EP3017056A2 - Production d'oligomères de cellulose induite par endoglucanase - Google Patents

Production d'oligomères de cellulose induite par endoglucanase

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Publication number
EP3017056A2
EP3017056A2 EP14733666.3A EP14733666A EP3017056A2 EP 3017056 A2 EP3017056 A2 EP 3017056A2 EP 14733666 A EP14733666 A EP 14733666A EP 3017056 A2 EP3017056 A2 EP 3017056A2
Authority
EP
European Patent Office
Prior art keywords
cellulose
hydrolysis
endoglucanase
chain length
endoglucanases
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.)
Withdrawn
Application number
EP14733666.3A
Other languages
German (de)
English (en)
Inventor
Mari GRANSTRÖM
Alois Kindler
Antje Spiess
Stefanie KLUGE
Benjamin BONHAGE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Original Assignee
BASF SE
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by BASF SE filed Critical BASF SE
Priority to EP14733666.3A priority Critical patent/EP3017056A2/fr
Publication of EP3017056A2 publication Critical patent/EP3017056A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/163—Sugars; Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/731—Cellulose; Quaternized cellulose derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/20—Organic compounds containing oxygen
    • C11D3/22—Carbohydrates or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/20—Organic compounds containing oxygen
    • C11D3/22—Carbohydrates or derivatives thereof
    • C11D3/222—Natural or synthetic polysaccharides, e.g. cellulose, starch, gum, alginic acid or cyclodextrin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use

Definitions

  • the invention relates to a process for the preparation of cellulose oligomers (cellooligomers) using endoglucanases and to the use of the oligomers thus produced in various technical fields.
  • Cellulose is the most common polymer on earth [Pinkert, Marshet al. Chemical Reviews, 109 (12): 6712-6728, 2009] and occurs in the form of lignocellulose in plant cell walls [Teeri, T.T., Trends in Biotechnology, 15 (5): 160-167, 1997].
  • the primary and secondary plant cell wall consists of 10 to 90% lignocellulose fibers [Weiler, E.W., General and Molecular Botany, Vol. 1. Thieme, Stuttgart, 2008].
  • Cellulose forms cellulosic fibrils in plant cell walls together with lignin and hemicellulose.
  • Within a cellulose fibril is a crystalline core, which consists of 30 to 100 parallel to each other arranged cellulose molecules.
  • Cellulose serves as a structural component of the plant cell wall and is responsible for stability and tensile strength, but also for flexibility. Unlike sugar and starch, cellulose is not a food that is relevant to human nutrition and is therefore ethically acceptable as a renewable raw material for materials and energy production.
  • a novel and surprisingly advantageous process procedure for the preparation of insoluble cellooligomers by means of endoglucanases is provided.
  • the process of the invention exhibits particular advantages in terms of producing oligomers of defined chain length and narrow chain length distribution of the insoluble cellooligomers, low production of soluble sugars, and achieving a cellooligomer chain length near the aqueous solubility limit.
  • cellulose is a water-insoluble, partially crystalline biopolymer and is poorly enzymatically hydrolyzed in aqueous media [Dadi et al., Biotechnology and Bioengineering, 95 (5): 904-10, 2006], in particular, a suitable pretreatment, e.g. by ionic liquids.
  • a suitable pretreatment e.g. by ionic liquids.
  • purified endoglucanases in particular from A. niger, B. amyloliquefaciens, T. maritima, are used for the hydrolysis of the cellulose.
  • Figures 1 a to 1 e show the time course of DP W and DP n for the enzymatic hydrolysis of Avicel by means of endoglucanases from a) A. niger, b) B. amyloliquefaciens, c) 7. maritima, d) 7. longibrachiatum and e) 7. emersonii.
  • Figures 2a to 2e show the time course of the DP W and DP n for the enzymatic hydrolysis of alpha-cellulose by means of endoglucanases from a) A. niger, b) B. amyloliquefaciens, c) 7. maritima, d) 7. longibrachiatum and e) 7. emersonii.
  • FIGS. 3 a to 3 e show the time course of the degrees of polymerization DP W and DP n for the enzymatic hydrolysis of sigmacell by means of endoglucanases from a) A. niger, b) B. amyloliquefaciens, c) 7. maritima, d) 7. longibrachiatum and e). 7 emersonii.
  • Figures 4a to 4c show the time course of the degree of polymerization DP W and DP n for the two-stage enzymatic hydrolysis of Avicel by endoglucanases from a) A. niger, b) B. amyloliquefaciens, and c) 7. maritima, after intermediate treatment with ionic Liquid (IL Restart).
  • Figures 5a to 5c show the time course of the degree of polymerization DP W and DP n for the two-stage enzymatic hydrolysis of alpha-cellulose by endoglucanases from a) A. niger, b) B. amyloliquefaciens, and c) 7. maritima, after intermediate treatment with ionic liquid (IL restart).
  • IL restart ionic liquid
  • the "DP N value” refers to the amount-related degree of polymerization or number average chain length of an oligomer or polymer.
  • the "DP W value” denotes the mass-based degree of polymerization or the chain length of an oligomer or polymer.
  • the "DP N value” and the “DP W value” are determined experimentally, in particular using gel permeation chromatography (GPC) under the standard conditions (mobile phase, operating temperature, flow rate, column material) described in more detail in the experimental section
  • chain length distribution and the “molecular weight distribution” is meant the frequency distribution of the chain length or the molecular weight, which was determined by GPC.
  • the number-average molar mass M N and the mass-average molar mass M w can be used for the quantitative description of the width of the chain length or molar mass distribution.
  • the polydispersity is defined as the ratio of M w to M N and is greater than or equal to 1. The smaller the polydispersity, the narrower is the molecular weight distribution.
  • Cellulose is to be understood broadly in the context of the invention in the environment of lignin-poor and essentially hemicellulose-free cellulose materials, from pulp to pure alpha-cellulose they do not substantially adversely affect the enzymatic reaction, the celluloses may have completely inkristallin or completely crystalline, or an average degree of crystallinity (Crl%) according to the known methods of determination (for example, X-ray diffraction) the.. "DP N - value" employed cellulose z can. B. in the range of about 30 to 150 are.
  • the "DP W value” can be, for example, in the range from 120 to 500.
  • a “celloologomer” is an oligomer having a DP N value in the range from 10 to 70 and composed of several glucose units linked to ⁇ -1,4-glycosidically.
  • Endoglucanases are enzymes that cleave randomly within a cellulose molecule. They attack in the amorphous regions of cellulose. Due to the arbitrary chain cleavage two mostly different lengths of cellulose chains are formed. This leads to rapid reduction of DP W and increase in reducing ends [Lynd, LR et al., Microbiology and Molecular Biology Reviews, 66 (3): 506-577, 2002].
  • One unit of endoglucanase activity is defined as the amount of enzyme required to produce one mole of glucose equivalents of reducing sugars per minute of carboxymethylcellulose at 40 ° C and a pH of 4.5 and 6, respectively.
  • Beta-glucosidase (EC 3.2.1 .21, or beta-1, 6-glucosidase) is a glucosidase enzyme that acts on ⁇ 1 -> 4 bonds between two glucose or substituted glucose molecules. It is an exocellulase with specificity for a variety of beta-D-glucosidic substrates. It catalyzes the hydrolysis of non-reducing terminal residues in beta-D-glucosides to release glucose.
  • Bacillus amyloliquefaciens is a Gram-positive rod-shaped bacterium discovered by J. Fukomoto in 1943 [Fukomoto, J., J. Agric. Chem. SOC. Jpn., 19; 487-503; 1943]. It was not characterized until 1987 by a group of scientists and declared as a species of its own [Priest et al. International Journal of Systematic Bacteriology, 37 (1): 69-71, 1987].
  • B. amyloliquefaciens was isolated from the soil and has a size of 0.7 ⁇ to 0.9 ⁇ times 1, 8 ⁇ to 3.0 ⁇ on. The peritrich flagellated cells are mobile and form chains. The optimum temperature is between 30 ° C to 40 ° C.
