Fused protein containing plant cell wall-destroying enzymes and use thereof

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Disclosed is a cell wall-destroying fused protein containing feruloyl esterase and xylanase, which do not contain a C-terminal hydrocarbon-binding molecule (CBM). If necessary, said fused protein contains CBM from a third enzyme, wherein the enzymes and CBM are recombinant proteins corresponding to native fungal proteins. Also disclosed is use of said fused protein to realise methods of destroying plant cell walls when producing compounds of interest from plants or plant by-products. The invention also discloses nucleic acid which codes said fused protein, an expression vector transformed by said nucleic acid and a host cell containing said vector. Described is a method of producing said fused proteins, involving culturing host cells, extraction and, if necessary, cleaning the fused proteins produced by said host cells in the culture. Disclosed is a method of producing desired compounds from plant products, comprising the following steps: 1) enzymatic treatment of plant products with fused proteins or transformed fungus cells, 2) if necessary, treating plant products with steam combined with the action of fused proteins, 3) if necessary, biotransformation of compounds extracted from cell walls during said enzymatic treatment, 4) extraction and, if necessary, cleaning the desired compounds.

EFFECT: invention increases the effect of destroying plant cell walls.

24 cl, 5 dwg, 2 tbl

 

The present invention relates to the design and sverginate genetically engineered multifunctional enzymes that destroy the cell wall of plants, and their applications as enhanced enzymatic tool to increase value to agricultural by-products.

Agricultural residues are a major renewable resource for the bioconversion of lignocellulose. Daily production food industry generates a large number of such products that are polluting waste and must be destroyed. Many studies aimed at increasing the value of these products in the biotechnology sector. Among the valuable compounds ferulic acid (4-hydroxy-3-methoxy-cinnamic acid) is a very attractive phenolic compound which is known that it is the most common hydroxyurea acid in the plant world. Ferulic acid, for example, can be used as an antioxidant (23) or converted by bacteria in natural vanilla, which is an expensive additive in the food, cosmetic and pharmaceutical industries (4, 26). Among agricultural products corn and Pshenichny the e bran are potential substrates due to the presence of a large number of ferulic acid in their cell wall 3% and 1% (weight/weight), respectively (43). In plant physiology ferulic acid is the main structural component, which has important physical and chemical properties of the cell wall of plants. In particular, it can act as a crosslinking agent between lignin and hydrocarbons or between hydrocarbons (16), affecting the integrity of the cell membrane and thereby reducing the degree of biological destruction caused by microbial enzymes (6).

Microorganisms have developed enzymes, such as feruloylated (EC 3.1.1.73), capable of the hydrolysis of ester bonds between ferulic acid and polysaccharides of the cell wall of plants. These enzymes improve accessibility to other lignocellulosic enzymes to the polysaccharide backbone (as an overview, see 12). Previous studies show that feruloylated act synergistically with destroying the main polysaccharide chain enzymes, such as β-(1,4)-endoxylanase, increasing yield of ferulic acid from cell membranes of plants and forming it in a quantity sufficient for several applications (2, 15, 50). Filamentous fungi, such asAspergillus nigerare well known producers of enzymes that destroy the cell wall of plants. Two different gene coding feruloylated fromA. nigeralready have a clone of the levels (10, 11), and the corresponding recombinant proteins sverhnegativny inPichia pastoris and A. niger(24, 30, 41). Several feruloylated of fungi were purified and characterized (18, 47), but other genes have not been cloned. In the previous work described receiving fromPenicillium funiculosumthe first cinnamon esterase mushrooms (type B) with C-terminal domain is most similar to group 1 uglevodorodokislyayuschih modules (Carbohydraye-Binding-Module, CBM) (27). A large number helicoiling hydrolases of anaerobic and aerobic microorganisms have a modular structure. In addition to the catalytic domain of one or more non-catalytic CBM can be located either at the N-terminal region or the C-terminal region, or in both areas. CBM have been classified into families based on their amino acid sequence and three-dimensional structure (http://afmb.cnrs-mrs.fr/CAZY/index.html). The main function of CBM is the destruction of the insoluble substrate (45). For example, they are responsible for maintaining the location of the catalytic domain in proximity to the substrate, increasing the duration and closeness of their contact. Moreover, in some cases, CBM can also change microfibrillar cellulose structure, weakening the hydrogen bonds together the cellulose chains(13, 14, 33).

In the present invention contains considerations about the synergistic effect between the individual and merged by the enzymes of fungi (such as A. niger)disrupting the cell wall of plants. In a recent study, based on genetically engineered bacterial cellulosome, the physical proximity of the two catalyst components were allowed to observe increased synergies in relation to evolutionary conservative substrates (17). Based on this, the authors have constructed a chimeric binding protein mushroom feruloylated with clostridiales doctrinalism domain, which together with another enzyme were transplanted into the bacterial CBM-containing anchor protein (31). However, the yield of recombinant protein was not high enough to test applications on an industrial scale, and in this regard was the introduction of the new strategy. In this work the authors have done a hybrid (protein) of the two fungal enzymes: feruloylated A (FAEA) and xylanase B (XYNB) fromA. nigerseparated hyperglycosylated of the linker peptide, the formation of a bifunctional enzyme (FLX), which has increased efficiency in the allocation of ferulic acid. In addition, the inventors have added a second construct containing CBM mushrooms fromA. nigercellobiohydrolase B (CBHB) at the C-terminal site of the specified bifunctional enzyme (FLXLC). Both hybrid enzyme were successfully synthesized inA. nigerand fully described in relation to biochemical and kinetics is their aspects, and also used to produce ferulic acid from natural substrates: corn and wheat bran. The aim of this work was to compare the efficiency of the allocation of ferulic acid using a separate or fused enzymes for studies of enzymatic synergies that occur due to the physical proximity of the enzymes of fungi. Moreover, in the design FLXLC was investigated the effect of joining CBM to the C-terminal part of the bifunctional enzyme.

The present invention is based on the demonstration of a synergistic effect from merging into one chimeric protein enzymes that destroy the cell wall of plants, when these enzymes do not contain a CBM, when compared with the use of single enzymes that destroy the cell wall of plants.

Thus, the main purpose of the present invention is the creation of new fused proteins, including not containing RAS-domain enzymes that destroy the cell wall of plants.

Another objective of the present invention is to develop a new method of obtaining interest compounds associated with the membranes of plant cells by use of these fused proteins in plants and mainly on agricultural by-products as substrates.

The present invention Rel is referring to the use of fused proteins comprising at least two enzymes that destroy the membranes of plant cells, and these enzymes do not contain uglevodorodokislyayuschih molecules (Carbohydraye-Binding-Molecule, SVM) in the C-terminal region, and additionally RAS; these enzymes and CBM are recombinant proteins corresponding native proteins in fungi or their mutated forms. Slit proteins are used in the processes of destruction of plant cell membranes in the framework of the preparation of plants or vegetal by-products, the target compounds in the membrane of plant cells, or bleaching paper pulp and paper.

The expression of the enzymes that destroy plant cell membrane" refers to enzymes that are capable of splitting components of cell membranes, such as cellulose, hemicellulose and lignin. Deplete membrane of plant cells the enzymes in these fused proteins are the same or different from one another.

The expression "C-terminal uglevodorodnaya molecule" refers to a molecule that has affinity to the cellulose, which directs the associated enzyme to the cellulose.

In more detail, the invention relates to defined above application, in which the enzymes that destroy plant cell membrane and not containing CBM, I is controlled by hydrolases, choose from:

- cellulases, such as cellobiohydrolase, endoglucanase and ekzoplanety or β-glucosidase,

- hemicellulase, such as xylanase,

- lignins able to destroy lignins, such as laccase, manganese peroxidase, lignin peroxidase, multilateral peroxidase, or auxiliary enzymes, such as cellobiose of deshydrogenase and aryl alcohol oxidase,

- hydrolases cinnamic esters, are able to select cinnamic acids, such as ferulic acid and hydrolyze communication diferuloyl acid between chains of hemicelluloses, such as feruloylated, cinnamon esterase and hydrolases chlorogenic acid.

In more detail, the invention relates defined above application, in which the enzymes that destroy plant cell membrane and not containing CBM, choose from feruloylated and xylanases.

In more detail, the invention relates to defined above application, in which the enzymes that destroy plant cell membrane and does not contain a CBM correspond to native enzymes, or their mutated forms, mushrooms, chosen from:

* records of Ascomycetes, such as strains ofAspergillusand , more specificallyAspergillus niger,

* basidiomycetes, such as strains ofPycnoporusorHalociphinaand , more specificallyPycnoporus cinnabarinus, Pycnoporus sanguineusorHalociphina villosa.

