited non-patent literature

• - Kamm et al., 2006 [0001]
• - Saha, 2005 [0003]
• - Lawford and Rousseau, 2003 [0006]
• - Pentose- (Lawford and Rousseau, 2003 [0007]
• - Lynd et al., 2005 [0007]
• - Saha, 2005 [0008]
• - Ramos et al., 2005 [0008]
• - Saha, 2005 [0008]
• - http://www1.eere.energy.gov/biomass/analytical procedures.html # samples [0030]
• - http://www1.eere.energy.gov/biomass/feedstock databases.html [0032]
• - http://wwwl.eere.energy.gov/biomass/analytical procedures.html # samples [0040]
• - http://www.sigmaaldrich.com/Area of Interest / Biochemicals / Enzyme Explorer / Key Resources / Assay Library / Assays by Enzyme Name.html [0054]
• Wood, TM and Bhat, K., 1988 [0056]
• Taylor et al., 2006 [0057]
• Chen et al., 1986 [0058]
• - Smirnov et al. 2001 [0059]
• - Kmaitisuji et al., 2005 [0060]
• Puls et al., 1985 [0065]
• - Ramos et al., 2005 [0065]
• Kinley et al., 2005 [0065]
• Itoh et al., 2003 [0085]
• - Demirbas, A., 1998 [0085]
• Puls et al., 1985 [0090]
• - Harms, 1989 [0090]
• - Foody et al., 1998 [0090]
• Taylor et al., 2006 [0091]
• Sorensen et al., 2006 [0091]
• Irwin et al., 1993 [0093]
• Kim et al., 1998 [0093]
• - Igarashi et al., 1998 [0094]
• Rosgaard et al., 2006 [0094]
• Ward et al., 2003 [0095]
• - formation of component III; Wariishi and Gold, 1990 [0095]
• - Kersten, 1990 [0095]
• Ferapontova et al., 2006 [0095]
• Barr et al., 1993 [0095]
• d'Acunzo et al., 2002 [0096]
• - Arias et al., 2003 [0096]
• Hamsen, G. et al. (1989) Process for the treatment of biomass with steam. [0096]
• - Chief, WP, Matsuo, M., Yasui, T. (1986) Agric. Biol. Che ,. 50, pp. 1183-1194 [0096]
• - Arias, ME, Arenas, M., Rodriguez, J., Solviveri, J., Ball, RS, Hernandez, M. (2003) Kraft pulp biobleaching and mediated oxidation of a non-phenolic substrate by laccase from Streptomyces cyaneus CECT 3335. Appl. Envir. Microbiol. 69, pp. 1953-1958 [0096]
• D'Acunzo, F. Galli, C., Masci, B. (2002) Oxidation of phenols by laccase and laccase-mediator systems. Solubility and steric issues. Eur. J. Biochem. 269, pp. 5330-5335 [0096]
• - Barr, D., Sha, MM, Aust, SD (1993) Veratryl alcohol-dependent production of molecular oxygen by lignin peroxidase. J. Biol. Chem. 268, pp. 241-244 [0096]
• - Currie, HA, Perry, CC (2006) Resolution of complex monosaccharide mixtures from plant cell wall isolates by high pH anion exchange chromatography. J. Chromatography. 1128 (1-2), pp. 90-96 [0096]
• Demirbas, A. (1998) Aqueous glycerol delignification of wood chips and ground wood. Bioresource Technol. 63 (2), pp. 179-185 [0096]
• Irwin, DC, Spezio, M., Walker, LP, Wilson, DB (1993) Biotech. Bioengineer. 42, pp. 1002-1013. [0096]
• - Itoh, H., Wada, M., Honda, Y., Kuwahara, M., Watanabe, T. (2003) Bioorganosolve pretreatments for simultaneous saccharification and fermentation of beech wood by ethanolysis and white rot fungi. J. Biotechnol. 103, pp. 273-280 [0096]
• - Igarashi, K., Samejima, M. Eriksson, K.-L. (1998) Cellobiose dehydrogenase enhances Phanerocaete chrysosporium cellobiohydrolase I activity by relieving product inhibition. Eur. J. Biochem. 253, pp. 101-106 [0096]
• - Ferapontova, EE, Castillo, J., Gorton, L. (2006) Bioelectrocatalytic properties of lignin peroxidase from Phanerocaete chrysosporium in reactions with phenols, catechols and ligninmodel compounds. Biochem. Biophys. Acta 1760 (9): pp. 1343-54 [0096]
• - Foody, B. et al. (1998) Pretreatment process for the conversion of cellulose to fuel ethanol. [0096]
• - Kamm, B., Gruber, PR, Kamm, M. (2006) Industrial processes and products [0096]
• - Status quo and future direction. Biorefineries 1, pp. 1-39 Kamitsuiji, H., Watanabe, T., Honda, Y., Kuwahara, M. (2005) Direct oxidation of polymeric substrates by multifunctional manganese peroxidase isoenzymes from Pleurotus ostreatus without redox mediators. Biochem. J. 386, pp. 387-393. [0096]