  • B. amyloliquefaciens has the number 23350 in the American Type Culture Collection.
  • Aspergillus niger is a filamentous fungus that grows aerobically [Schuster et al. Applied Microbiology and Biotechnology, 59 (4-5): 426-435, 2002]. In nature, A. niger occurs in soil, waste, compost and rotten plant material. A. niger grows at temperatures of 6 ° C to 47 ° C and in a pH range of 1.4 to 9.8 [Reiss, J., Schimmelpilze lifestyle, benefits, harm, fight. Springer, 1986]. It produces black, shaded conidiospores that spread across the air. Indus- trially, A.
  • Thermotoga maritima is a rod-shaped gram negative strictly anaerobic bacterium with a free outer cell membrane.
  • T. maritima was discovered in 1986 by R. Huber in a volcano off Italy. The growth optimum of this bacterium is at 80 ° C. Below 55 ° C no growth occurs.
  • T. maritima is 1 .5 ⁇ to 1 1 ⁇ long and 0.6 ⁇ wide. It is subpolar monotrich flagellated and thus freely mobile [Huber et al.
  • T. maritima grows on simple and complex carbohydrates such as glucose, sucrose, starch, cellulose and xylan [Nelson, KE et al., Nature, 399 (6734): 323-329, 1999].
  • Talaromyces emersonii belongs to the family of Trichocomaceae, which belongs to the Ascomycota Division and was discovered by Stolk in 1965 [Stolk, A. C, et al., Journal of Microbiology and Serology, 31 (3): 262, 1965].
  • the current name is Rasam- sonia emersonii [Houbraken, Spierenburg, Frisvad. Antonie van Leeuwenhoek, 101 (2): 403-21, 2012].
  • Trichoderma longibrachiatum belongs to the family Hypocreaceae. This belongs to the department of Ascomycota. T. longibrachiatum was discovered by Rifai in 1969 [Rifai, M.A., Mycological Pat. 1 16, Washington Mycological Institute, 1969].
  • Reaction medium with at least one endoglucanase (EC3.2.1 .4), in particular a microbial EG, such as from bacteria or fungi, hydrolytically cleaves and b) the reaction product comprising one or more cellooligomers, ie a cellooligomer fraction, isolated from the reaction medium.
  • EC endoglucanase
  • the formed cellooligomer (the cellooligomers formed) have a number average chain length or a number average degree of polymerization; DP N in the range of 10 to 100.
  • the cellooligomer formed (the cellooligomers formed) has a DP N value in the range of 15 to 50, such as 20 to 45, 25 to 40, or 30 to 35.
  • the chain length distribution of cellooligomers prepared according to the invention can be determined, e.g. but not limited to, in the range of 5 to 500, 10 to 4000 15 to 300 20 to 2000 or 25 to 150 are.
  • the EG is a naturally or recombinantly produced, possibly genetically modified enzyme from microorganisms of the genus Bacillus, Aspergillus or Thermotoga, in particular the species Bacillus amyloliquefaciens, Aspergillus niger or Thermotoga maritima, or a combination of at least two of these natural or recombinant enzymes.
  • Method according to one of the preceding embodiments wherein at least one EG is used in a concentration of about 0.01 to 100, such as 1 to 50, 1, 5 to 30 or 2 to 10, U / ml reaction mixture.
  • Method according to one of the preceding embodiments wherein cellulose is used in a concentration in the range of 0.1 to 5 or 1 to 4 or 2 to 3% (w / v) based on the total volume of the reaction mixture.
  • a1 is subjected to a pretreatment step by which the crystallinity of the cellulose is reduced
  • the ionic liquid is selected from salts which are liquid below a temperature of 100 ° C, in particular 1-ethyl-3-methylimidazolium acetate (EMIM Ac) and 3-methyl-N-butylpyridinium chloride ([C4mpy] CI).
  • EMIM Ac 1-ethyl-3-methylimidazolium acetate
  • [C4mpy] CI 3-methyl-N-butylpyridinium chloride
  • step a1) cellulose is initially introduced in the ionic liquid, optionally under the action of temperature, e.g. 20 to 80, 25 to 60 or 30 to 50 ° C dissolves and then precipitated by addition of water, an organic solvent or a mixture thereof, the precipitate separates off if necessary washes and optionally removed liquid.
  • temperature e.g. 20 to 80, 25 to 60 or 30 to 50 ° C dissolves and then precipitated by addition of water, an organic solvent or a mixture thereof, the precipitate separates off if necessary washes and optionally removed liquid.
  • step a1) the acid treatment is carried out with concentrated phosphoric acid.
  • step a1) the mechanical treatment takes place by means of a ball mill, for example with 1 mm glass balls, and / or a power consumption in the range of about 200 to 600 or 300 to 500 or about 400 W. 14.
  • Method according to one of the preceding embodiments wherein the treatment steps, in particular the steps a1) and a2), are repeated one or more times before the reaction product is isolated. 15. The method according to any one of the preceding embodiments, wherein the reaction is also carried out in the presence of a beta-glucosidase.
  • the present invention is not limited to the specifically disclosed or used enzymes having endoglucanase activity, but rather extends to functional equivalents thereof.
  • amino acid sequences of the invention used in the literature are given below: Thermotoga maritima: (SEQ ID NO: 1)
  • Bacillus amyloliquefaciens (SEQ ID NO: 3):
  • “functional equivalents” is understood to mean enzymes which, in the test used for endoglucanase activity, have an activity which is at least 1%, for example at least 10% or 20%, such as at least 50% or 75% or 90% higher or lower activity
  • functional equivalents are preferably stable between pH 2 to 1 and advantageously have a pH optimum in the range of pH 3 to 10, and a temperature optimum in the range of 25 ° C to 95 ° Coder 20 ° C to 70 ° C, such as about 45 to 60 ° C or about 50 to 55 ° C.
  • Endoglucanase activity can be detected by several known tests. Without being limited to this, let a test be performed using a reference substrate, such as a reference substrate. As carboxymethylcellulose, under standardized conditions at 40 ° C and a pH of 4.5 and 6, respectively.
  • “functional equivalents” are understood to mean, in particular, “mutants” which, in at least one sequence position of the abovementioned amino acid sequences, have a different amino acid than the one specifically mentioned but nevertheless possess one of the abovementioned biological activities.
  • “Functional equivalents” thus include the mutants obtainable by one or more amino acid additions, substitutions, deletions, and / or inversions, which changes can occur in any sequence position as long as they result in a mutant having the property profile of the invention Equivalence is also given in particular if the reactivity patterns between the mutant and the unchanged polypeptide match qualitatively, ie, for example, identical substrates are reacted at different speeds. the. Examples of suitable amino acid substitutions are summarized in the following table:
  • Precursors are natural or synthetic precursors of the polypeptides with or without the desired biological activity.
  • Salts are understood as meaning both salts of carboxyl groups and acid addition salts of amino groups of the protein molecules of the invention
  • Salts of carboxyl groups can be prepared in a manner known per se and include inorganic salts such as, for example, sodium, calcium, ammonium, iron and zinc salts, as well as salts with organic bases such as amines such as triethanolamine, arginine, lysine, piperidine and the like, acid addition salts such as salts with mineral acids such as hydrochloric acid or sulfuric acid and salts with organic acids such as acetic acid and oxalic acid also the subject of the invention.
  • inorganic salts such as, for example, sodium, calcium, ammonium, iron and zinc salts
  • organic bases such as amines such as triethanolamine, arginine, lysine, piperidine and the like
  • acid addition salts such as salts with mineral acids such as hydrochloric acid or sulfuric acid and salts with organic acids such as acetic acid and
  • “Functional derivatives” of polypeptides of the invention may also be produced at functional amino acid side groups or at their N- or C-terminal end by known techniques
  • Such derivatives include, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups obtainable by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups prepared by reaction with acyl groups; or O-acyl derivatives of free hydroxy groups prepared by reacting with acyl groups.