Over concr the IDT, the invention relates defined above application, in which the enzymes that destroy plant cell membrane and does not contain a CBM correspond to native enzymes, or their mutated forms, from strains ofAsperagillussuch asAsperagillus niger.

More specifically, the invention relates to defined above application, in which at least one of the enzymes that destroy plant cell wall, is feruloylated chosen from:

- feruloylated AndA. nigerrepresented by SEQ ID NO: 2, encoded by a nucleic acid represented by SEQ ID NO: 1,

- feruloylated fromA. nigerrepresented by SEQ ID NO: 4, encoded by a nucleic acid represented by SEQ ID NO: 3.

More specifically, the invention relates to defined above application, in which at least one of the enzymes that destroy plant cell wall, is a xylanase defined above, such as xylanase from A. niger represented by SEQ ID NO: 6, encoded by a nucleic acid represented by SEQ ID NO: 5.

More specifically, the invention relates defined above application, in which the enzymes that destroy plant cell membrane and does not contain a RAS correspond to native enzymes, or their mutated forms, mushrooms, selected from the records of Ascomycetes, such strains as Asperagillus and more specifically, Asperagillus niger.

SVM, which can be used in accordance with the present invention, belong to group 1.

More specifically, the invention relates defined above application, in which RAS is a RAS present in cellobiohydrolase from A. niger, and represented by SEQ ID NO: 8 encoded by a nucleic acid represented by SEQ ID NO: 7.

The invention also relates to a specific higher use of fused proteins containing linkers between at least two proteins containing these fused proteins, and these linkers are polypeptides ranging in length from 10 to 100 amino acids, predominantly about 50 amino acids.

More specifically, the invention relates defined above application of the fused protein in which the linker inserted between each of the proteins in the composition of these fused proteins.

More specifically, the invention relates defined above application, in which the linker is hyperglycosylated polypeptide, such as having the sequence represented by SEQ ID NO: 10, contained in cellobiohydrolase from A. niger and encoded by a nucleic acid represented by SEQ ID NO: 9.

More specifically, the invention also relates to a specific higher use of fused proteins between feruloylated and xylanase and advanced RAS.

More specific is about, the invention relates defined above application of fused proteins, including feruloylated And of A. niger represented by SEQ ID NO: 4, or feruloylated In a of A. niger represented by SEQ ID NO: 4, and the xylanase from A. niger represented by SEQ ID NO: 6.

More specifically, the invention relates to a specific higher use of fused protein comprising feruloylated And of A. niger represented by SEQ ID NO: 2, and the xylanase from A. niger represented by SEQ ID NO: 6, and the specified protein contains the sequence represented by SEQ ID NO: 10 as hyperglycosylated linker between the two primary proteins, and represented by SEQ ID NO: 12.

The invention relates also to a certain higher use of fused proteins, including feruloylated And of A. niger represented by SEQ ID NO: 2, a xylanase from A. niger represented by SEQ ID NO: 6, and the CBM represented by SEQ ID NO: 8 contained in cellobiohydrolase from A. niger.

More specifically, the invention relates defined above application of the fused protein comprising feruloylated And of A. niger represented by SEQ ID NO: 2, a xylanase from A. niger represented by SEQ ID NO: 6, and the CBM represented by SEQ ID NO: 8, and the specified protein contains the sequence represented by SEQ ID NO: 10 as hyperglycosylated linker between each of the three primary proteins, and presents SEQ ID NO: 14.

More con is specific, the invention relates to a specific higher use of fused protein comprising feruloylated In a of A. niger represented by SEQ ID NO: 4, and the xylanase from A. niger represented by SEQ ID NO: 6, and the specified protein contains the sequence represented by SEQ ID NO: 10 as hyperglycosylated linker between the two primary proteins, and represented by SEQ ID NO: 16.

More specifically, the invention relates defined above application of fused proteins, including feruloylated In a of A. niger represented by SEQ ID NO: 4, a xylanase from A. niger represented by SEQ ID NO: 6, and the CBM represented by SEQ ID NO: 8 contained in cellobiohydrolase from A. niger.

More specifically, the invention relates to a specific higher use of fused protein comprising feruloylated In a of A. niger represented by SEQ ID NO: 4, a xylanase from A. niger represented by SEQ ID NO: 6, and the CBM represented by SEQ ID NO: 8, and the specified protein contains the sequence represented by SEQ ID NO: 10 as hyperglycosylated linker between each of the three initial protein and represented by SEQ ID NO: 18.

The invention also relates to a specific above application for the implementation of the methods of destruction of plant cell membranes within the following interest connections:

- bioethanol,

- antioxidants, such as fer the gross acid or caffeic acid, related to cinnamic acid and hydroxytyrosol, or gallium acid,

- flavoring additives, such as vanillin or p-hydroxybenzaldehyde obtained by biotransformation of ferulic acid or p-coumarin acid, respectively,

- or bleaching paper pulp and paper.

The invention also relates to a specific higher applications in which these fused proteins are directly added to plants or plant byproducts as substrates or are secreted by the fungal cell, transformed with nucleic acids encoding these fused proteins, such as mushrooms, above, and particularlyA. nigerandPycnoporus cinnabarinus; these mushrooms are in direct contact with these plants or plant byproducts as substrates.

The invention also relates to a method of destruction of plant cell membranes to obtain from plants and plant by-products of the target compounds present in the membranes of plant cells, characterized in that it comprises the following stages:

- enzymatic treatment of plants or vegetal by-products and industrial wastes fused proteins defined above, or transformed cells of fungi, defined above,

- updat the additional physical steam treatment plants or plant products in combination with the action of fused proteins,

additionally biotransformation of compounds that are released from cell membranes in the process of the above-mentioned enzymatic processing, using the appropriate microorganisms or enzymes

- selection and, if necessary, cleaning of interesting compounds that are released from cell membranes during the indicated enzyme treatment or received on the specified stage biotransformation.

Preferably the plants treated fused proteins in the method according to the invention, are selected from sugar beet, wheat, corn, rice, or from any of the trees used in the paper industry.

Preferably vegetable by-products, or industrial waste, treated fused proteins in the method according to the invention, are selected from wheat straw, corn bran, wheat bran, rice bran, Apple husks, coffee husks, coffee by-products, waste water olive pressing.

More specifically, the invention relates defined above method of obtaining antioxidants as target compounds, including

- treatment of plants or vegetal by-products fused proteins containing at least two of the following damaging the cell wall enzymes that do not contain CBM: feruloylated And feruloylated the SHL, hydrolases chlorogenic acid, xylanase, in which the protein is preferably selected from feruloylated And-xylanase, feruloylated In-xylanase, feruloylated And-feruloylated And-xylanase, hydrolases chlorogenic acid - xylanase.

- selection and, if necessary, cleaning of the antioxidants that are released from the cell walls of these plants or plant products.

More specifically, the invention relates to the method defined above, obtaining cinnamic acids, such as ferulic acid as interesting antioxidant, which used protein contains feruloylated and xylanase and may contain CBM, as defined above, and, in particular, selected from SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18.

Mainly in the framework of obtaining antioxidants such as ferulic acid, plants, processed above fused proteins, which are selected from the following group: sugar beet, wheat, corn, rice, or vegetable by-products or industrial waste processed by the above-described fused proteins, which are selected from the following group: wheat straw, corn bran, wheat bran, rice bran, Apple bagasse, coffee husk, coffee by-products, waste water from olive pressing.

Izobreteny is also relates to a method, defined above, to obtain a flavoring additives as target compounds, including:

- treatment of plants or vegetal by-products fused proteins used in the framework of obtaining antioxidants, as defined above,

the biotransformation of compounds secreted from the cell membranes at the previous stage using the contact of these compounds with uncertain enzymes produced by microorganisms, selected from the records of Ascomycetes or basidiomycetes, such asA. nigerorP. Cinnabarinusaccordingly,

- selection and, if necessary, clean flavour, obtained at the previous stage biotransformation.

More specifically, the invention relates to the method defined above, obtaining vanillin as the target, flavour, which used a protein selected from proteins, used to obtain ferulic acid as defined above, and the stage biotransformation carried out by bringing into contact the ferule acid released from cell membranes, with uncertain enzymes produced by the records of Ascomycetes or basidiomycetes, such asA. nigerorP. Cinnabarinusrespectively.

