Our Beverage area covers enzymatic solutions for the Brewing -, Beverage Alcohol -, Wine Industry and for Fruit &Vegetable Processing.
Brewing
Beer is an alcoholic, effervescent beverage traditionally made from malted barley, water, hops and yeast. Malting, mashing and fermentation/maturation are enzymatic processes within production of beer.
Malt, the main raw material (contains extract components like starch, proteins etc and enzymes like amylases, proteases etc) can be substituted partially or totally by unmalted cereals like barley, rye, oats, wheat, maize and also sorghum, known as adjuncts.
Novozymes range of enzymes for the brewing industry and applications (adjunct liquefaction, filtration, increased attenuation, maturation, troubleshooting and innovations) offers economical and technical benefits, such as
- better control of the brewing process
- faster production and so increased capacity
- potential for new and different beer types
- same quality for cheaper and unconventional raw material mixtures
Beverage Alcohol Industry
The production of fermented alcoholic drinks from starch and non-starch containing raw materials, like sugars, molasses, plant raw materials, wine and fruit mashes, have been practised for centuries. Starch is composed of long chains of glucose molecules and these have to be broken down into smaller molecules that the yeast can transform into alcohol. Our enzymes, used as processing aids, can carry out these liquefaction and saccharification processes. Starch containing cereals, in particular maize (corn), tend to be low in soluble nitrogen compounds. This results in poor yeast growth and in-creased fermentation time. Herefore the free amino nitrogen content can be regulated by protein splitting enzymes added to the mash to overcome this problem.
Wine Industry
Novozymes offers enzyme based solutions for producers of wine, grape juice and wine specialities, giving our customers a competitive advantage. Tailored solutions are offered for a number of applications as maceration (mash treatment) to release colour and aroma compounds, for clarification (must treatment) to speed up settling; for wine maturation - aroma liberation, wine stabilization and filtration.
Informations on market needs, process solutions and customer needed knowhow complete together with the relevant tailored enzymatic solutions the package offered to the winemakers in order to ensure or improve their final product quality and increase production flexibility.
Fruit & Vegetable Processing
Enzymatic solutions for extraction and modification of plant materials are needed to modify or break down plant cell walls in order to give e.g. higher juice yields and better process capacity, improved release of colour, aroma and other healthy components and clear or turbid stable juices and bever-ages.
Plant material is widely used for the production of valuable food and feed products. Many ingredients used for beverages, food and feed are produced by the extraction or modification of plant raw materi-als. Examples are proteins, starch and other polysaccharides (like pectins, gums, alginate, carra-geenan, agar, celluloses, hemicelluloses), sugars, juices (from fruit and vegetables), oils, flavours, colour components, phenolic and astringent components, and fibres. All these components are found intracellularly in plant material such as seeds, fruit and vegetables.
Our technologies include processing of fruit and vegetable juices using pectin enzyme preparations and fruit preparations using pectinesterase. Using the latter technology, fruit firming, it is possible to ensure the intact structure of fruit pieces in fruit preparations, which are incorporated into yoghurts, ice cream and pastries. Intact fruit pieces clearly improve the visual appearance and mouthfeel of these products. Moreover, our enzyme preparations are used in a number of applications: maceration, mashing, tailored customer specific fruit processing (called AFP = Advanced Fruit Processing), clarifi-cation, fining, filtration and stabilization of beverages.
Below are a few of our patents covering the Brewing area. If you are interested in one of the patents do not hesitate to contact us for more details.
Title of Technology: DNA construct comprising a DNA sequence encoding an enzyme with endoglucanase activity, which is especially useful in brewing proc-esses.
Abstract: The present technology relates to a DNA construct encoding an enzyme with endoglucanase activity, a method of producing the enzyme, an enzyme produced by the method, and an enzyme preparation containing the enzyme. DNA encoding an endoglucanase from Trichoderma harzianum is disclosed. The endoglucanase has activity toward mixed .beta.-1,3-1,4 glucans and is especially useful in brew-ing processes.
The novel DNA sequence from a strain of Trichoderma harzianum, encodes an endoglucanase enzyme the deduced amino acid sequence of which has been found to be without homology to any of the known endoglucanases (EC no. 3.2.1.4) or any other known enzymes. The DNA sequence shown in SEQ ID No. 1 (see US 5,817,499) encodes an enzyme which in the following disclosure is re ferred to as Endoglucanase I.