  • “functional equivalents” also include polypeptides that are accessible from other organisms, as well as naturally occurring variants. For example, regions of homologous sequence regions can be determined by sequence comparison and, based on the specific requirements of the invention, equivalent enzymes can be determined.
  • “Functional equivalents” also include fragments, preferably single domains or sequence motifs, of the polypeptides of the invention having, for example, the desired biological function.
  • Fusion equivalents are also fusion proteins which contain one of the abovementioned polypeptide sequences or functional equivalents derived therefrom and at least one further functionally different heterologous sequence in functional N- or C-terminal linkage (ie without substantial functional impairment of the fusion protein portions
  • heterologous sequences are, for example, signal peptides, histidine anchors or enzymes.
  • Homologs to the concretely disclosed proteins according to the invention include homologs with at least 60%, preferably at least 75%, in particular at least 85%, such as 90, 91, 92, 93, 94, 95, 96, 97 , 98 or 99%, homology (or identity) to one of the specifically disclosed amino acid sequences calculated according to the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sci. (USA) 85 (8), 1988, 2444-2448.
  • a percentage homology or identity of an invented The homologous polypeptide according to the invention means, in particular, percent identity of the amino acid residues relative to the total length of one of the amino acid sequences specifically described herein. The percent identity values can also be determined using BLAST alignments, blastp algorithm (protein-protein BLAST), or by applying the Clustal settings below.
  • “functional equivalents” include proteins of the type described above in deglycosylated or glycosylated form as well as modified forms obtainable by altering the glycosylation pattern.
  • Homologs of the proteins or polypeptides of the invention may be generated by mutagenesis, e.g. by point mutation, extension or shortening of the protein.
  • Homologs of the proteins of the invention can be identified by screening combinatorial libraries of mutants such as truncation mutants.
  • a variegated library of protein variants can be generated by combinatorial mutagenesis at the nucleic acid level, such as by enzymatic ligation of a mixture of synthetic oligonucleotides.
  • methods that can be used to prepare libraries of potential homologs from a degenerate oligonucleotide sequence. The chemical synthesis of a degenerate gene sequence can be carried out in a DNA synthesizer, and the synthetic gene can then be ligated into a suitable expression vector.
  • degenerate gene set allows for the provision of all sequences in a mixture that encode the desired set of potential protein sequences.
  • Methods of synthesizing degenerate oligonucleotides are known to those skilled in the art (eg, Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al., (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acids Res. 1 1: 477).
  • Several techniques for screening gene products of combinatorial libraries made by point mutations or truncation and for screening cDNA libraries for gene products having a selected property are known in the art.
  • REM Recursive ensemble mutagenesis
  • the person skilled in the art can introduce completely random or even more targeted mutations into genes or non-coding nucleic acid regions (which are important, for example, for the regulation of expression) and then generate gene banks.
  • the molecular biological methods required for this purpose are known to the person skilled in the art and are described, for example, in Sambrook and Russell, Molecular Cloning. 3rd Edition, Cold Spring Harbor Laboratory Press 2001.
  • DNA shuffling in which a pool of closely related genes is formed and digested, and the fragments are used as templates for a polymerase chain reaction in which repeated strand separation and recapture ultimately generate full-length mosaic genes (Stemmer WPC (1994) Nature 370: 389; Stemmer WPC (1994) Proc Natl Acad. USA 91: 10747).
  • first gene libraries of the respective proteins are generated, using, for example, the abovementioned methods
  • the gene banks are expressed in a suitable manner, for example by bacteria or by phage display systems.
  • the respective genes of host organisms expressing functional mutants with properties which largely correspond to the desired properties can be subjected to another round of mutation.
  • the steps of the mutation and the selection or screening can be repeated iteratively until the functional mutants present have the desired properties to a sufficient extent.
  • a limited number of mutations such as 1 to 5 mutations, can be carried out step by step and evaluated and selected for their influence on the relevant enzyme property.
  • the selected mutant can then be subjected in the same way to a further mutation step. This significantly reduces the number of single mutants to be studied.
  • appropriate expression constructs can be used in particular.
  • the invention also relates to expression constructs comprising, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for an enzyme according to the invention; and vectors comprising at least one of these expression constructs.
  • an "expression unit” is understood as meaning a nucleic acid with expression activity which comprises a promoter as defined herein and, after functional linkage with a nucleic acid or a gene to be expressed, regulation of expression, ie transcription and translation, of said nucleic acid or gene
  • a promoter as defined herein and, after functional linkage with a nucleic acid or a gene to be expressed, regulation of expression, ie transcription and translation, of said nucleic acid or gene
  • regulatory elements e.g. Enhancers
  • an expression cassette or “expression construct” is understood according to the invention to mean an expression unit which is functionally linked to the nucleic acid to be expressed or to the gene to be expressed.
  • an expression cassette comprises not only nucleic acid sequences that regulate transcription and translation, but also the nucleic acid sequences that are to be expressed as a protein as a result of transcription and translation.
  • expression in the context of the invention describe the production or increase of the intracellular activity of one or more enzymes in a microorganism which are encoded by the corresponding DNA, for example by introducing a gene into an organism replace existing gene with another gene, increase the copy number of the gene (s), use a strong promoter, or use a gene encoding a corresponding enzyme with a high activity and, if desired, combine these measures 5'-upstream of the respective coding sequence, a promoter and 3'-downstream, a terminator sequence and optionally further customary regulatory elements, in each case operatively linked to the coding sequence.
  • promoter a "nucleic acid with promoter activity” or a " Promotorsequ enz” is understood according to the invention as a nucleic acid which regulates the transcription of this nucleic acid in functional linkage with a nucleic acid to be transcribed.
  • a “functional” or “operative” linkage in this context means, for example, the sequential arrangement of one of the nucleic acids with promoter activity and a nucleic acid sequence to be transcribed and, if appropriate, further regulatory elements, for example nucleic acid sequences which ensure the transcription of nucleic acids, and, for example a terminator, such that each of the regulatory elements can fulfill its function in the transcription of the nucleic acid sequence.
  • further regulatory elements for example nucleic acid sequences which ensure the transcription of nucleic acids, and, for example a terminator, such that each of the regulatory elements can fulfill its function in the transcription of the nucleic acid sequence.
  • Genetic control sequences such as enhancer sequences, may also exert their function on the target sequence from more distant locations or even from other DNA molecules.
  • nucleic acid sequence to be transcribed is positioned behind (ie at the 3 'end) of the promoter sequence, so that both sequences are covalently linked to one another.
  • the distance between the promoter sequence and the transgenic nucleic acid sequence to be expressed may be less than 200 base pairs, or less than 100 base pairs or less than 50 base pairs.
  • examples of other regulatory elements include targeting sequences, enhancers, polyadenylation signals, selectable markers, amplification signals, origins of replication, and the like. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • Nucleic acid constructs according to the invention comprise, in particular, those in which the coding sequence with one or more regulatory signals is advantageously used for the control, e.g. Increased, the gene expression was operatively or functionally linked.
  • the natural regulation of these sequences may still be present before the actual structural genes and may have been genetically altered so that natural regulation is eliminated and expression of genes increased.
  • the nucleic acid construct can also be simpler, ie no additional regulatory signals have been inserted before the coding sequence and the natural promoter with its regulation has not been removed. Instead, the natural regulatory sequence is mutated so that regulation stops and gene expression is increased.
  • a preferred nucleic acid construct advantageously also contains one or more of the already mentioned “enhancer” sequences, functionally linked to the promoter, which allow increased expression of the nucleic acid sequence. Additional advantageous sequences can also be inserted at the 3 'end of the DNA sequences, such as further regulatory elements or terminators.
  • the nucleic acids of the invention may be contained in one or more copies in the construct.
  • the construct may also contain further markers, such as antibiotic resistance or auxotrophic complementing genes, optionally for selection on the construct.
  • suitable regulatory sequences are promoters such as cos-, tac-, trp-, tet-, trp-tet, Ipp-, lac-, Ipp-lac-, laclq " T7-, T5-, T3-, gal-, trc, ara, rhaP (rhaP B AD) SP6, lambda P R - or contained in the lambda P L promoter, which are advantageously in gram- find negative bacteria application.