Preferably plants and plant by-products or industrial waste used in RA is Kah obtain the flavoring such as vanilla, selected from those mentioned above to obtain antioxidants.

The invention also relates to a method as defined above, for ethanol production as the target compounds, including:

- treatment of plants or vegetal by-products fused proteins containing at least two destructive plant cell wall enzyme, which do not have the CBM from the following group: cellulase, hemicellulase, esterase, a laccase, peroxidase, arylalkyl oxidase and protein preferably selected from endoglucanase-ekzoplanety, laccase-xylanase, xylanase-cellulase (endo - or ekzoplanety), it is preferable that this treatment was combined with physical treatment of these plants or plant products,

the biotransformation of treated plants or plant by-products obtained in the previous phase, in formatiruem sugar using fused proteins described above, or transformed fungi, secreting fused proteins, in combination with enzymes selected from cellulases, hemicellulase, or esterase, or microorganisms, selected from the records of Ascomycetes, such asA. nigerorTrichoderma reesei.

the biotransformation formatiruem sugars into ethanol using yeast.

More specifically, the image is a buy above refers to a specific method of obtaining formatiruem sugars to produce ethanol, in which the protein is selected from endoglucanase-ekzoplanety, laccase-xylanase, xylanase-cellulase (endo-or ekzoplanety).

Mostly plants and plant by-products or industrial waste used in the framework for ethanol production, choose from the following: wood, annual plants or agricultural by-products.

The invention also relates to a method of bleaching paper pulp and paper, including:

chemical and physical treatment of plants or vegetal by-products in combination with fused proteins containing at least two destructive plant cell wall enzyme, which do not have the CBM from the following group: feruloylated And feruloylated, xylanase, laccase, arylalkyl oxidase, manganese peroxidase, lignin peroxidase, versatilely peroxidase or cellobiose dehydrogenase.

additional softening of the treated plants or plant products, obtained on the previous stage, transformed mushrooms, secreting fused proteins containing at least two destructive plant cell wall enzyme, which do not have the CBM from the following group: feruloylated And feruloylated, xylanase, laccase, arylalkyl oxidase, peroxidase manganese, p is oxidase lignin, versatilely peroxidase or cellobiose dehydrogenase;

- bitalian of treated plants or plant by-products obtained in the previous stage, transformed mushrooms, secreting fused proteins containing at least two destructive plant cell wall enzyme, which do not have the CBM from the following group: feruloylated And feruloylated, xylanase, laccase, arylalkyl oxidase, manganese peroxidase, lignin peroxidase, versatilely peroxidase or cellobiose dehydrogenase.

The invention more specifically relates to a method as described above, the bleaching of pulp and paper, in which the protein used in the first stage of processing plants and plant products, choose from feruloylated And-xylanase, feruloylated In-xylanase, feruloylated And-feruloylated And-xylanase, feruloylated And-feruloylated In-xylanase, laccase-xylanase, arylalkyl xylanase-peroxidase manganese; protein, secretory transformed mushrooms and used under birthmate, choose from: feruloylated And-xylanase, feruloylated In-xylanase, feruloylated And-feruloylated And-xylanase, feruloylated And-feruloylated In-xylanase, laccase-xylanase, arylalkyl xylanases is-peroxidase manganese, overproducing P. Cinnabarinus or A. niger, and protein, used under bootblue, choose from: feruloylated And-xylanase, feruloylated In-xylanase, feruloylated And-feruloylated And-xylanase, feruloylated And-feruloylated In-xylanase, laccase-xylanase, arylalkyl xylanase-peroxidase manganese.

The invention also relates to fused proteins comprising at least two destructive plant cell wall enzyme, which do not contain C-terminal uglevodorodokislyayuschih molecule (CBM) and advanced RAS, these enzymes and CBM are recombinant proteins corresponding native proteins in fungi or their mutated forms, as defined above.

More specifically, the invention relates to fused proteins, as defined above, comprising linkers between at least two of the proteins in these fused proteins, and these linkers defined above.

More specifically, the invention relates to fused proteins, as defined above, including feruloylated and xylanase and advanced RAS.

The invention more specifically relates to fused proteins as defined above, including feruloylated And of A. niger represented by SEQ ID NO: 2, or feruloylated In a of A. niger represented by SEQ ID NO: 4, and the xylanase from A. niger represented by SEQ ID NO: 6./p>

The invention more specifically relates to fused proteins, as defined above, including feruloylated And of A. niger represented by SEQ ID NO: 2 and a xylanase from A. niger represented by SEQ ID NO: 6, and these fused proteins contain sequence represented by SEQ ID NO: 10 as hyperglycosylated linker between the two primary proteins, and represented by SEQ ID NO: 12.

The invention also relates to fused proteins, as defined above, including feruloylated And of A. niger represented by SEQ ID NO: 2, a xylanase from A. niger represented by SEQ ID NO: 6, and the CBM represented by SEQ ID NO: 8 contained in cellobiohydrolase from A. niger.

The invention more specifically relates to fused proteins, as defined above, including feruloylated And of A. niger represented by SEQ ID NO: 2 and a xylanase from A. niger represented by SEQ ID NO: 6, and the CBM represented by SEQ ID NO: 8, and these integral proteins contain sequence represented by SEQ ID NO: 10 as hyperglycosylated linker between each of the three primary proteins, and presents SEQ ID NO: 14.

The invention more specifically relates to fused proteins, as defined above, including feruloylated In a of A. niger represented by SEQ ID NO: 4, and the xylanase from A. niger represented by SEQ ID NO: 6, and these fused proteins contain sequence represented by SEQ ID NO: 10 in quality is TBE hyperglycosylated linker between the two primary proteins, and represented by SEQ ID NO: 16.

The invention also relates to fused proteins, as defined above, including feruloylated In a of A. niger represented by SEQ ID NO: 4, a xylanase from A. niger represented by SEQ ID NO: 6, and the CBM represented by SEQ ID NO: 8 contained in cellobiohydrolase from A. niger.

The invention more specifically relates to a fused protein, as defined above, including feruloylated In a of A. niger represented by SEQ ID NO: 4 and a xylanase from A. niger represented by SEQ ID NO: 6, and the CBM represented by SEQ ID NO: 8, and these fused proteins contain sequence represented by SEQ ID NO: 10 as hyperglycosylated linker between each of the three primary proteins, and presents SEQ ID NO: 18.

The invention also relates to nucleic acids coding for a protein defined above.

The invention more specifically relates to nucleic acids represented by SEQ ID NO: 11, 13, 15 and 17, the encoding fused protein represented by SEQ ID NO: 12, 14, 16 and 18, respectively.

The invention also relates to nucleic acids represented by SEQ ID NO: 19 and 21 corresponding to SEQ ID NO: 11 and 13, where the sequence of SEQ ID NO: 1 is replaced by the sequence SEQ ID NO: 23 encoding a pre-feruloylated And corresponding to SEQ ID NO: 24, and these nucleic acid SEQ ID NO: 19 and 21 encode pre-proteins fused proteins, the relevant PEFC is deuternomy SEQ ID NO: 20 and 22.

The invention also relates to nucleic acids represented by SEQ ID NO: 25 and 27 corresponding to SEQ ID NO: 15 and 17, where the sequence of SEQ ID NO: 3 is replaced by the sequence SEQ ID NO: 29, encoding the pre-feruloylated In corresponding to SEQ ID NO: 30, and these nucleic acid SEQ ID NO: 25 and 27 encode pre-proteins fused proteins, corresponding to the sequences SEQ ID NO: 20 and 22.

The invention also relates to vectors, such as pAN 52.3 transformed by the nucleic acid defined above.

The invention also relates to cells of the host, such as records of Ascomycetes or basidiomycetes, and more specificallyA. nigerandP. Cinnabarinustransformed by the nucleic acid defined above, using the above vectors.

The invention also relates to a method for producing a fused protein as defined above, including in vitro culture of host cells as defined above, allocation and, if necessary, clean the slit proteins produced by these cells-host culture.

The invention is also illustrated by the following detailed description of the production and properties of chimeric enzymes FLX (SEQ ID NO: 12) and FLXLC (SEQ ID NO: 14).