• Kaschemekat, J. Klose, M. (1985) Separation of the components of a liquid mixture. [0096]
• - Kersten, PJ (1990) Glyoxal oxidase of Phanerocaete chrysosporium: its characterization and activation by lignin peroxidase proc. Natl. Acad. Sci. 87, pp. 2936-2940 [0096]
• - Kim, E., Irwin, DC, Walker, LO, Wilson, DB (1998) Factorial optimization of a six-cellulase mixture. Biotech. Bioengineer. 58 (5), pp. 494-501 [0096]
• - Kinley, M.T., Krohn, B. Biomass conversion to alcohol using ultrasonic energy. [0096]
• - Lawford, HG, Rousseau, JD (2003) Cellulosic fuel ethanol. Appl. Biochem and Biotechnol. 105, pp. 457-469 [0096]
• - Lawford, HG, Rousseau, JD (2003) Cellulosic fuel ethanol. Alternative fermentation process designs with wild-type and recombinant Zymomonas mobilis. Appl. Biochem. Biotechnol. 105, pp. 457-469 [0096]
• - Lynd, LR, van Zyl, WH v., McBride, JE, Laser, M. (2005) Consolidated bioprocessing of cellulosic biomass: an update. Curr. Opin. Biotechnol. 16, pp. 577-583 [0096]
• - Mammela, P. (2001) Phenolics in selected European hardwood species by liquid chromatography-electrospray ionization mass spectrometry. Analyst 126 (9), pp. 1535-1538 [0096]
• - Palla, G. (1981) C18 reversed-phase liquid chromatography determination of invert sugar, sucrose and raffinose. Anal. Chem. 53, pp. 1966-1967 [0096]
• Pulp, J., Poutanen, K., Körner, H.-U., Viikari, L. (1985) Biotechnological utilization of wood carbohydrates after steaming pretreatment. Appl. Microbiol. Biotechnol. 22, pp. 416-423 [0096]
• - Ramos, LP, Silva, TA, Martins, LF, Satyanarayana, KG (2005) Conversion of lignocellulose to ful, chemicals and environmentally-friendly materials. Metals and Processes 117, pp. 299-318 [0096]
• - Rosgaard, L., Peterson, S., Cherry, JR, Harris, P., Meyer, AS (2006) Efficiency of new fungal cellulose systems in boosting enzymatic degradation of barley straw lignocellulose. Biotechnol. Prog. 22 (2), pp. 493-498 [0096]
• - Saha, BC, Enzymes as biocatalysts for conversion of lignocellulosic biomass to fermentable sugars (2005) in Handbook of industrial biocatalysis, ed. Ching T. Hou, CRC Press, Chapter 24, pp. 1-12 [0096]
• - Smirnov, SA, Korovela, OV, Gavrilova, VP, Belova, AB, Klyachko, NL (2001) Laccases for basidomyces: Physicochemical characteristics and substrate specificity towards methoxyphenolic compounds. Biochem. (Moscow) 66 (7), pp. 774-779 [0096]
• Sorensen, H., Pederson, S., Viksoe-Nielsen, A. et al. (2006) Hydrolysis of arabinoxylan. [0096]
• Taylor, E. Smith, N., Turkenburg, J. et al. (2006) Structural insights into the ligand specificity of a thermostable family 51 arabinofuranosidase, Araf51, from Clostridium thermocellum. Biochem. J. 395, pp. 31-37 [0096]
• Ward, G., Hadar, Y., Bilkis, I., Dosoretz, C. (2003) Mechanistic features of lignin peroxidase-catalysed oxidation of substituted phenols and 1,2-dimethoxyarenes. J. Biol. Chem. 278, pp. 39726-39734 [0096]
• Wariishi, H., Gold, MH (1990) Lignin Peroxidase Compound III. Mechanism of formation and decompo sition. J. Biol. Chem. 265, pp. 2070-2077 [0096]
• - Wood, TM and Baht, KM, Methods for Measuring Cellulose Activities. Methods in Enzymology. 160, pp. 87-112 [0096]

Claims (15)