  • Further advantageous regulatory sequences are included, for example, in the gram-positive promoters amy and SP02, in the yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH. It is also possible to use artificial promoters for regulation.
  • the nucleic acid construct, for expression in a host organism is advantageously inserted into a vector, such as a plasmid or a phage, which allows for optimal expression of the genes in the host.
  • a vector such as a plasmid or a phage
  • Vectors other than plasmids and phages are also all other vectors known to those skilled in the art, ie viruses such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be autonomously replicated in the host organism or replicated chromosomally. These vectors represent a further embodiment of the invention. Suitable plasmids are described, for example, in E.
  • plasmids mentioned represent a small selection of the possible plasmids. Further plasmids are well known to the person skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018 ).
  • the vector containing the nucleic acid construct according to the invention or the nucleic acid according to the invention can also advantageously be introduced in the form of a linear DNA into the microorganisms and integrated into the genome of the host organism via heterologous or homologous recombination.
  • This linear DNA can consist of a linearized vector such as a plasmid or only of the nucleic acid construct or of the nucleic acid according to the invention.
  • the "codon usage" can be easily determined by computer evaluations of other known genes of the organism concerned.
  • An expression cassette according to the invention is produced by fusion of a suitable promoter with a suitable coding nucleotide sequence and a terminator or polyadenylation signal.
  • common recombination and cloning techniques are used, as described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusion, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).
  • the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector for expression in a suitable host organism, which enables optimal expression of the genes in the host.
  • Vectors are well known to those skilled in the art and can be found, for example, in "Cloning Vectors” (Pouweis P.H. et al., Eds. Elsevier, Amsterdam-New York-Oxford, 1985).
  • recombinant microorganisms can be produced, which are transformed, for example, with at least one vector according to the invention and can be used to produce the endoglucanases which can be used according to the invention.
  • the above-described recombinant constructs according to the invention are introduced into a suitable host system and expressed.
  • prokaryotic or eukaryotic organisms are suitable as recombinant host organisms for the nucleic acid or nucleic acid construct according to the invention.
  • microorganisms such as bacteria, fungi or yeast are used as host organisms.
  • gram-positive or gram-negative bacteria preferably bacteria of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae or Nocardiaceae, more preferably bacteria of the genera Escherichia, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium, Clostridium or Rhodococcus used , Very particularly preferred is the genus and species Escherichia coli. Further beneficial bacteria are also found in the group of alpha-proteobacteria, beta-proteobacteria or gamma-proteobacteria
  • the host organism or the host organisms according to the invention preferably contain at least one of the endoclucanase-encoding nucleic acid sequences, nucleic acid constructs or vectors which encode an enzyme having an endoglucanase activity as defined above.
  • Microorganisms are generally contained in a liquid medium which contains a carbon source mostly in the form of sugars, a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese, magnesium salts and optionally vitamins. at temperatures between 0 ° C and 100 ° C, preferably between 10 ° C to 60 ° C attracted under oxygen fumigation.
  • the pH of the nutrient fluid can be kept at a fixed value, that is regulated during the cultivation or not.
  • the cultivation can be done batchwise, semi-batchwise or continuously.
  • Nutrients can be presented at the beginning of the fermentation or fed in semi-continuously or continuously.
  • a microorganism producing this enzyme is cultured, optionally the expression of the enzyme is induced and this is isolated from the culture.
  • the polypeptides can thus also be produced on an industrial scale, if desired.
  • the microorganisms produced according to the invention can be cultured continuously or batchwise in the batch process (batch cultivation) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process).
  • batch cultivation is in the textbook by Chmiel (Bioreatechnik 1st Introduction to bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook of Storhas (bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)) Find.
  • the culture medium to be used must suitably satisfy the requirements of the respective strains. Descriptions of culture media of various microorganisms are contained in the Manual of Methods for General Bacteriology of the American Society for Bacteriology (Washington D.C, USA, 1981).
  • These media which can be used according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.
  • Preferred carbon sources are sugars, such as mono-, di- or polysaccharides.
  • Very good sources of carbon are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose.
  • Sugar can also be added to the media via complex compounds, such as molasses, or other by-products of sugar refining. It may also be advantageous to add mixtures of different carbon sources.
  • Other possible sources of carbon are oils and fats such.
  • Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds.
  • Exemplary nitrogen sources include ammonia gas or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soybean meal, soybean protein, yeast extract, meat extract and others.
  • the nitrogen sources can be used singly or as a mixture.
  • Inorganic salt compounds which may be included in the media include the chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
  • sulfur source inorganic sulfur-containing compounds such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides but also organic see sulfur compounds, such as mercaptans and thiols can be used.
  • Phosphoric acid potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source.
  • Chelating agents can be added to the medium to keep the metal ions in solution.
  • Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.
  • the fermentation media used according to the invention usually also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine. Growth factors and salts are often derived from complex media components, such as yeast extract, molasses, corn steep liquor, and the like.
  • suitable precursors can be added to the culture medium.
  • composition of the media compounds will depend heavily on the particular experiment and will be decided on a case by case basis.
  • Information about the media optimization is available from the textbook "Applied Microbiol. Physiology, A Practice”. Growth Approaches can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and the like.
  • All media components are sterilized either by heat (20 min at 1, 5 bar and 121 ° C) or by sterile filtration.
  • the components can either be sterilized together or, if necessary, sterilized separately. All media components may be present at the beginning of the culture or added randomly or in batches as desired.
  • the temperature of the culture is usually between 15 ° C and 45 ° C, preferably 25 ° C to 40 ° C and can be kept constant or changed during the experiment.
  • the pH of the medium should be in the range of 5 to 8.5, preferably around 7.0.
  • the pH for cultivation can be controlled during cultivation by addition of basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid.
  • foam anti-foaming agents such as. As fatty acid polyglycol, are used.
  • suitable selective substances such as e.g. As antibiotics, are added.
  • oxygen or oxygen-containing gas mixtures such. B. ambient air, registered in the culture.
  • the temperature of the culture is usually 20 ° C to 45 ° C and.
  • the culture is continued until a maximum of the desired product has formed. This goal is usually reached within 10 hours to 160 hours.
  • the fermentation broth is then further processed.
  • the biomass can be wholly or partly by separation methods, such. Centrifugation, filtration, decantation or a combination of these methods are removed from the fermentation broth or left completely in it.
  • the cells may be disrupted and the product digested by known protein isolation techniques obtained from the lysate.
  • the cells may be disrupted by high frequency ultrasound, by high pressure such as in a French pressure cell, by osmolysis, by detergents, lytic enzymes or organic solvents, by homogenizers or by a combination of several of the listed procedures.
  • Purification of the polypeptides may be accomplished by known chromatographic techniques such as molecular sieve chromatography (gel filtration) such as Q-Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, as well as other conventional techniques such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, T.G., Biochemische Harvey Methoden, Verlag Walter de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.
  • anchors such as anchors.
  • hexa-histidine anchors which can be recognized as antigens of antibodies (described, for example, in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor (NY) Press ).
  • anchors may be used to attach the proteins to a solid support, e.g. a polymer matrix, which may for example be filled in a chromatography column, or used on a microtiter plate or on another carrier.
  • these anchors can also be used to detect the proteins.
  • conventional markers such as fluorescent dyes, enzyme markers which upon reaction with a substrate form a detectable reaction product, or radioactive markers, alone or in combination with the anchors, can be used to identify the proteins for the derivation of the proteins.
  • the endoglucanase can be used freely or immobilized.
  • An immobilized enzyme is an enzyme which is fixed to an inert carrier. Suitable support materials and the enzymes immobilized thereon are known from EP-A-1 149849, EP-A-1 069 183 and DE-OS 100193773 and from the references cited therein. The disclosure of these documents is hereby incorporated by reference in its entirety.