Two chimeric enzymes were constructed and successfully sverhnegativny inAspergillus niger. Design FLX consists of sequences encoding feruloylated is A (FAEA), connected with endonuclease (XYNB) from A. niger. C-terminal hydrocarbon-binding module (CBM family 1) was attached to FLX, forming a second hybrid enzyme: FLXLC. Between each of the proteins was inserted by the linker to stabilize the structure. Hybrid proteins were purified to homogeneity, and assessed their molecular masses: 72 kDa and 97 kDa for FLX and FLXLC respectively. The integrity of the hybrid enzymes was checked by immunodeciencies, which showed a single form using antibodies to FAEA and polyhistidine label. Physico-chemical properties of each of the catalytic module of these bifunctional enzymes corresponded to the properties of the individual enzymes. In addition, the authors it has been verified that FLXLC showed high affinity towards microcrystalline cellulose (Avicel) with binding parameters corresponding to Kdto 9.9×10-8M for the dissociation constants and 0.98 µmol/g cellulose for binding capacity. Both bifunctional enzyme were investigated to determine their ability to release ferulic acid from natural substrates: corn and wheat bran. Compared with the individual enzymes FAEA and XYNB was received higher synergistic effect when using FLX and FLXLC for both substrates. Moreover, synergies excretion of ferulic acid was increased to FLLC compared with FLX using corn bran as a substrate, confirming the positive role of the CBM. In conclusion, these results show that the formation of composite bifunctional enzymes from natural individual cell wall hydrolases and CBM from A. niger can increase the synergistic effect of the destruction complex substrates.

Materials and methods

Strains and culture medium

The Escherichia coli strain JM109 (Promega) was used for design and cultivation vectors, whileA. nigerstrain D15#26 (pyrg-) (22) - for the production of recombinant proteins. After co-transformation vectors containing the pyrG gene and the expression cassette FLX or FLXLC respectively (figure 1),A. nigerwere grown for selection on solid minimal medium (without uridine), containing 70 mm NaNO3, 7 mm KCl, 11 mm KH2HPO4, 2 mm MgSO4, 1% glucose (wt/vol), and trace elements [1000x blend: 76 mm ZnSO4, 25 mm MnCl2, 18 mm FeSO4, 7.1 mm CoCl2, 6.4 mm CuSO4, 6.2 mm Na2MoO4, 174 mm ethylenediaminetetraacetic acid (EDTA)]. For screening of transformants for the production of recombinant proteins in a liquid medium spores at a concentration of 1×106/ml were seeded in 100 ml of culture medium containing 70 mm NaNO3, 7 mm KCl, 200 mm Na2HPO4, 2 mm MgSO4a 6% glucose (wt/vol) and trace elements in a 500 ml flask with baffles. For isolation of genomic DNA using what was little more than a strain BRFM131 (Banque de Ressources Fongiques de Marseille), and the resulting DNA was used as template for PCR amplification strategy linker-CBM sequence.

Construction of expression vectors and transformation of fungi

The fusion of sequences encoding FAEA (SEQ ID NO: 19; Y09330), XYNB (SEQ ID NO: 5; AYl 26481), and CBM (SEQ ID NO: 7; AFl 56269) fromA. Nigerproduced by the method of PCR with overlapping elongation (41)using 5'-GGACTCATGAAGCAATTCTCTGCAAAATAC-3' (BspHl) as a forward primer and 5'-ACTGGAGTAAGTCGAGCCCCAAGTACAAGCTCCGCT-3' as reverse primer. Genomic DNA encoding a linker region CBHB (SEQ ID NO: 9; AF156269)was obtained by amplification of the strain BRFM131A. nigerusing the direct primer 5'-AGCGGAGCTTGTACTTGGGGCTCGACTTACTCCAGT-3' and reverse primer 5'-GGTCGAGCTCGGGGTCGACGCCGCCGATGTCGAACT-3'. Finally, xynB gene was amplified from cDNA (29) using the direct primer 5'-AGTTCGACATCGGCGGCGTCGACCCCGAGCTCGACC-3' and reverse primer GGCTAAGCTTTTAGTGGTGGTGGTGGTGGTGCTGAACAGTGATGGACGAAG-S' (Hind III) (His-tag is underlined by the dashed line in all sequences). The obtained overlapping fragments were mixed, and the composite sequence was synthesized in one-step reaction using two external primers. The construct was cloned in the vector pGEM-T (Promega), and cloned PCR fragment was verified by sequencing. Composite fragment was cloned into the NcoI-HindIII linearized and dephosphorylated pAN52.3 vector to obtain pELX p is asmady (figa). Plasmid FLXLC was constructed using pFLX as template to amplify a fragment encoding the recombinant sequence FAEA-linker-XYNB, using the direct primer 5'-GGACTCATGAAGCAATTCTCTGCAAAATAC-3' (BspHI) and reverse primer 5'-ACTGGAGTAAGTCGAGCCCTGAACAGTGATGGACGA-3'. Genomic DNA from strainA. nigerBRFM131 was used as a substrate for PCR amplification of the sequence of the linker-CBM using two specific primers designed on the basis of available sequence cbhB (AF156269): forward primer 5'-TCGTCCATCACTGTTCAGGGCTCGACTTACTCCAGT-3' and reverse primer 5'-ATGCAAGCTTTTAGTGGTGGTGGTGGTGGTGCAAACACTGCGAGTAGTAC-3' (HindIII). Composite fragment was synthesized by PCR with overlapping elongation using two external primers tested sekvenirovanie and cloned in the vector pAN52.3 to obtain vector pFLXLC (pigv). In both expression vectors for expression of recombinant sequences were used the promoter of the gene of glyceraldehyde-3-phosphate dehydrogenase (gpdA)A. nidulansa 5' untranslated region of mRNA gene gpdA and the trpC terminatorA. nidulans. In addition to targeting secretion of both recombinant proteins were used signal peptide (21 amino acids) of FAEA.

Both co-transformation of fungi was carried out as described by the authors Punt and van den Hondel (39), using expression vectors pFLX and pFLXLC appropriate to estwenno and pAB4-1 (48), containing the marker pyrG selection, in relation to 10:1. In addition, the strain D15#26 A. niger was transformed pyrG gene without an expression vector for the control experiment. Selection of co-transformants on oridinary phototropins was carried out on plates with selective minimal medium without uridine), which were incubated for 8 days at 30°C. For screening transformants were cultured and were checked daily 40 individual clones from each transformation.

Screening activities feruloylated and xylanase

Surveillance cultures were performed within 14 days at 30°C in an incubator with shaking (130 rpm). pH daily summed up to 5.5 with 1M citric acid solution. Every culture conditions were repeated twice. Daily from liquid culture medium were selected aliquots (1 ml), from which the mycelium was harvested by filtration. The esterase activity was measured as described above using methylfolate (MFA) as a substrate (40), while the activity of xylanase was determined by the amount of xylose released from 1% (weight/volume) of xylan from birch wood, according to the method proposed by Bailey et al (1). The enzymes were incubated with a solution of xylan [1% (weight/volume) solution of xylan from birch wood, 50 mm buffer citrate pH 5.5] at 45°C for 5 minutes Allocated to restore the pouring sugar was determined by DNS method, using xylose as standard (35). All analyses were performed using control samples to account for corrections to the background in the samples of enzyme and substrate.

The activity was expressed in Natal (nkat) units; 1 nkat was defined as the amount of enzyme that catalyzes the allocation of 1 nmol Frolovich acid or 1 nmol reducing sugars per second at specified conditions. Each experiment was repeated twice and the measurement three times and the standard deviation was less than 2% from the mean esterase activity and less than 5% of the activity of xylanase.

Purification of recombinant proteins

The best selection for each of the constructs was planted using the same conditions as in the screening procedure. The culture was collected after 8 days of growth, filtered (0.7 micron) and concentrated by ultracentrifugation through polyethersulfone membrane (boundary molecular weight was 30 kDa) (Millipore). Concentrated fractions were subjected to dialysis against 30 mm Tris-HCl, pH 7.0 (buffer binding) and the selection of proteins with His-tag was performed on Heleroosa Sepharose column bystrogo flow (13×15 cm) (Amersham Biosciences) (38). As for single proteins, recombinant xylanase, which contains a fragment of the His-tag, was also purified on Heleroosa Sepharose column rapid stream, as if the about described (29). Finally, recombinant protein FAEA was purified by one-step procedure, using phenyl-sepharose column, as described (41).