The technology relates also to an expression vector harbouring a DNA construct, a cell comprising the DNA construct or expression vector and a method of producing an enzyme with endoglucanase activity which method comprises culturing said cell under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
Furthermore, the technology relates to an enzyme with endoglucanase activity, which enzyme a) is encoded by a DNA construct as defined above, b) produced by the method, and/or c) is immunologically reactive with an antibody raised against a purified endoglucanase en-coded by the DNA sequence shown in SEQ ID No. 1 (see US 5,817,499) and derived from Trichoderma harzianum, CBS 243.71.
Moreover, the technology relates to an enzyme preparation compris-ing an enzyme and the use of the enzyme or enzyme preparation for various purposes in which modification or degradation of plant cell wall containing material is desirable.
Finally, the technology relates to an enzyme preparation useful for the degradation of plant cell wall components, said preparation being en-riched in an enzyme exhibiting endoglucanase activity as described above.
Detailed Description: The DNA sequence encoding an enzyme with endoglucanase activity may be isolated by a general method involving cloning, in suitable vectors, a DNA library from Trichoderma sp., transforming suitable yeast host cells with said vectors, culturing the host cells under suit-able conditions to express any enzyme of interest encoded by a clone in the DNA library, and screening for positive clones by determining any endoglucanase activity of the enzyme produced by such clones.
A more detailed description of this screening method is given in Example 1 in US 5,817,499 and in WO 93/11249.
The DNA sequence coding for the enzyme may for instance be isolated by screening a cDNA library of Trichoderma harzianum, e.g strain CBS 243.71, and selecting for clones expressing the appropriate enzyme activity . The appropriate DNA sequence may then be isolated from the clone by standard procedures, e.g. as described in Example 1 in US 5,817,499.
Alternatively, the DNA coding for an endoglucanase may, in accor-dance with well-known procedures, conveniently be isolated from DNA from any of the above mentioned organisms by use of oligonu-cleotide probes, such as 20mer probes, prepared on the basis of a DNA sequence shown in SEQ ID No. 1 (see US 5,817,499).
The DNA sequence may subsequently be inserted into a recombinant expression vector. This may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Al-ternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
In the vector, the DNA sequence encoding the endoglucanase should be operably connected to a suitable promoter and terminator sequence. The promoter may be any DNA sequence which shows tran-scriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
The host cell which is transformed with the DNA sequence encoding the enzyme is preferably a eukaryotic cell, in particular a fungal cell such as a yeast or filamentous fungal cell. In particular, the cell may belong to a specie s of Aspergillus or Trichoderma, most preferably Aspergillus oryzae or Aspergillus niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. The use of Aspergillus as a host microorganism is described in EP 238 023 (of Novo Nordisk A/S). The host cell may also be a yeast cell, e.g. a strain of Saccharomyces, in particular Sac-charomyces cerevisiae, Saccharomyces kluyveri or Saccharomyces uvarum, a strain of Schizosaccharomyces sp., such as Schizosaccharomyces pombe, a strain of Hansenula sp. Pichia sp., Yarrowia sp. such as Yarrowia lipolytica, or Kluyveromyces sp. such as Kluy-veromyces lactis.
The technology also relates to a method of producing an enzyme according to the technology, wherein a suitable host cell transformed with a DNA sequence encoding the enzyme is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.
The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question. The expressed endoglucanase may conveniently be secreted into the culture medium and may be recovered therefrom by wellknown procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
Furthermore, the technology relates to an enzyme preparation useful for the degradation or modification plant cell wall containing materials, said preparation being enriched in an enzyme with endoglucanase activity as described above.
The enzyme preparation having been enriched with an enzyme may e.g. be an enzyme preparation comprising multiple enzymatic activities, in particular an enzyme preparation comprising multiple plant cell wall degrading enzymes such as Pectinex.RTM., Pectinex Ultra SP.RTM., Celluclast or Celluzyme (all available from Novo Nordisk A/S). Alternatively, the enzyme preparation enriched in an enzyme with endoglucanase activity may be one which comprises an enzyme as the major enzymatic component, e.g. a mono-component enzyme preparation.