  • Suitable support materials include, for example, clays, clay minerals such as kaolinite, diatomaceous earth, perlite, silica, alumina, sodium carbonate, calcium carbonate, cellulose powders, anion exchange materials, synthetic polymers such as polystyrene, acrylic resins, phenolformaldehyde resins, polyurethanes and polyolefins such as polyethylene and polypropylene.
  • the support materials are usually used to prepare the supported enzymes in a finely divided, particulate form, with porous forms being preferred.
  • the particle size of the carrier material is usually not more than 5 mm, in particular not more than 2 mm (grading curve).
  • Carrier materials are, for example, calcium alginate and carrageenan.
  • Enzymes as well as cells can also be cross-linked directly with glutaraldehyde (cross-linking to CLEAs). Corresponding and further immobilization processes are described, for example, in J. Lalonde and A. Margolin "Immobilization of Enzymes" in K. Drauz and H. Waldmann, Enzyme Catalyst in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim described.
  • the preparation of the celloologomers is carried out by single or multiple enzymatic hydrolysis by means of endoglucanases optionally combined with a treatment of the cellulose by ionic liquids and / or mechanical treatment.
  • the optimal sequence can be determined by the skilled person by simple preliminary tests. a) Treatment of cellulose with ionic liquids
  • the cellulose is suspended, for example, in cold EMIM Ac.
  • 0.02 g of cellulose per ml of EMIM Ac are heated until the liquid becomes clear.
  • the cellulose is precipitated with water.
  • the precipitated cellulose is passed through a vacuum filtered from the ionic liquid and the water used for precipitation.
  • the cellulose is washed with water.
  • Freshly pretreated cellulose is used for enzymatic hydrolysis.
  • a suitable buffer acetate, phosphate or Tris buffer, 0.01 to 0.25 M, pH 4 to 7
  • a suitable buffer e.g. 0.1 M Na acetate buffer pH 4.5 and 0.1 M Na-K phosphate buffer pH 6.
  • the buffer concentration and the pH value can be optimally adjusted by a few preliminary experiments.
  • the cellulases are dissolved in buffer in the desired concentration.
  • the weighed cellulose is added to the cellulase solution and mixed for a few seconds in advance. Unless otherwise stated, about 10 mg (dry weight) of cellulose are used in 2 ml Eppendorf tubes with 1 ml cellulase solution for hydrolysis, which are incubated in a thermomixer at 40 ° C. and 800 min -1 for the desired time. To stop the reaction, the samples are centrifuged for 3 min at 14000 min -1 in a bench top centrifuge. A corresponding implementation in larger batches is also possible after some routine preliminary tests.
  • Areas of application - are e.g. in the field of fiber reinforcement or the modification of surface-active substances or formulations (defoamers, rheology-modifying agents, etc.).
  • cellulose substrates with different property profiles were used according to the invention.
  • the cellulose substrates differ in terms of their degree of polymerization or their chain length distribution and their degree of crystallinity and the available surface. Table 1 shows an overview of the substrates used.
  • the degree of polymerization and the chain length distribution of a cellulose substrate are determined by GPC.
  • the samples were derivatized according to previous methods and then measured by GPC [Rinaldiet al. Angewandte Chemie International Edition, 47 (42): 8047-8050, 2008, Rinaldi et al., Chemsuschem, 3 (2): 266-276, 2010].
  • the samples analyzed according to the invention have to be analyzed for GPC by an alternative GPC method [Engel et al. 2012, Biotechnology for Biofuels 5:77] are no longer derivatized.
  • Table 1 shows the determined degree of polymerization of the substrates with the GPC measuring system used according to the invention. enzymes:
  • the cellulose is suspended in cold EMIM Ac. For this, 0.75 g of cellulose per 50 mL of EMIM Ac are heated to 80 ° C for at least 40 minutes until the liquid becomes clear. The cellulose is then precipitated with 400 ml of water. The precipitated cellulose is separated from the ionic liquid and the water used for precipitation by means of a vacuum filtration in which a 0.2 .mu. ⁇ pore size cellulose acetate filter is used. Subsequently, the cellulose is washed 3 times with 500 ml of water. 2. Mechanical pretreatment of the cellulose
  • cellulose 10% (w) of cellulose is dissolved in dist. Water suspended.
  • the ball mill (Beadbeater, Biospec Products) is prepared according to instructions with 1 mm glass beads, filled with the cellulosic suspension and ground for 3 min. Thereafter, the ball mill must cool for 3 minutes until the cellulose can be ground again. This process is performed on ice five times. Subsequently, the cellulose with dist. Wash water out of the glass balls until the wash water clears. The washed suspension is collected and then alternates 3 min at 4000 min -1 (Rotina 35R) centrifuge. The water is decanted from the pretreated cellulose and the cellulase used for the enzymatic hydrolysis.
  • the endoglucanases from A. niger, T. longibrachiatum and T. emersonii, and A. niger ⁇ -glucosidase are dissolved in the desired concentration in Na acetate buffer.
  • the endoglucanases from T. maritima and B. amyloliquefaciens are dissolved in the desired concentration in Na-K phosphate buffer.
  • the weighed cellulose is added to the cellulase solution and vortexed for a few seconds. Unless otherwise stated, 10 mg (dry weight) of cellulose in 2 ml Eppendorf tubes with 1 ml cellulase solution are used for the hydrolysis, which are incubated in a thermomixer at 40 ° C.
  • the samples are centrifuged for 3 min at 14000 min -1 in a bench top centrifuge, the supernatant removed and transferred to another Eppendorf reaction vessel. The pellet and the supernatant are frozen at -80 ° C and can be analyzed later.
  • the determined cellulose loss is used to calculate the amount of cellulose after hydrolysis (dry weight).
  • the pellet obtained after the hydrolysis is dissolved in EMIM Ac at 80 ° C for 2 h.
  • the amount of EMIM Ac required depends on the amount and moisture content of the cellulose because the water present in the cellulose samples decreases the cellulose solubility of EMIM Ac.
  • the cellulose is precipitated from EMIM Ac by addition of water.
  • the cellulose is washed three times with water. To separate the liquid, the cellulose is centrifuged for 3 min at 4000 min -1 (Rotina 35R) and the liquid is then decanted off. Weighing determines the wet weight of the cellulose samples.
  • the determined wet weight of the samples and the calculated dry weights after hydrolysis are used to determine the swelling factor of the samples.
  • the cellulose purified by cellulase using this method is then used for a second hydrolysis.
  • the previously determined swelling factor is used.
  • GPC 0 Organic gel permeation chromatography determines the chain length distribution of cellulose samples.
  • the cellulose samples obtained from the hydrolysis are lyophilized and the lyophilizate is subsequently dissolved at 80 ° C. in DMF / 19% (v / v) EMIM Ac.
  • the dissolved cellulose is filtered through a 0.1 ⁇ PT-FE filter and transferred to HPLC glassware.
  • a GPC 0 system was used with the devices listed above (Table 3) under the following conditions.
  • aqueous gel permeation chromatography (GPC W )
  • the composition of the sugar present in the aqueous supernatant of the hydrolysis is determined.
  • the supernatants obtained from the hydrolysis are sterile-filtered (0.2 ⁇ ) and transferred into HPLC glass vessels.
  • a GPC system with the components according to Table 4 under the following conditions
  • glucose and cellooligomers (cellobiose [C2] to cellohexaose [C6]) from Megazyme (Ireland) are used.
  • the retention time is 27 to 31, 5 min.
  • Dry Mass Determination In order to subsequently determine the exact amount of cellulose used for the hydrolysis, a dry mass determination of the pretreated cellulose is carried out. For this Eppendorf reaction vessels are labeled and dried at 80 ° C for 3 d. After listing the weight, the Eppendorf reaction tube is filled with a certain amount of wet cellulose and the weight noted. Then the filled Eppendorf reaction vessel is dried at 80 ° C. After drying the Eppendorf reaction vessel is weighed again. From the curb weight, the wet weight and the dry weight, the swelling factor will be determined. Since freshly pretreated cellulose is used for all tests, the determined swelling factor merely serves to retrospectively check the values calculated for the hydrolysis tests.