Characterization of recombinant proteins

Protein analysis and identification of N-terminal amino acid sequence. Protein concentration was determined by the method of Lowry (34) using bovine serum albumin as standard. Preparation and purification of the protein was monitored by staining Kumasi SDS-PAGE polyacrylamide (11% polyacrylamide) gels. Determination of N-terminal sequence was produced by the method of degradation of Edman after electroblotting samples FLX and FLXLC (100 ág) on the membrane of polyvinylidene of diferida (Millipore). Analyses were performed on the unit Applied Biosystem 470A.

Analysis Western blotting. After electrophoresis the total and purified proteins in 11% SDS-PAGE polyacrylamide gel according to the method Laemmli (28) were electroblotting proteins to BA85 nitrocellulose membrane (Schleicher and Schuell) at room temperature for 45 minutes, the Membranes were incubated in blocking solution (50 mm Tris, 150 mm NaCl and 2% (V/V) milk pH 7.5) overnight at 4°C. then the membranes were washed in a solution of TBS-0.2% of tween and processed blocking solution containing anti-FAEA serum diluted 1/8000 or serum antiprogestogen-peroxidase (Sigma). In the case of anti-FAEA anti who ate the membrane was then incubated with anti-rabbit immunoglobulin G goats, conjugated with horseradish peroxidase (Promega). Signal detection was performed using chemiluminescence set for Western blotting (Roche) according to manufacturer's recommended procedure.

Temperature and pH stability of recombinant proteins. thermostability of the purified recombinant proteins was tested in the range from 30° to 70°C. Aliquots pre-incubated at the same temperature, and after cooling to 0°C, the activity of esterase and xylanase was tested as described above under standard conditions. After incubation was carried out analysis of samples for SDS-PAGE polyacrylamide gel to check the integrity of the bifunctional protein. The influence of pH on the stability of the proteins was investigated by incubation of purified recombinant proteins in citrate-phosphate buffer (pH 2.5 to 7.0) and the phosphate (pH 7.0 to 8.0). All incubation was carried out for 90 min, after which aliquots were transferred to a standard reaction mixture for determination of residual activity. Activity specific to the pre-incubation was considered as 100% activity.

The effect of temperature and pH on the activity of esterase and xylanase. To determine the optimal temperature conditions used aliquots of recombinant proteins were incubated at different temperatures (from 30° to 70°C) determines which of the n activity esterase and xylanase. Optimal pH was determined using a citrate-phosphate buffer (pH 2.5 to 7.0) and sodium phosphate buffer (pH 7.0 to 8.0) under standard conditions.

Determination of pulp-binding capacity and dissociation constant. Samples treated FLX and FLXLC was added in 2-ml centrifuge tubes containing cellulose in 25 mm potassium phosphate buffer (pH 7), to a total volume of 2 ml Capacity FLX (control) and FLXLC for binding to Avicel PH101 cellulose (Fluka) was determined using different amounts of recombinant protein (from 30 to 170 µg) and a constant number of cellulose (2 mg). Both recombinant protein were incubated with cellulose for 1 hour at 4°C With mild shaking. After centrifugation (4000g, 10 min) was determined by the number of remaining proteins in the supernatant (unbound enzyme). The amount of enzyme bound to cellulose was calculated by subtracting the value of the unbound FLX or FLXLC of the total amount of added enzyme. Analysis of the results was carried out by constructing a double-inverse relationship (1/(linked enzyme) 1/(unbound enzyme)). The dissociation constant is defined as 1/B=(Kd/Bmax× 1/F)+1/Bmax, where B is the concentration of bound peroxidase enzyme, and F is the concentration of free enzyme (21, 37).

The test application

Enzymatic hydrolysis. Wheat bran (ON) and corn is bran (KO) obstructively. These substrates were then subjected to heat treatment at 130°C for 10 min Enzymatic hydrolysis produced in 0,1M buffer 3-N-morpholinepropanesulfonic (MOPS)containing 0.01% of sodium azide at pH 6.0 in a thermostatically controlled incubator with vshrapyvaniem at 40°C. or KOH (200 mg) were incubated with purified FAEA (SEQ E) NO: 2), XYNB (SEQ ID NO: 6), FAEA+XYNB, FLX and FLXLC independently, in the final volume of 5 ml Concentration of purified enzyme for individual and bifunctional enzymes were as follows: 11 Natal activity of esterase and 6496 has Natal activity of xylanase on 200 mg of dry bran for each analysis. This ratio corresponds to the molar-molar ratio, specific for purified bifunctional enzyme. Each analysis was made twice, and the standard deviation was less than 5% from the mean for and TO.

Obtaining alkali-extractable hydroxycortisol acid. The total content of the alkali-extractable hydroxycortisol acid was determined by adding 20 mg or TO to 2n NaOH and incubation for 30 min at 35°C in the dark. the pH was brought to 2 with 2n HCl. Phenolic acids were extracted three times with 3 ml of ether. The organic phase was transferred into a test tube and dried at 40°C. Then to the dry extract was added one milliliter of a solution of methanol-water (50:50) (V/V) and samples sent to you is whether in HPLC system as described in the next section. The total content of the alkali-extractable ferulic acid was taken as 100% for enzymatic analysis.

Determination of ferulic acid. Enzymatic hydrolysates were diluted by half with 100% methanol, centrifuged at 12000g for 5 min and supernatant filtered through 0.2 μm nylon filter (Gelman Sciences Acrodisc 13, Ann Arbor, MI). Then the filtrates were analyzed by HPLC (25 µl per application). HPLC analysis was made at 280 nm and 30°C in installation model HP1100 (Hewlett-Packard, Rockville, MD)equipped with a variable UV/VIS detector and auto sampler-injector 100 samples. Separation was performed on a column of reverse phase Merck RP-18 (Chromolith 3.5 µm and 4.6×100 mm, Merck). The flow rate was 1.4 ml/min as mobile phase used 1% acetic acid and 10% acetonitrile in water (A) against acetonitrile 100% (C) when the total operation time of 20 min, the gradient was changed as follows: the solvent In started from 0% for 2 min, then increased to 50% for 10 min, to 100% for 3 min before the end of the process. The results were processed on the HP 3365 ChemStation and quantitative analysis was made by using external standard calibration.

RESULTS

Design and research developments bifunctional enzymes

Sequence ofA. nigerencoding feruloylated A (FAEA) and the xylanase B (XYNB), was the genetically merged by adding between two protein sequences from gene cellobiohydrolase In (cbhB), coding hyperglycosylated linker (figa). To obtain the second design FLXLC corresponding merged FLX was further merged with the partial sequence of a gene cbhB fromA. nigerthat encodes a linker-CBM (pigv). Both broadcast composite protein were placed under the control of a strong constitutive gpdA promoter and trpC terminator with the endogenous signal sequence faeA for targeting secretion. The protoplastsA. nigerD15#26 were co-transformed with a mixture of expression vector pFLX or pFLXLC and plasmid pAB4-1 containing the pyrG gene. Selection of transformants was carried out by their growth on the Cup with minimal medium without uridine. 40 transformants for each construct were sowed in the environment with minimal glucose, suppresses endogenous gene expression faeA and xynB. For the control host cells transformed pAB4-1, esterase activity, or a xylanase was not detected. For both structures the activity of esterase and xylanase detected in the extracellular medium of transformed after 2 days of growth (figure 2). Activity feruloylated and xylanase was determined as synchronous during growth in culture. Activity was increased up to 10 days and up to 11 days for the best transformants FLXLC and FLX, respectively, and has reached a level that has remained more or less stable up to 14 days. Maximum activity e is teraz was 13,0 ncat/ml for FLX and 9.8 ncat/ml for FLXLC of transformants. The maximum values obtained for the activity of xylanase was 2870 ncat/ml for FLX and 2038 ncat/ml for FLXLC transformations. Taking into account the corresponding specific activity partner FAEA in the bifunctional enzyme, the product yield was estimated as 1.4 g/l and 1.5 g/l FLX and FLXLC of transformants, respectively.

Biochemical and kinetic characterization of bifunctional enzymes

SDS-PAGE electrophoresis and analysis by Western blotting. In both cases the resulting proteins was monitored by electrophoresis on SDS-PAGE polyacrylamide gel (figure 3, lanes 1 and 3). The most intense band of approximately 72 kDa and 97 kDa observed among the total extracellular proteins, consistent with FLX and FLXLC the transformants, respectively. The difference between the observed and theoretical molecular masses FLX and FLXLC supposedly says about the glycosylation of about 12% (weight/weight) and 26% (weight/weight), respectively. Recombinant enzymes were purified on a column of Heleroosa Sepharose Fast Flow (Chelating Sepharose Fast Flow), and the homogeneity of the fractions were monitored by SDS-PAGE electrophoresis (lanes 2 and 4).