The additional enzyme(s) may be producible by means of a microor-ganism belonging to the genus Aspergillus, preferably Aspergillus ni-ger, Aspergillus aculeatus, Aspergillus awamori or Aspergillus oryzae.
The endoglucanase mentioned herein may also be produced essen-tially free from other plant cell wall degrading enzyme. This makes it possible to use the enzyme alone or together with other monocomponent enzymes to give the optimal combination of enzymes for a particular application. It is thereby possible to design enzyme combinations, which only degrade specific parts of the plant cell. This specific degradation has not previously been possible to obtain with commercially available cellulase, hemicellulase and/or pectinase preparations. The endoglucanase has been found to have a high specificity towards beta-glucan.
This activity towards mixed beta -1,3-1,4 glucans makes the endoglucanase and homologues thereof useful for brewing purposes as the enzymes degrades the barley betaglucan and thereby reduces the viscosity and improves the filterability of the wort. In brewing the high specificity for betaglucans is an advantage as compared to other en-doglucanases as the viscosity caused by betaglucan can be reduced without simultaneous degradation of the cellulose structures which are essential for the filterability of the wort where brewers spent grains act as filter-aid. Furthermore, the activity towards mixed beta-1,3-1,4 glucans makes the enzyme useful for processing of or for addition to food or feed to improve the feed-uptake and/or digestibility.
Furthermore, the endoglucanase may be used to improve the quality of baked products or other cereal products. The endoglucanase may also be used to produce oligosaccharides from e.g. plant material with mixed beta-1,3-1,4 glucan. The resulting oligosaccharides may be used as bulking agents in e.g. food. Furthermore, the endoglucanase may be used for extraction of aro-matic compounds from plant materials.
Glossary of Terms
| Term: |
Definition: |
| Derived from |
The term "derived from" is intended not only to indicate an endoglu-canase produced by strain CBS 243.71, but also an endoglucanase encoded by a DNA sequence isolated from strain CBS 243.71 and produced in a host organism transformed with said DNA sequence. |
| Analogue |
The term "analogue" used to define the DNA construct is understood to include any DNA sequence which encodes an enzyme with en-doglucanase activity and which is at least 70% homologous to the DNA sequence shown in SEQ ID No. 1 (see US 5,817,499) |
| Enriched |
The term "enriched" is intended to indicate that the endoglucanase activity of the enzyme preparation has been increased, e.g. with an enrichment factor of at least 1.1, conveniently due to addition of an enzyme prepared by the method described above. |
Patent References
| Patent Number: |
Title of Patent: |
Year of Issue: |
| US 5,817,499 |
DNA encoding an enzyme with endogluca-nase activity from Trichoderma harzianum |
1998
|
| WO 9502043 |
DNA encoding an enzyme with endogluca-nase activity from Trichoderma harzianum |
1995 (International publication year) |
Title of Technology: DNA construct comprising a DNA sequence encoding an enzyme with glucanase activity, which opens up the cells in a gently way, making large and porous pores in the cell walls.
Abstract: The technology relates to a novel enzyme exhibiting .beta.-1,3-glucanase activity. More specifically a DNA construct encoding the novel enzyme, an expression vector comprising said DNA construct, a cell comprising said DNA construct or said recombinant expression vector, a method of producing said novel enzyme, an enzyme prepa-ration comprising said novel enzyme, and use of the enzyme for deg-radation or modification of .beta.-glucan containing material.
By using the novel .beta.-1,3-glucanase to get access to the desired intercellular component facilitates the recovery process. The novel enzyme opens up the cells in a gently way, which involves making large and porous pores in the cell walls. This will further lead to a reduced amount of contaminants.
The gene encoding the novel .beta.-1,3-glucanase is expressed in a suitable heterologous host cell. Specifically in a strain of Bacillus subtilis, suitable for large scale industrial production. The yield of the novel .beta.-1,3-glucanase is increased in comparison to the parent cell from which the gene originates.
Detailed Description: The DNA sequence encoding an enzyme exhibiting .beta.-1,3-glucanase activity may be isolated by a general method involving cloning, in suitable vectors, a DNA construct from Oerskovia xanthineolytica LLG109, transforming suitable host cells with said vectors, culturing the host cells under suitable conditions to express any en-zyme of interest, creening for positive clones by determining any .beta.-1,3-glucanase activity of the enzyme produced by such clones, selection of clones, and isolating the enzyme encoding DNA from such clones.