  • the cellulose loss determination can be used to determine how much of the cellulose is broken down into soluble sugars and cellooligomers. To determine the cellulose loss, 30 mg dry mass of cellulose are hydrolyzed by the method described above.
  • the cellulose is not separated from the buffer by centrifugation.
  • the weight of a cellulose acetate filter having a pore size of 0.2 ⁇ m is recorded and the hydrolysed cellulose suspension is filtered off via the filter.
  • the filter with the hydrolyzed cellulose is washed three times with 10 ml. Washed water and dried at 80 ° C for 3 d. After drying, weigh the cellulose acetate filter with the dried cellulose and record the weight. From the difference in weight between the cellulose used for the hydrolysis and the weight after the drying of the cellulose, the cellulose loss can be determined.
  • High performance liquid chromatography can be used to determine the concentration of glucose and cellobiose present in the aqueous supernatant of the hydrolysis reaction.
  • HPLC High-performance liquid chromatography
  • the supernatants obtained from the hydrolysis are sterile filtered and transferred into HPLC glass vessels.
  • an HPLC (Table 5) under the following conditions
  • the 4-hydroxybenzoic acid hydrazide test can be used to determine the molar concentration of the reducing sugar present in the aqueous supernatant of the hydrolysis reaction.
  • the reducing soluble sugars react with the PAH BAH reagent to form a yellow dye that can be measured photometrically at 410 nm.
  • glucose calibration series are made with the buffers used at a concentration range of 0.025 g to 0.5 g / L.
  • For the PAH BAH test two reagents have to be prepared (A and B) whose recipes are listed in the following table. Before carrying out the PAHBAH test, the reagents A and B (ratio 1:10) are used to prepare the working reagent.
  • Trisodium citrate 12.45 g
  • Reagent B calcium chloride 1, 1 g
  • SDS gel electrophoresis sodium dodecyl Sulfate polyacrylamide gel electrophoresis (SDS gel electrophoresis) using an SDS gel system manufactured by Life Technologies (California, USA) was used.
  • Example 1 GPC Q of the cellulosic substrates Avicel, ⁇ -cellulose and Sigmacell To GPCo of the three enzymatically unhydrolyzed cellulose substrates Avicel, o cellulose and Sigmacell samples of these three substrates were incubated for 1 d at 40 ° C in buffer. Subsequently, they were prepared for the GPCo analogously to the samples of the hydrolysis and analyzed in the GPCo.
  • the chain length distribution determination for Avicel gave the same result for the acetate and phosphate buffers.
  • the chain length distribution of the ⁇ -cellulose and Sigmacell samples incubated in phosphate buffer show a proportional shift of the curve towards longer chains. The cause of this observation is unclear.
  • the samples incubated in acetate buffer are not shifted to longer chain lengths as compared to samples not incubated in buffer.
  • for the preparation of the chain length distribution of o cellulose and Sigmacell without enzymatic degradation substrate samples are used as a reference, which were previously incubated in acetate buffer and then lyophilized.
  • composition of the hydrolysis batch :
  • the chain length distribution of the sample without endoglucanase is between 10 and 1000 glucose units, with a maximum at 250. After hydrolysis with the A. niger endoglucanase for 5 min, a shift of the upper end of the chain length distribution by 700 glucose units towards shorter chain lengths is observed. The chain length distribution after this reaction time is between 10 and 300 glucose units. The maximum of the distribution is 90 glucose units. In the Time span between 5 min and 24 h shifts the upper end of the chain length distribution by 100 glucose units towards shorter chain lengths. After 24 h, the curve is between 10 and 200 glucose units and has a maximum at 70 glucose units.
  • composition of the hydrolysis batch :
  • the chain length distribution of the sample without endoglucanase is between 10 and 700 glucose units, with a maximum at 250. Also in the hydrolysis of Avicel by B. amyloliquefaciens endoglucanase, a shift of the upper end of the chain length distribution to shorter chains can be observed after 20 min , The chain length distribution of the 2 h sample is shifted by 150 glucose units towards shorter chain lengths after 20 min. The curves of the samples between 2 h and 24 h are congruent. The substrate is therefore not further degraded. After hydrolysis for 24 h, a chain length distribution is achieved which is between 10 and 200 glucose units and has a maximum at about 65 glucose units
  • composition of the hydrolysis batch :
  • the GPCo measurement of the sample without endoglucanase has a chain length distribution of 10 to 700 glucose units.
  • the 5 min sample has a chain length distribution between 15 and 300 glucose units.
  • the chain length distribution after this reaction time is between 15 and 300 glucose units, with a maximum at 90 glucose units.
  • composition of the hydrolysis batch :
  • the upper end of the chain length distribution after hydrolysis for 5 minutes is between 300 and 400 glucose units. This is the case with T. longibrachiatum endoglucanase only after 20 min.
  • composition of the hydrolysis batch :
  • the chain length distribution of the sample without endoglucanase is between 10 and 700 glucose units, with a maximum at 250 glucose units.
  • the upper end of the chain length distribution shifts to shorter chain lengths during the first 4 h of hydrolysis. After 4 h, the curve of the chain length distribution lies between 15 and 200 glucose units, with a maximum at 70 glucose units.
  • the test results are shown graphically in the attached Figures 1 a to e.
  • the initial value of the DP W of the sample, before addition of cellulase (0 h sample), is between 160 and 220 with an average value of 190.
  • the DP W drops by 37% to 53% during the first 5 min and thus lies between 90 and 120. After 24 h, it decreases the DP W in the hydrolysis experiments with the endoglucanases from A niger, T. maritima and T. longibrachiatum by another 10% to 21% to 50% to 37% of the initial value.
  • the DP W of the hydrolysis with B. amyloliquefaciens endoglucanase is 40% of the initial value after 20 minutes. After 24 h of hydrolysis, the DP W is lowered by a further 5% to 35% of the initial value.
  • the DP W of hydrolysis of the endoglucanase from T. emersonii is lowered after 20 minutes of hydrolysis of 52% of the initial value. After 2 h of hydrolysis, the DP W with a value of 70 is 36% of the initial value. Thereafter, the DP W increases from 120 h to 24 h by the end of the reaction time. Also on hydrolysis of Sigmacell with the T. emersonii endoglucanase, both the DP W and the DP N increase after 3 h of hydrolysis. Therefore, it can not be assumed that the observed increase is measurement error. A possible cause would be an increased reduction of short chains, whereby the already existing longer chains, in the ratio more significant.
  • the DP W is reduced most in the first 5 min trial period compared to the rest of the trial period relative to baseline. In the first 1 to 2 h, the DP W drops to approx. 50%. Compared to the other endoglucanases, the lowest DP W of 65 can be achieved using B. amyloliquefaciens endoglucanase.
  • the polydispersity decreases with decreasing DP W.
  • polydispersity decreases with decreasing DP W.
  • Example 4 Mass balance of the hydrolysis of Avicel
  • soluble cellooligomers and glucose also accumulate in the hydrolysis of cellulose.
  • This example is devoted to the quantification and evaluation of the loss of cellulose employed by production of soluble cellooligomers and glucose.
  • the dissolved cellooligomers and glucose were quantified by HPLC and PAHBAH assay.
  • HPLC analysis the glucose and cellobiose concentration certainly. The concentration of these two dissolved sugars increases with time for all endoglucanases used
  • T. emersonii endoglucanase produces the most unknown soluble cellooligomers with 11% unknown soluble cellooligomers.
  • B. amyloliquefaciens endoglucanase produces 6.1% of unknown soluble cellooligomers.
  • T. longibrachiatum endoglucanase is used, 2% of unknown soluble cellooligomers are formed.
  • T. maritima and A. niger endoglucanases at 0.5% and 0% results in a very small or immeasurable amount of unknown soluble cellooligomers. Since the endoglucanases from T. emersonii and B.
  • amyloliquefaciens produce the most soluble cellooligomers compared to the other endoglucanases, these two endoglucanases could be used to produce soluble cellooligomers.