Both chimeric enzyme from supernatants total proteins and purified fractions were immunodetectable using antibodies to FAEA (figa). A single band was evident from the total protein extract (track 1, 3) and etchemendy samples (tracks 2, 4) for FLX and FLXLC of transformants, respectively. For immunodetection was also used antibodies to C-terminal his-tag label for monitoring the integrity of the recombinant proteins as antibodies to the xylanase or CBM were unavailable (pigv). In this case also for FLX and FLXLC detected a single band, confirming that the bifunctional enzymes were obtained as intact proteins without any forms of degradation (lanes 5 and 8). Negative control (C) confirmed that both antibodies were specific for the epitope and that endogenous FAEA were not formed under these culture conditions.

N-terminal sequencing. The first five amino acids FLX and FLXLC were sekvenirovan (ASTQG) and compared with amino acids from native FAEA. The comparison showed 100% identity between the recombinant proteins and native FAEA. These results confirmed that FLX and FLXLC were processionary correctly.

Analysis of epinasty bifunctional proteins to cellulose and binding capacity. In contrast FLX, FLXLC contains a C-terminal region of CBM gene cellobiohydrolase In (cellobiohydrolase B)A. niger. Carried out analysis of the interaction FLXLC with cellulose to determine the binding parameters CBM. The affinity of binding to cellulose and capacity binding FLXLC were defined relative to microcrystalline cellulose Avicel PH101. P is obtained values for the dissociation constants and binding capacity was 9.9×10 -8M and 0.98 mmol/g Avicel respectively. As expected, the interaction for the chimeric enzyme FLX (without CBM) was not observed.

Biochemical and kinetic parameters. For control, did not change the characteristics of each enzyme in the result of joining two enzymatic partners in the presence or in the absence of CBM, biochemical and kinetic parameters FLX FLXLC were compared with those for free recombinant FLX and FLXLC in accordance with the activities of esterase and xylanase (table 1). In relation to the optimum pH and stability found no significant differences between the two bifunctional enzymes and separate FLX and FLXLC. In relation to the optimum temperature and stability the only difference concerned the small shift for the activity of xylanase. In addition, the integrity of FLX and FLXLC was monitored using SDS-PAGE electrophoresis after incubation at different temperatures. Both bifunctional enzyme was completely stable up to 45°C and partially split at 50°C. the Initial amino acids cleaved forms were sekvenirovan and were as follows: GSGSS. Comparison of the sequence of FLX and FLXLC pokazivaet 100% identity with the sequence in the linker. These results showed that hyperglycosylated the linker is stable up to 45°C and that the cleavage occurs at 50°C in about the Asti after GSGSS sequence. Chimeric protein FLXLC containing two of the linker sequence was cut only in the C-terminal of the linker (between FLX and FLXLC). Was searching for possible places proteasome splitting the amino acid sequences of both hybrid proteins using peptide-cutting (19), however, the splitting in the field of sequence GSGSS was not found.

In respect of kinetic characteristics were measured Michaelis constants for FLX and FLXLC on schedule, Leinweber Berka using methylfolate and birch wood xylan as the substrate. The values found for FLX and FLXLC were in accordance with values found for recombinant FAEA and XYNB separately (table 1). The specific activity of bifunctional enzymes were determined based on activities feruloylated and xylanase, and compared with values for FAEA and XYNB separately (table 1). The values found for bifunctional enzymes were close to values for the FAE and XYNB separately.

In conclusion, these results confirmed that the biochemical and kinetic parameters for enzymatic compound modules (FAEA and XYNB) was mainly retained in the bifunctional complexes FLX and FLXLC.

Enzymatic selection of ferulic acid from wheat and corn bran

To study the synergistic e is the antecedent, due to physical proximity of the two enzymes in bifunctional proteins, and the impact of adding CBM proteins FLX and FLXLC were compared with single enzymes FAEA and XYNB in relation to the efficiency of the allocation of ferulic acid. All enzymes were purified to homogeneity and were incubated with and TO, as the latter contain large amounts of ferulic acid in their cell membranes. In the case of individual FAEA 41% and 51% of the total alkali-extractable ferulic acid was separated from for 4 and 16 hours, respectively (figa). These values are slightly increased to 51% (4 h) and 54% (16 h) adding XYNB separately. As for the effect of FLX and FLXLC there was complete selection of ferulic acid in only 4 h of incubation. Using CO as a substrate (pigv) free FAEA released 4.2% and 4.8% of ferulic acid for 4 h and 16 h, respectively, and adding XYNB did not increase this value. At the same time FLX and FLXLC were able to release of 6.2% and 7.2% in 4 h of hydrolysis, respectively. If the enzymatic treatment was carried out for 16 h, the yield was increased to 6.3% and 7.9% for FLX and FLXLC respectively. Were also determined and compared factors of synergism between single (FAEA + XYNB) and compound enzymes (FLX and FLXLC). As calculated in table 2, the estimated ratio was greater than 1, showing that for both substrates synergistic efficiency which is higher for bifunctional enzymes, than for the corresponding single enzymes. Regarding the allocation of ferulic acid from TO a higher synergy was observed for FLXLC (1,80 and 1,62)than FLX (1,53 and 1,30) for 4 h and 16 h, respectively. In conclusion, these results showed that for both substrates bifunctional enzymes FLX and FLXLC was more effective to highlight ferulic acid compared with the corresponding enzymes separately (FAEA + XYNB). In addition, from these results we can conclude that FLXLC is more suitable for the selection of ferulic acid using KOH as the substrate, which indicates a possible positive effect produced by CBM.

Conclusion

For birthrate cell membrane of plants due to their heterogeneity in composition and structure requires a large number of different enzymes. Certain combinations of different hydrolytic enzymes of the primary circuit with the auxiliary enzymes showed an important synergistic effect, resulting in the effective destruction of the cell membrane (9, 12). In this study, two enzymes that destroy the cell wall of plants, and using hydrocarbon-binding module fromA. nigerwere merged into a hybrid molecule to study the synergistic effect in the process of destruction of the natural substrates. The design of such hybrid proteins are the two which is one of the main aspects of protein engineering, enabling a large number of applications. In this case, the concept is to use two functional units for advanced education bifunctional proteins (3, 36). Such multi-modal patterns can often be observed in nature, which form the enzymes with more than one enzymatic activity or protein function. In biotechnology, some proteins have already been explored, including, for example such as a hybrid between α-amylase and glucoamylase (44). The results of this study indicate an increase in enzymatic efficiency in comparison with the individual enzymes in the process of destruction of raw starch. In other work was designed chimeric xylanase/endoglucanases (XynCenA) c internal CBM, but further results showed that the effect of hydrolysis of the hybrid enzyme in relation to homogeneous Xylenol or cellulose substrates was negligible compared with single enzyme, however, the test application on natural substrates were not performed (46).

To evaluate the effect arising due to the physical proximity of the two hydrolases cell membranes, FAEA and XYNB were merged together (design FLX). In the second design (FLXLC) for targeting bifunctional enzyme to the cellulose in the C-terminal region was annexed CBM mushrooms is C A. nigerCBHB. For both designs hyperglycosylated linker peptide was inserted between each molecule (FAEA, XYNB or CBM) for three main reasons. First, it is known that the linker leaves the possibility for modules independently to form tertiary structure and save conformational freedom relative to each other (36). In this case, as feruloylated and xylanase were able to adopt this conformation, and designed a bifunctional protein was active with the biochemical and kinetic properties that correspond to the individual enzymes. Secondly, the high degree of glycosylation of the linker allows you to increase the stability of the protein sequence that prevents the linker from by activity and, finally, avoiding common problems associated with splitting between component modules (8, 36). Such an effect was indeed observed, as both bifunctional protein were stable, as shown by SDS-PAGE electrophoresis and Western blotting analysis. At the same time it was shown that the stability of the hybrid enzyme has certain boundaries during heat treatment. In fact, the influence of thermal processing on the integrity of FLX and FLXLC showed that they remained stable up to 45°C, and then were split in the field linker at 50°C. Finally, glycosylated linker can in order to play a positive role in secretion, increasing the product yield, as shown for hyperglycosylated linker fromA. nigerglucoamylase (32). In fact, sites of glycosylation due to the presence of one or two linkers for FLX and FLXLC consequently could increase the residence time of recombinant proteins in the endoplasmic reticulum, by adding the time for the formation of the correct tertiary structure and leading to an increase in production (42). This last hypothesis could explain why the yield for both bifunctional enzymes was higher than the yield obtained for the respective individual recombinant enzymes (29, 41).