The general method is further disclosed in WO 93/11249. A more detailed description of the method is given below.
The DNA sequence coding for the enzyme may for instance be isolated from Oerskovia xanthineolytica strain LLG109 and selecting for clones expressing the appropriate enzyme activity.
The DNA construct comprises a DNA sequence, which a) comprises the DNA sequence shown in SEQ ID No. 1 (see US 5,919,688), or b) comprises an analogue of the DNA sequence shown in SEQ ID No. 1 (see US 5,919,688), which i) hybridizes with the same oligonucleotide probe as the DNA sequence shown in SEQ ID No. 1 (see US 5,919,688), and ii) encodes a polypeptide which is homologous with the polypeptide encoded by a DNA sequence com-prising the DNA sequence shown in SEQ ID No. 1 (see US 5,919,688), or iii) encodes a polypeptide which is immunologically reactive with an antibody raised against a purified .beta.-1,3-glucanase encoded by the DNA sequence shown in SEQ ID No. 1 (see US 5,919,688) derived from Oerskovia xanthineolytica LLG109.
A preferred method of amplifying specific DNA sequences are by the use of polymerase chain reaction (PCR) using degenerate oligonucleotide probes synthesized. For instance, the PCR may be carried out using the techniques described in U.S. Pat. No. 4,683,202 or by R. K. Saiki et al. (1988), Science, 239, 487-491.
It is expected that a DNA sequence coding for a homologous enzyme, i.e. an analogous DNA sequence, is obtainable from other microorganisms. For instance, the DNA sequence may be derived from another microorganism, in particular either a fungus or bacteria.
Such DNA sequences may originates from fungi, comprising a strain of an Aspergillus sp., in particular a strain of A. aculeatus or A. niger, a strain of Trichoderma sp., in particular a strain of T. reesie, T. viride, T. longibrachiatum or T. koningii, T. harzianum or a strain of a Fusarium sp., in particular a strain of F. oxysporum, or a strain of a Humicola sp.. Further a DNA sequence encoding a homologous enzyme may be expected to derive from bacteria, such as another strain of a Oerskovia sp., or a strain of an Arthrobacter sp., Cytophaga sp., Rhodothermus sp., in particular a strain of Rh. marinus, or a strain of a Clostrium, in particular strains of Cl. thermocellum, or a strain of Bacillus sp., in particular strains of B. lichenifornis., B. amyloliquefaciens, or B. circulars.
Alternatively, the DNA coding for a .beta.-1,3-glucanase may, in ac-cordance with well-known procedures, conveniently be isolated from DNA from any of the above mentioned organisms by use of synthetic oligonucleotide probes prepared on the basis of a DNA sequence dis-closed herein. For instance, a suitable oligonucleotide probe may be prepared on the basis of a partial nucleotide sequence of the se-quence shown in SEQ ID No. 1.
The DNA sequence may subsequently be inserted into a recombinant expression vector. This may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Al-ternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
In the vector, the DNA sequence encoding the .beta.-1,3-glucanase should be operably connected to a suitable promoter and terminator sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
It is preferred to use a vector under control of the promoter for the maltogenic .beta.-1,3-amylase from Bacillus stearothermophilus and/or the signal of Bacillus stearothermphilus.
The host cell which is transformed with the DNA sequence encoding the enzyme may be either eukaryotic cells or prokaryotic cells.
Suitable prokaryotic host cells are in particular bacterial cells. Examples of such bacterial host cells which, on cultivation, are capable of producing the novel enzyme are grampositive bacteria such as strains of Bacillus, such as strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium or B. thuringiensis, or strains of Streptomyces, such as S. lividans or S. murinus, or gramnegative bacteria such as Escherichia coli. The transformation of the bacteria may be effected by protoplast transformation or by using competent cells in a manner known per se (cf. Sambrook et al., supra, 1989).
When expressing the enzyme in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the polypeptide is refolded by diluting the denaturing agent. In the latter case, the polypeptide may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the poypeptide.
Suitable eukaryotic cells are in particular fungal cells such as yeasts or filamentous fungal cells.