  • the endoglucanase from T. emersonii produces mainly glucose, cellobiose (51%) and soluble cellooligomers (11%).
  • the endoglucanases from B. amyloliquefaciens with 75%, A. niger with 81% and T. maritima with 96.6% insoluble cellulose after hydrolysis the best cellulosic yields are achieved compared to the other endoglucanases.
  • the endoglucanases from B. amyloliquefaciens and A. niger are of particular interest if the insoluble cellulose is relatively short-chain cellooligomers.
  • Example 5 Enzymatic hydrolysis of ⁇ -cellulose by means of endoglucanases from A. niger, B. amyloliquefaciens, T. maritima, T. longibrachiatum and T. emersonii
  • composition of the hydrolysis batch :
  • composition of the hydrolysis batch :
  • the chain length distribution of the hydrolysis of ⁇ -cellulose was determined by means of B. amyloliquefaciens endoglucanase. After hydrolysis for 5 minutes with A. niger endoglucanase, only a flattening of the slope between 10 and 30 glucose units of the chain length distribution can be seen instead of the shoulder. After 20 min reaction time, this flattening is no longer visible, but instead a nearly Gaussian chain length distribution is observed. At the end of the experiment, the distribution of the chain lengths is between 10 and 400 glucose units with a maximum at 80 glucose units. By the enzymatic hydrolysis a reduction of the maximum by 80% and the upper end by 75% is achieved.
  • composition of the hydrolysis batch :
  • the chain length distribution of the hydrolysis of ⁇ -cellulose was determined by T. maritima endoglucanase.
  • the GPC 0 analyzed 5 min sample of the hydrolysis of ⁇ -cellulose by T. maritima endoglucanase shows a chain length distribution between 10 and 1800 glucose units. Compared to the sample Without enzyme, cellooligomers are present in the range 10 to 20 glucose units. The maximum after 5 minutes has shifted from 450 to 350. A shoulder is no longer pronounced, but a slight flattening of the slope at about 120 glucose units visible.
  • the slope of the chain length distribution is in the range between 10 and 30 glucose units compared to the slope in the range of 30 glucose units to the maximum, flatter.
  • the upper end of the chain length distribution and the maxima of the curves shift towards shorter chain lengths during hydrolysis. After 19 h reaction time, the curve is between 10 and 600 and the maximum at 150 glucose units. Thus, the maximum of the chain length distribution is reduced by 65% and the upper end of the chain length distribution by 65%.
  • composition of the hydrolysis batch :
  • the chain length distribution of the hydrolysis of ⁇ -cellulose was determined by T. longibrachiatum endoglucanase.
  • the upper end of the chain length distribution of the hydrolysis of ⁇ -cellulose by T. longibrachiatum endoglucanase shifts to shorter chain lengths after hydrolysis for 5 minutes.
  • the chain length distribution after this reaction time is between 10 and 1500 glucose units.
  • the maximum has shifted from 450 to 200 glucose units.
  • the shoulder has also shifted to shorter chain lengths and is about 25 glucose units.
  • the upper end of the chain length distribution shifts ever further towards shorter chain lengths.
  • the maxima of the curves also shift towards shorter chains.
  • the chain length distribution becomes narrower in the course of the experiment, the height of the maxima increases in the course of the experiment.
  • the chain length distribution is between 10 and 400 and the maximum at 75 glucose units.
  • composition of the hydrolysis batch :
  • the chain length distribution of the hydrolysis of ⁇ -cellulose by T. emersonii endoglucanase was determined by GPCo.
  • the chain length distribution of the sample without enzyme is between 20 and 1800 glucose units, with a maximum at 450 glucose units. Between 90 and 200 glucose units, the chain length distribution has a shallower area (shoulder).
  • the upper end of the chain length distribution of the hydrolysis of ⁇ -cellulose by T. emersonii endoglucanase shifts to shorter chain lengths after hydrolysis for 5 minutes.
  • the chain length distribution is after this reaction time between 10 and 1500 glucose units.
  • the maximum has shifted from 450 to 280 glucose units.
  • the shoulder has also shifted towards shorter chain lengths and is about 30 glucose units.
  • Example 6 Determination of the degree of polymerization DP of enzymatically hydrolysed alpha cellulose For the determination of the DP of enzymatically hydrolysed alpha-cellulose (from Example 5), GPC analyzes were carried out.
  • the DP W of the A. niger samples drops to 180. This results in a reduction of the DP W of at least 50%.
  • the DP W continues to decrease and, after 19 h, has a value of 1 to about 25% of the initial value. This shows that the reduction of the DP W slows down over time.
  • the DP N drops from 240 to 140 after a reaction time of 5 min. Until the end of the test after 19 h, the DP N drops to 50.
  • the DP W of the B. amyloliquefaciens sample drops to 140 within 5 min.
  • the DP N is 70 after this reaction time. After 19 h, the DP W is 90 and the DP N is 50.
  • the DP is located W of the sample using T. emersonii endoglucanase at 150.
  • T. longibrachiatum endoglucanase a DP W of 170 to 40 minutes is achieved.
  • values in this range are already reached after 5 min. Accordingly, the reduction of DP W using the endoglucanases from T. emersonii and T. longibrachiatum proceeds more slowly than when using the endoglucans from A. niger and B. amyloliquefaciens.
  • T. emersonii and T. longibrachiatum proceeds more slowly than when using the endoglucans from A. niger and B. amyloliquefaciens.
  • a DP W of 90 and a DP N of 50 are achieved.
  • a DP W of 90 and a DP N of 40 are achieved after hydrolysis for 19 h.
  • the DP W and DP N increase after approx. 1 h of hydrolysis with the T. emersonii endoglucanase.
  • the increase in DP is not observed when using ⁇ -cellulose.
  • T. maritima and A. niger endoglucanases 87.1% and 94.5% insoluble cellulose were measured after hydrolysis.
  • these two enzymes are of particular interest when the insoluble cellulose is short-chain cellooligomers.
  • the DP W from 150 to 19 h by T. maritima endoglucanase is approximately 50 units above the DP W of the other endoglucanases after the same hydrolysis time.
  • DP W is not reduced to the same extent as using the other endoglucanases.
  • a DP W T. emersonii and T. lon gibrachiatum is using the endoglucanase from A. niger achieved in the same range as when using the endoglucanases from B. amyloliquefaciens.
  • the reduction of DP W in the same proportion and the low production of glucose and soluble cellooligomers compared to the other endoglucanases makes A. niger endoglucanase the favored endoglucanase for the enzymatic hydrolysis of ⁇ -cellulose.
  • the endoglucanases from B. amyloliquefaciens and T. maritima produce little or no soluble cellooligomers at 0.7% and 0%, respectively.
  • the A. niger and T. longibrachiatum endoglucanases produce 2.7% and 3.1% unknown cellooligomers, respectively.
  • T. emersonii endoglucanase produces the most unknown soluble cellooligomers (27.4%).
  • the endoglucanase from T. emersonii is probably suitable for the production of soluble cellooligomers, which are larger than cellobiose.
  • Example 8 Enzymatic hydrolysis of Sigmacell using endoglucanases from A. niger, B. amyloliquefaciens, T. maritima, T. longibrachiatum and T. emersonii With the aid of GPCo, the chain length distribution of the hydrolysis samples of o cellulose and of sigmacell was determined.
  • composition of the hydrolysis batch :
  • the chain length distribution of Sigmacell without enzymatic degradation is in the range between 10 and 1000 glucose units, with a maximum at 300-400 glucose units and has a smaller slope in the range between 10 and 50 glucose units in the range between 50 glucose units and the maximum.
  • the flattening after 5 minutes of hydrolysis with A. niger endoglucanase is between 10 and 20 glucose units.
  • the chain length distribution is between 10 and 500 glucose units, with a maximum at 100 glucose units.
  • the upper end of the chain length distribution is between 10 and 350 glucose units and is thus shifted by 150 glucose units towards shorter chain lengths in comparison to the chain length distribution of the 5 min sample.
  • the 6 h hydrolysis sample shows no flattening in the left part of the curve.