To study the synergistic effect arising due to the proximity of both enzymatic modules, rather than a simple increase due to the modification of enzyme properties as a result of changes in protein conformation during the formation of the tertiary structure, biochemical and kinetic characteristics of each module has been carefully investigated. All major biochemical and kinetic properties of both bifunctional proteins FLX and FLXLC, i.e. temperature and pH stability, optimum temperature and pH, Kmand the specific activity was in the same range as for the individual enzymes. Regarding CBM taken fromA. nigerCBHB, analyses linking was carried out on cellulose, as it is not what, had been characterized in the past (20). Avicel cellulose has an important degree of polymerization, characterized 100-250 glycopyranosyl units and 50-60% crystalline form with a crystalline phase composed mainly of type Iβtypical of higher plants (49). The results showed that FLXLC has affinity towards Avicel, confirming that the structure of the CBM is not changed and that CBM has retained its functions in a hybrid enzyme.

Finally, both bifunctional protein were analyzed to study the effect of physical proximity of two complementary enzymes of fungi on enzymatic synergies and the impact of adding CBM. The test application was based on the allocation of ferulic acid from two natural and model substrates, and CO, known for its high content of ferulic acid in their plant cell walls is about 1% and 3% (weight/weight), respectively (43). Both substrates were obtained from agriculture and can have value in the agricultural industry, cosmetic and pharmaceutical sectors (4, 26). Individual enzymes were able to free up 54% and 4.8% of ferulic acid from and TO, respectively. In contrast bifunctional enzymes effectively freed all Frolovo acid from and to 6.3% or 7.9% from TO depending on the presence or absence of CBM. So far, the previous results is udeleniu ferulic acid from were obtained using xylanase from Trichoderma virideand FAEA fromA. niger, which was released 95% maximum (weight/weight) of the total ferulic acid (15). In case a significant amount of ferulic acid (to 13.6%) were released fromHumicola insolenswhen using a commercial preparation Novozym 342 (5). At the same time should take into account the fact that this commercial drug contained various types of enzymatic activity. In this analysis, using the processing bifunctional enzymes ferulic acid was released completely from the software, while less than 8% were formed from KOH. Despite the fact that the content of ferulic acid in maize bran is higher than in wheat bran, xylan in corn bran is more than replaced by traces of xylose, arabinose and galactose (7, 15). Thus, the observed difference can be explained by the number of substitutions in heteroxylan the basis of corn by the presence of highly branched xylose side chains and the links between arabinose and xylose near groups of ferulic acid, which significantly limits the availability of the enzyme. Finally, when considering hydrolysis TO using FLXLC positive effect of CBM on the release of ferulic acid is likely to occur due to (i) targeting to the cellulose, which increases the concentration of the enzyme near substr is the and/or (ii) destabilization of the cellulose structure, making the substrate more accessible. As a conclusion to the tests applied using FLX and FLXLC it can be noted that for both substrates was obtained best synergies for selection of ferulic acid in comparison with individual enzymes FAEA and XYNB. An assumption was made that the total increase synergy was due to the physical proximity of each enzymatic partner in bifunctional enzymes, as all major biochemical and kinetic properties for each partner in a hybrid proteins was not changed. If FLXLC positive effect on the synergy produced C-terminal addition of CBM. Moreover, one can also assume that the spatial orientation of the active sites did not change between the constituent modules.

As a General conclusion it was shown that the design of new enzymatic tools for the destruction of the cell walls of plants using complementary cell wall hydrolases, such as auxiliary enzyme FAEA and the enzyme that breaks down the main chain (XYNB), is noteworthy strategy to increase the synergistic effect of enzymatic partners. For biotechnological applications, the use of such hybrid proteins is an alternative to costly is m and polluting chemical treatments or used for improvements to existing enzymatic processes to enhance the value of plant by-products in the manufacture of pulp and paper, agro-industries and sectors biofuel production.

SHAPES

Figure 1 Expression cassettes used in this study. To construct FLX insert (A) sequence ofA. nigerencoding FAEA, the linker region of CBHB and XYNB were merged together. In the second design (In) the matrix FLX was merged with the sequence cbhb, the coding sequence of the linker and CBM, forming FLXLC box. Expression cassettes were under the control of the gpdA promoter and trpC terminator. Both constructs contained a sequence encoding six histidines on the 3' end.

(1) a Sequence encoding a linker: GSTYSSGSSSGSGSSSSSSSTTTKATSTTLKTTSTTSSGSSSTSAA.

Figure 2 time dependence of the activity of extracellular feruloylated and xylanase from A. niger. Measurement activities feruloylated (a) and xylanase () was made for the best FLX (♦) and FLXLC (■) transformants. Activity was determined using methylfolate and xylan from birch wood as substrates for esterase and xylanase, respectively.

3 Analysis in SDS-PAGE polyacrylamide gel of extracellular proteins produced FLX and FLXLC the transformants. On SDS-PAGE 11% polyacrylamide gel was applied to the total and purified proteins from FLX (tracks 1 and 2, respectively) and FLXLC (tracks 3 and 4, respectively). The gel was stained Kumasi blue (Coomassie blue). SD: one hundred is the molecular weight standards.

Figure 4. Western-blot hybridization of total and purified proteins produced FLX and FLXLC the transformants. In immunodetection total extracellular and purified proteins from FLX and FLXLC of transformants was used antibodies to FAEA (A) or His-Tag (B). Tracks 1,5: total extracellular proteins from FLX of transformant. Tracks 2,6: cleaned FLX. Tracks 3,7: total extracellular proteins from FLXLC of transformant. Tracks 4,8: cleaned FLXLC. With the control strain D15#26, transformed pAB4-1. Detection was performed using chemiluminescence.

Figure 5. Comparison of the effectiveness of the release of ferulic acid under the action of an individual or a bifunctional enzymes. For hydrolysis of ferulic acid, private or bifunctional enzymes were used wheat bran or corn bran (In). The release of ferulic acid was determined using HPLC for 4 h (white bars) and 16 h (black bar). Activity was expressed as a percentage of the total ferulic acid contained in the substrate. The standard deviation was less than 5% from the average for wheat bran and corn bran.

Table 1
Physico-chemical and kinetic parameters for partners feruloylated xylanase
Activity feruloylatedThe activity of xylanase
FAEA(1)FLXFLXLCXYNB(2)FLXFLXLC
Tpoptimal60°C55-6055-60504545
Tpstability-4545504545
the pH optimum5555,566
pH stability5-65-65-64-74-74-7
Km(3)0,750,800,786,67,57,5
Specific activity(4)0,720,660,63386394368
(1); (2) (References 41 and 29, respectively).
(3) Kmexpressed in mol for activity feruloylated and milliliters for activity of xylanase.
(4) the Specific activity was expressed in Natal on nmol protein to facilitate comparison between the individual and bifunctional proteins.

For pH and temperature stability incubation was carried out for 90 minutes.

Table 2
Comparison of the synergistic effect on the allocation of ferulic acid in the case of separate and merged enzymes
ONTO
4 h6 h 4 h16 h
FLX1,951,851,531,30
FLXLC1,951,851,801,62

The factor of synergism was defined as follows:

(ferulic acid, selected using bifunctional enzymes FLX or FLXLC)/(ferulic acid, selected by individual enzymes FAEA+XYNB).

Sources of information

1. The use of fused protein comprising feruloylated and xylanase, these enzymes are such that they do not contain C-terminal uglevodorodokislyayuschih molecule (carbohydrate-binding-molecule, SVM), and, if necessary, SVM from the third enzyme, and these enzymes and CBM are recombinant proteins corresponding native proteins in fungi, for the implementation of the means of destruction of the cell walls of plants within getting from plants or vegetal by-products, the compounds of interest and contained in CL is accurate membranes of plants.

2. The use of fused protein comprising feruloylated and xylanase, these enzymes are such that they do not contain C-terminal uglevodorodokislyayuschih molecule (carbohydrate-binding-molecule, SVM), and, if necessary, SVM from the third enzyme, and these enzymes and CBM are recombinant proteins corresponding native proteins in fungi, for the implementation of the means of destruction of the cell walls of plants under the bleaching of pulp and paper.