Process for the enzymatic treatment of a polymeric raw substrate, comprising the following steps: a. Treatment of the polymeric raw substrate with an enzyme system to defined monomeric or oligomeric building blocks from the polymeric raw substrate release; b. Separation of in step a. generated defined monomeric or oligomeric building blocks from the remainder of the polymeric raw substrate. The method of claim 1, wherein in step a. used enzyme system not more than 50%, preferably not more as 20%, more preferably not more than 10%, particularly preferred not more than 5%, more preferably not more than 2%, especially preferably not more than 1% of other enzymatic activities contains, besides the enzymatic activity, which is the release of the defined monomeric or oligomeric Building blocks from the polymer raw substrate according to step a. causes. Method according to one of the preceding claims, wherein steps a. and b. one or more times with different ones Enzyme systems are repeated to other defined monomers or release oligomeric building blocks from the polymeric raw substrate. Method according to one of the preceding claims, wherein the enzymatic treatment in an aqueous medium carried out, which released from the polymeric raw substrate defined monomeric or oligomeric building blocks in the aqueous Medium are soluble and the separation according to step b. in the aqueous medium by solid / liquid separation the soluble building blocks of the insoluble polymeric raw substrate is carried out. Method according to one of the preceding claims, wherein the according to step a. defined monomers or oligomeric building blocks are selected from the group consisting from glucose, xylose, arabinose, galactose, mannose, amino acids or phenolic compounds or other products listed in Table 1. Method according to one of the preceding claims, where at a certain enzymatic treatment step used enzyme system not more than 50%, preferably not more as 20%, more preferably not more than 10%, particularly preferred not more than 5%, more preferably not more than 2%, especially preferably not more than 1% of contaminating enzymatic activities, not in a previous enzymatic treatment step were used when using a different enzyme system, or for the release of other defined monomers or oligomeric building blocks that are not in preceding enzymatic Treatment steps were released, lead, or the only lead to the release of products from polymeric substrates, not originally in the polymeric starting material are essentially present. Method according to one of the preceding claims, the polymeric raw substrate being cellulose and hemicellulose being polymeric Includes substrates and that at a certain enzymatic treatment step enzyme system used cellulase activity, and optionally Beta-glycosidase, glucohydrolase and / or alpha or beta-amylase activity but essentially free of hemicellulase activity is. Method according to one of the preceding claims, where at a certain enzymatic treatment step used enzyme system as contaminating enzymatic activities contains one or more such enzymatic activities, in a previous enzymatic treatment step Using a different enzyme system were used or only for the release of products from polymeric substrates originally, in the polymeric starting material are not essentially present. Method according to one of the preceding claims, where at a certain enzymatic treatment step used enzyme system a first enzymatic activity and at least one further enzymatic activity which has the same defined from the polymeric raw substrate monomeric or oligomeric building blocks such as the first enzymatic activity of the enzyme system, in particular from a different, in the polymeric raw substrate contained polymeric substrate. Method according to one of the preceding claims, wherein the polymeric raw substrate prior to step a. or before repeating step a. according to claim 6 a selective or nonselective physi subjected to a chemical-chemical treatment step. The method of claim 10, wherein the physicochemical Treatment step a treatment with aqueous solvents, organic solvents or any combination or any Mixture of these, preferably with ethanol or glycerol. Method