  • composition of the hydrolysis batch :
  • the chain length distribution of the 5 min sample of the hydrolysis of Sigmacell by means of B. amyloliquefaciens endoglucanase is between 10 and 400 glucose units. After this reaction time, no flattening between 15 and 50 glucose units can be seen. Until the end of the experiment after 20 h, the upper end of the shifts Chain length distribution continuously on to shorter chains. The distribution is after this reaction time between 10 and 300 glucose units. The maximum also shifts towards shorter chain lengths and is at 65 glucose units. T. maritima
  • composition of the hydrolysis batch :
  • the chain length distribution is in the same range as untreated Sigmacell.
  • the maximum of the chain length distribution is shifted by 100 glucose units to shorter chain lengths and an increase of short cellulose chains in the range of 10 to 200 glucose units can be seen.
  • the chain length distribution shifts further towards shorter chain lengths and then lies between 10 and 400 glucose units.
  • a slower degradation of sigmacell is observed in comparison to Sigmacell hydrolysis experiments using A. niger and B. amyloliquefaciens endoglucanases.
  • composition of the hydrolysis batch :
  • the chain length distribution is between 15 and 800 glucose units.
  • the flattening at the front of the chain length distribution is still visible between 15 and 60 glucose units at this point, but in a shorter range than untreated Sigmacell.
  • the curve is between 10 and 300 glucose units with a maximum at 80 glucose units
  • composition of the hydrolysis batch :
  • the chain length distribution of the 20 h sample shows an unexpected course compared to the other hydrolysis samples.
  • the chain length distribution ranges from 10 to 300 glucose units, with a maximum at 70 glucose units. Thereafter, an increase in the longer chains can be observed, with the range of the chain length distribution narrowing more and more.
  • the maximum of the curve also shifts towards longer chain lengths.
  • the chain length distribution is after 6 h hydrolysis between 10 and 250 glucose units, with a maximum at 100 glucose units.
  • the DP W drops to 130.
  • the DP N drops from 1 10 to 60. This reduces both the DP W and the DP N by at least 55%.
  • a DP W of 100 and a DP N of 40 are reached. The hydrolysis slows down thus. With a DP W of 120, the DP W of the 1 h and 3 h samples increases slightly.
  • the DP N shows an analogue course.
  • the DP W of 100 and DP N of 50 after 6 h reaction time support the assumption that the increase in DP W and DP N after 1 h and 3 h hydrolysis time is probably also due to measurement disturbances.
  • the DPw is at 150 using the T. emersonii endoglucanase. After 1 h reaction time decreases the DP W is still at 75. The DP N is at 40 at this point. After 3 h of hydrolysis, an increase in the DP W is observed. Also, upon hydrolysis of ⁇ -cellulose with T. emersonii endoglucanase, both DP W and DP N increase after 2 h of hydrolysis. A possible cause would be an increased dismantling of short chains, as a result of which the longer chains that already exist fall more in proportion.
  • a DP W of 200 is reached after a reaction time of 5 min. After 1 h of reaction time, the DP W further drops to 130. The DP N is at this point at 65. After 1 h, the cellulose is further degraded using T. longibrachiatum endoglucanase and after completion of the experiment a DP W of 80 and reached a DP N of 50.
  • the hydrolysis of Sigmacell by T. maritima endoglucanase is slower than sigmacell hydrolyses using A. niger, B. amyloliquefaciens, T. emersonii and T. longibrachiatum endoglucanases.
  • the DP W can be reduced to less than half of the initial value.
  • the endoglucanases used differ in their reaction rate and in DP W , which is reached at the end of the experiment.
  • the lowest DP values can be achieved using B. amyloliquefaciens and T. longibrachiatum endoglucanase.
  • B. amyloliquefaciens endoglucanase stagnation of DP W at 80 is observed. It is therefore achieved a mining stop.
  • the DP W decreases until the end of the experiment. It is thus achieved no loss of construction. Because of the faster reaction rate of the A. niger and B. amyloliquefaciens endoglucanases compared to the other endoglucanases, these endoglucanases are of particular interest for further experimental investigations.
  • the polydispersity of DP W and DP N was also calculated for Sigmacell and can be seen in the following table.
  • the DP and polydispersity results of enzymatic hydrolysis using A. niger and T. emersonii endogiucanases do not correlate. In these endoglucanases, however, no DP results after 1 d were used due to measurement errors in the hydrolysis samples of the A. niger endogiucanase and an unusual DP increase in the hydrolysis samples of T. emersonii endogiucanase.
  • the DP W and the polydispersity of the other endoglucanases with a hydrolysis time of 1 d correlate, as already observed with Avicel and ⁇ -cellulose.
  • Example 11a Hydrolysis of Avicel to IL Restart:
  • composition of the hydrolysis batch :
  • the chain length distributions resulting from the second hydrolysis differ depending on the endoglucanase used.
  • the endoglucanase from A. niger and B. amyloliquefaciens is used, an increase in cellooligomers with a size of 18 glucose units can be seen after performing an IL restart.
  • the hydrolysis by A. niger endoglucanase gives a further maximum at 18 glucose units after carrying out an IL restart.
  • T. maritima endoglucanase an increase in cellooligomers of 15 glucose units is observed after performing an IL restart.
  • Example 11b Determination of the degree of polymerization DP of enzymatically hydrolyzed Avicel after IL restart
  • the DP W of the samples is between 55 and 65, except for the Restart experiments.
  • a DP W of 35 is achieved
  • the DP W of the 1 d and 2 d samples is 80.
  • the IL restarts method results in a DP W of 55. With this method, the DP W can be reduced by at least 30% reduced to the simple hydrolysis.
  • Example 11c Hydrolysis of ⁇ -cellulose after IL restart
  • composition of the hydrolysis batch :
  • the chain length distributions of the hydrolysis experiments of ⁇ -cellulose with the enzymes used essentially give an analogous picture of Avicel hydrolysis. Only the IL restart experiments lead to significantly reduced molecular weights.
  • A. niger endoglucanase is used, an increase in cellooligomers with a size of 18 glucose units can be seen after performing an IL restart.
  • the chain length distribution shifts to shorter chains after carrying out an IL restart and is between 10 and 200 glucose units.
  • B. amyloliquefaciens endoglucanase is used for the hydrolysis of ⁇ -cellulose, the maximum of the chain length distribution shifts by 30 glucose units to shorter chain lengths after carrying out an IL restart. After carrying out an IL restart using the T. maritima endoglucanase, no shift of the upper end of the chain length distribution or of the maximum of the chain length distribution towards shorter chain lengths can be observed.
  • Example 11 d Determination of the degree of polymerization DP of enzymatically hydrolysed alpha-cellulose after IL restart To determine the DP of enzymatically hydrolysed alpha-cellulose (from Example 11c), GPC analyzes were carried out.
  • the DP W can be reduced by 36% compared with the simple hydrolysis after 2 days.
  • a DP W of 120 is achieved after 1 d of hydrolysis of DP W of 140 and after 2 d of hydrolysis.
  • ⁇ -cellulose By the end of the experiment, the DP W decreases in the case of simple hydrolysis; it is thus achieved no loss of construction.
  • ⁇ -cellulose With the IL Restart method ⁇ -cellulose can be produced with a DP W of 55.
  • the ⁇ -glucosidase used in the examples represents an optional further embodiment of the invention. In the context of investigations according to the invention it has been possible to show that their use is not absolutely necessary. It is known that ⁇ -glucosidase can prevent possible product inhibition of the endoglucanases by the presence of cellulose degradation products (in particular cellobiose). The actual need to use this enzyme, however, can be determined by a few routine preliminary experiments.

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Abstract

L'invention concerne un procédé de production d'oligomères de cellulose (cello-oligomères) faisant appel à des endoglucanases ainsi que l'utilisation des oligomères ainsi produits dans divers domaines techniques.
EP14733666.3A 2013-07-01 2014-06-30 Production d'oligomères de cellulose induite par endoglucanase Withdrawn EP3017056A2 (fr)

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