3. The use of fused protein according to claim 1 or 2, in which the enzymes that destroy the cell wall of plants and does not contain SVM correspond to the native enzymes in fungi chosen from:
the records of Ascomycetes, such as:
- strains of Aspergillus, and more specifically, Aspergillus niger,
- strains of Trichoderma, and more specifically, Trichoderma reesei,
basidiomycetes, such as strains of Pycnoporus or Halocyphina, and more specifically, Pycnoporus cinnabarinus, Pycnoporus sanguineus or Halocyphina villosa.

4. The use of fused protein according to claim 1 or 2, in which the enzymes that destroy the cell wall of plants and does not contain SVM correspond to the native enzymes from strains of Aspergillus, such as Aspergillus niger.

5. The use of fused protein according to claim 1, in which at least one enzyme that destroys the cell wall of plants is feruloylated and is chosen from:
- feruloylated And of A. niger represented by SEQ ID NO:2,
- Il is feruloylated from A. niger represented by SEQ ID NO:4.

6. The use of fused protein according to claim 1 or 2, in which at least one enzyme that destroys the cell wall of plants is a xylanase, such as xylanase from A. niger represented by SEQ ID NO:6.

7. The use of fused protein according to claim 1 or 2, in which the protein, which is a RAS-choose from RAS present in the native enzymes of fungi selected from the records of Ascomycetes, such as strains of Aspergillus, and more specifically, Aspergillus niger.

8. The use of fused protein according to claim 1 or 2, in which SVM SVM is present in cellobiohydrolase from A. niger and represented by SEQ ID NO:8.

9. The use of fused protein according to claim 1 or 2, containing linkers between at least two proteins that belong to the specified protein, and the linkers are polypeptides ranging in length from 10 to 100 amino acids primarily about 50 amino acids.

10. The use of fused protein according to claim 1 or 2, in which the linker is included between each protein included in the protein.

11. The use of fused protein according to claim 1 or 2, in which the linker is hyperglycosylated polypeptide, such as having the sequence represented by SEQ ID NO:10, contained in cellobiohydrolase from A. niger.

12. The use of fused protein according to claim 1 or 2, comprising feruloylated and xylanase and, if necessary, SVM, such as:
- fused protein comprising feruloylated And A. niger represented by SEQ ID NO:2, and the xylanase from A. niger represented by SEQ ID NO:6, with the specified protein contains the sequence represented by SEQ ID NO:10, as hyperglycosylated linker between the two preceding proteins, and represented by SEQ ID NO:12,
- fused protein comprising feruloylated And of A. niger represented by SEQ ID NO:2, and the xylanase from A. niger represented by SEQ ID NO:6, and the CBM represented by SEQ ID NO:8 and is present in cellobiohydrolase from A. niger,
- fused protein comprising feruloylated And of A. niger represented by SEQ ID NO:2, and the xylanase from A. niger represented by SEQ ID NO:6, and the CBM represented by SEQ ID NO:8, with the specified protein contains the sequence represented by SEQ ID NO:10, as hyperglycosylated linker between each of the three preceding proteins, and represented by SEQ ID NO:14,
- fused protein comprising feruloylated In a of A. niger represented by SEQ ID NO:4, and the xylanase from A. niger represented by SEQ ID NO:6, with the specified protein contains the sequence represented by SEQ ID NO:10, as hyperglycosylated linker between the two preceding proteins, and represented by SEQ ID NO:16,
- fused protein comprising feruloylated In a of A. niger represented by SEQ ID NO:4, and the xylanase from A. niger represented by SEQ ID NO:6, and the CBM represented by SEQ ID NO:8 and prisutstvuiu is in cellobiohydrolase from A. niger,
- fused protein comprising feruloylated In a of A. niger represented by SEQ ID NO:4, and the xylanase from A. niger represented by SEQ ID NO:6, and the CBM represented by SEQ ID NO:8, with the specified protein contains the sequence represented by SEQ ID NO:10, as hyperglycosylated linker between each of the three preceding proteins, and represented by SEQ ID NO:18.

13. The use according to any one of claims 1 or 2 for the implementation of the means of destruction of the cell walls of plants within the following target compounds:
- bioethanol,
- antioxidants such as ferulic chilota or caffeic acid, which are cinnamic acid and hydroxytyrosol, or gallium acid,
- flavoring additives, such as vanillin or p-hydroxybenzaldehyde obtained by biotransformation of ferulic acid or p-coumarin acid, respectively.

14. The use according to any one of claims 1 or 2 for the implementation of the means of destruction of the cell walls of plants under the bleaching of pulp and paper with a concomitant production of bioethanol or without it.

15. Destroying the cellular membrane protein, including feruloylated and xylanase, which do not contain C-terminal uglevodorodokislyayuschih molecule (CBM), and the specified protein, if necessary, includes RAS from the third f is rment, while enzymes and CBM are recombinant proteins corresponding native proteins of fungi.

16. Destroying the cell membrane protein indicated in paragraph 15, in which:
- enzymes that destroy the cell wall of plants that do not contain a RAS defined in any of p-5,
- RAS defined in claim 7 or 8.

17. Destroying the cell membrane protein indicated in paragraph 15, including linkers between at least two proteins contained in these fused proteins, these linkers identified in any of PP-11.

18. Destroying the cell membrane protein according to item 15 or 16, comprising feruloylated and xylanase, and, if necessary, RAS, such as:
- fused protein comprising feruloylated And of A. niger represented by SEQ ID NO:2, and the xylanase from A. niger represented by SEQ ID NO:6, with the specified protein contains the sequence represented by SEQ ID NO:10, as hyperglycosylated linker between the two preceding proteins, and represented by SEQ ID NO:12,
- fused protein comprising feruloylated And of A. niger represented by SEQ ID NO:2, and the xylanase from A. niger represented by SEQ ID NO:6, and the CBM represented by SEQ ID NO:8 and is present in cellobiohydrolase from A. niger,
- fused protein comprising feruloylated And of A. niger represented by SEQ ID NO:2, and the xylanase from A. niger represented by SEQ ID NO:6, and the VM represented by SEQ ID NO:8, with the specified protein contains the sequence represented by SEQ ID NO:10, as hyperglycosylated linker between each of the three preceding proteins, and represented by SEQ ID NO:14,
- fused protein comprising feruloylated In a of A. niger represented by SEQ ID NO:4, and the xylanase from A. niger represented by SEQ ID NO:6, with the specified protein contains the sequence represented by SEQ ID NO:10, as hyperglycosylated linker between the two preceding proteins, and represented by SEQ ID NO:16,
- fused protein comprising feruloylated In a of A. niger represented by SEQ ID NO:4, and the xylanase from A. niger represented by SEQ ID NO:6, and the CBM represented by SEQ ID NO:8 and is present in cellobiohydrolase from A. niger,
- fused protein comprising feruloylated In a of A. niger represented by SEQ ID NO:4, and the xylanase from A. niger represented by SEQ ID NO:6, and the CBM represented by SEQ ID NO:8, with the specified protein contains the sequence represented by SEQ ID NO:10, as hyperglycosylated linker between each of the three preceding proteins, and represented by SEQ ID NO:18.

19. Nucleic acid encoding a protein represented by the amino acid sequence SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:18.

20. The expression vector, transformed nucleic acid, Oprah is elenai in claim 19.

21. A host cell producing a protein by p transformed using a vector as defined in claim 20.

22. The method of obtaining from plants or vegetal by-products of the target compounds in plant cell walls, by destroying cell membranes, which is characterized by the fact that it comprises the following stages:
- enzymatic treatment of plants or vegetal by-products fused protein as defined in any of PP-18, or transformed cells mushrooms defined in item 21,
- if necessary, the physical treatment of plants or vegetal by-products by steam in combination with the action of fused proteins,
- if necessary, the biotransformation of compounds released from cell membranes in the process of the above-mentioned enzymatic processing, using the appropriate microorganisms or enzymes
- extraction and, if necessary, purification of the target compounds released from cell membranes in the process of the above-mentioned enzymatic processing or obtained in the above stage biotransformation.

23. The method according to item 22 to obtain antioxidants such as ferulic acid, or caffeic acid, which are cinnamic acid and hydroxytyrosol, or gallium acid; flavoring additives such as vanillin or p-hydroxybenzaldehyde obtained by biotransformation of ferulic acid or p-coumarin acid, respectively, bioethanol,
or for bleaching paper pulp and paper.

24. The method of obtaining the fused proteins identified in PP-18, comprising culturing in vitro host cells according to item 21, allocation and, if necessary, clean the slit proteins produced by these cells-host culture.



 

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