In a still further aspect, the technology relates to a method of producing an enzyme, wherein a suitable host cell transformed with a DNA sequence encoding the enzyme is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.
The medium used to culture the cells may be any conventional me-dium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Col-lection).
The expressed .beta.-1,3-glucanase produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the type of polypeptide in question.
The present technology also relates to a novel enzyme preparation useful for the modification or degradation of .beta.-glucan containing materials, said preparation being enriched in an enzyme exhibiting .beta.-1,3-glucanase activity as described above.
The enzyme preparation having been enriched with the novel .beta.-1,3-glucanase enzyme may e.g. be an enzyme preparation compris-ing multiple enzymatic activities, in particular an enzyme preparation comprising different enzyme activities required for the modification or degradation of microbial cell walls.
Alternatively, the enzyme preparation enriched in an enzyme exhibit-ing .beta.-1,3,-glucanase activity may be one which comprises an enzyme as the major enzymatic component, e.g. a monocomponent enzyme composition.
The enzyme preparation may be prepared in accordance with meth-ods known in the art and may be in the form of a liquid or a dry preparation. For instance, the enzyme preparation may be in the form of a granulate or a microgranulate. The enzyme to be included in the preparation may be stabilized in accordance with methods known in the art.
The enzyme preparation may, in addition to a .beta.-1,3-glucanase, contain one or more other cell wall degrading enzymes, for instance those with cellulytic, mannanolytic, chitinolytic or proteolytic activities such as endo- and exoglucanase, mannanase, endo- and exo- chiti-nase, protease, or .alpha.- or .beta.-mannosidase.
The enzyme preparation described herein is preferably used as an agent for degradation or modification of .beta.-glucan containing ma-terial such as microbial cell walls. In particular, the enzyme prepara-tion may be used for rupturing or lysing cell walls of microorganisms thereby enabling recovery of desirable products produced by the mi-croorganism.
It will be understood that the specific composition of the enzyme pre-paration to be used should be adapted to the composition of the cell wall to be ruptured or lysed. For instance, yeast cell walls have been found to comprise two main layers, an outer layer of protein-mannan complex and an inner glucan layer. In order to efficiently rupturing the cell wall of yeast it is desirable that the enzyme preparation comprises at least protease, mannanase and .beta.-glucanase activity.
The extract recovered after rupture of the microbial cell walls normally comprises a number of different components, such as pigments, vitamins, colorants and flavourants. Extracts obtained from rupture of yeast, i.e. yeast extracts, are used as such, e.g. for food or feed applications or components thereof may be recovered and optionally further processed.
Examples of such products include vitamins, colorants (e.g. carotenoids, Q-10 and astaxanthin), enzymes, proteins and flavour components or flavour enhancers (e.g. MSG, 5'-GMP and 5'-EMP). The products to be recovered may be inherent products of the microo rganism in question, or may be products which the microorganism has been constructed to produce, e.g. recombinant products.
In addition the enzyme preparation may be used in the production of protoplasts from yeasts (e.g. of Saccharomyces sp. or Schizosaccharomyces sp.) or from fungi (e.g. Aspergillus sp. or Penicillium sp.). Preparation and regeneration of protoplast from such organisms are important in fusion, transformation and cloning studies. The production of protoplasts may be performed in accordance with methods known in the art.
The technology may also be used for improving fungal transformation.
Further, the enzyme or enzyme preparation described herein may be used in the preparation of pharmaceuticals, especially products entrapped inside the cells in the cytoplasmic membrane, the peripiasmic space and/or the cell wall.
In addition, the enzyme preparation may be used in the modification of .beta.-glucans such as curdlan and laminarin.
Glossary of Terms
| Term: |
Definition: |
| Enriched |
The term "enriched" is intended to indicate that the .beta.-1,3-glucanase activity of the enzyme preparation has been increased, e.g. with an enrichment factor of at least 1.1, conveniently due to addition of an enzyme described herein prepared by the method described above. |
Patent References
| Patent or Application Number: |
Title of Patent: |
Year of Issue: |
| US 5,919,688 |
Enzyme with B-1, 3-glucanase activity |
1999 |
| WO 9612013 |
A novel enzyme with beta-1,3-glucanase activity |
1996 (International publication year) |