according to one of the preceding claims, wherein the enzyme systems to be used and the order of their use is determined by first the polymeric raw substrate is analyzed. A method according to claim 12, wherein in order to be used To determine enzyme systems and their order, the polymeric raw substrate a. first in separate enzymatic treatments with each a variety of enzyme systems (mixtures), as shown in Table 1 are treated; b. For any enzymatic treatment the defined monomeric or oligomeric Building blocks released from the polymeric raw substrate after separation of the (soluble) defined monomers or oligomeric building blocks of the (insoluble) polymer Raw substrate on purity of the defined monomeric or polymeric Blocks are tested; c. the enzyme system which the highest purity achieved, for the first enzymatic Treatment step according to step a. of claim 1 is selected; d. optionally the order of Steps a. to c. with the residue of the polymeric raw substrate is repeated to determine the enzyme system which is in the subsequent enzymatic treatment step used shall be. A method according to claim 12, wherein in order to be used To determine enzyme systems and their order, the polymeric raw substrate a. first in separate enzymatic treatments with each a variety of enzyme systems (mixtures), as shown in Table 1, is treated to the monomers or to determine oligomeric building blocks that are the largest Represent mass fraction of the composition of the polymeric raw substrate; b. the defined monomeric or oligomeric building blocks, the largest Represent the mass fraction in the polymeric raw substrate, after separation be examined for purity from the polymeric raw substrate; c. if the certain purity indicates that more than 75% by weight is preferred more than 90% by weight, more preferably more than 95% by weight, especially preferably more than 99 wt .-% of the total solids content of the defined monomeric or oligomeric building blocks, the relevant Enzyme system for the first enzymatic treatment step according to step a. selected from claim 1 becomes; d. if after step b. certain purity lower than the one after step c. required purity is the defined monomeric or oligomeric building blocks, the next largest Represent mass fraction of the polymeric raw substrate, after separation from the (insoluble) polymeric raw substrate to purity be examined until a purity meets the demands for step c. met and the enzyme system for the first enzymatic treatment step according to step a. selected from claim 1; e. optionally the Order of steps a. to a. with the backlog of the polymer raw substrate is repeated to determine the enzyme system, used in the subsequent enzymatic treatment step becomes. A method according to claim 12, wherein in order to be used To determine enzyme systems and their order, the polymeric raw substrate a. first in separate enzymatic treatments with each a variety of enzyme systems (mixtures), as shown in Table 1, is treated to the enzymatic Treatment to determine which yields the highest monomeric or oligomeric building blocks, which in the polymeric raw substrate are included; b. the enzymatic Treatments are selected which are a defined monomeric or oligomeric product having a purity of more than 75% by weight, preferably more than 90% by weight, more preferably more than 95% Wt .-%, more preferably more than 99 wt .-% of the total solids yield; c. among the remaining enzymatic treatments the treatment with the highest yield of monomeric or is determined oligomeric building blocks; d. optionally the order of steps a. to c. with the residue of the polymeric Raw substrate is repeated to determine the enzyme system, the used in the subsequent enzymatic treatment step becomes.