EP2609204A1

EP2609204A1 - Whole-cell biocatalyst

Info

Publication number: EP2609204A1
Application number: EP11749822.0A
Authority: EP
Inventors: Joachim Jose; Ruth Maas; Eva Kranen
Original assignee: Zyrus Beteiligungs GmbH and Co Patente I KG
Current assignee: Zyrus Beteiligungs GmbH and Co Patente I KG
Priority date: 2010-08-27
Filing date: 2011-08-26
Publication date: 2013-07-03
Also published as: ZA201302201B; BR112013004383A2; CN103140583A; AU2011295025A1; AU2011295025A2; US20130203118A1; CA2812390A1; DE102010035702A1; WO2012025628A1; JP2013537428A; RU2013110454A

Abstract

The invention relates to a nucleic acid molecule comprising a section that encodes a signal peptide, a section that comprises a heterologous redox factor-regenerating polypeptide, an optional section that encodes a protease detection site, a section that encodes a transmembrane linker, and a section that encodes a transporter domain of an autotransporter or a variant thereof. The nucleic acid molecule enables the expression of redox factor-regenerating enzymes.

Description

Ganzzeil biocatalyst

The invention relates to a nucleic acid molecule comprising (1) a portion encoding a signal peptide, (2) a portion which omits a heterologous redox factor-regenerating polypeptide or a variant thereof, (3) optionally, a portion encoding a protease recognition site (4) a portion encoding a transmembrane linker; and (5) a portion encoding a transporter domain of an autotransporter or a variant thereof.

About a quarter of all known enzymes are oxidoreductases. A wide range of applications has already been explored for this group of enzymes, including the synthesis of chiral compounds such as alcohols, aldehydes and amino acids, the production and modification of polymers, the production of biosensors for a wide variety of applications and the degradation of pollutants. Oxidoreductases are characterized by comprising not only at least one polypeptide chain but also one or more redox-reactive groups, which may be one or more redox-reactive amino acid side chains or prosthetic groups.

The substrates of the oxidoreductases include metabolic products and redox factors. In contrast to prosthetic groups of enzymes, these substrate redox factors do not act as catalysts, but participate in the reaction in stoichiometric amounts and undergo chemical reactions with other substrates. In view of the fact that redox factors are generally much more expensive than other organic substrates, the feasibility of industrial syntheses using oxidoreductases is limited because of the need to provide suitable reduced or oxidized factors. To solve this problem, scientists have proposed the use of redox factor regenerating enzymes. For example, the use of formate dehydroxygenase (FDH) and NADH oxidase or the use of glucose dehydrogenase (GDH) for the regeneration of NADH and NADPH has been proposed. Such enzymes should be immobilized on suitable supports for several cycles of catalysis to avoid having to repeatedly purify large quantities of the polypeptides. Unfortunately, redox factor regenerating enzymes are often unstable, especially when present as immobilized enzymes.

Sanjust ef al. (1997) immobilized the NADH oxidase from Thermus aquaticus on different supports and compared the kinetic properties of soluble and immobilized enzymes. It turned out that the immobilized enzyme has lower thermostability than the soluble enzyme. Hummel and Riebet (2003) describe the use of soluble, purified NADH oxidase from Lactobacillus brevis for the regeneration of NADH oxidase. They indicate that the enzyme is prone to rapid oxidative inactivation, most likely due to the presence of a cysteine residue susceptible to oxidation in the catalytic center of the protein. Therefore, the use of a reducing agent (1 mM DTT or mercaptoethanol) is described as essential for maintaining the catalytic capability of the enzyme. Ahmed and Claiborne (1992) purified the FAD-containing NADH oxidase from Streptococcus faecalis 10C1 and teach that the presence of 2 mM DTT is required to stabilize the holo-oxidase. El-Zahab et al, (2004), reviewed by Lee and Fing (2007), describe immobilizing redox factor-regenerating enzymes, such as glucose dehydrogenase (GDH), on a solid support, more specifically, on nanoporous silica glass. However, they do not describe how such enzymes can be stabilized.

Therefore, the object of the present invention is to provide an agent with a redox factor-regenerating activity, wherein the source of the activity is not only readily accessible to substrate molecules, but can be easily amplified, recycled and regenerated to allow multiple catalytic steps, without it being necessary to repeatedly prepare new agent. Another object of the present invention is to provide an agent which by itself or in the presence of potentially inactivating chemicals, such as inhibiting metal ions, or at acidic or basic pHs, has a redox factor regenerating activity with better properties than the original form of the corresponding Enzyme in terms of kinetics, thermal stability, shelf life and stability. In particular, an object of the present invention It is to provide an agent which, in the presence of oxygen or oxygen-releasing agents or under oxidizing conditions, exhibits a stable redox factor-regenerating activity, the stability of which is better than that of the original form of the corresponding enzyme.

The present inventors have surprisingly found that redox-factor regenerating enzymes can be expressed on the surface of an entire cell using the autotransporter system and are intrinsically surprisingly stable with respect to the presence of oxygen and various temperatures and conditions. In addition, the subject invention has surprisingly found that redox factor regenerating enzymes can be incubated and remain stable for a long time in various solutions and buffers under various conditions.

Autodisplay is an elegant tool for presenting recombinant proteins to the bacterial surface. This expression system is based on the secretory mechanism of the protein family of car transporters belonging to the type V secretion system.

In gram-negative bacteria, the autotransporter pathway has evolved both for the transport of proteins to the cell surface and for the secretion of proteins into the extracellular space (Jose and Meyer, 2007). Autotransporter proteins are synthesized as precursor proteins that fulfill all structural requirements for transport to the cell surface (Jose, 2006). They are synthesized with an N-terminal signal peptide, which is typical of the See pathway, which allows traversing the inner membrane. Once in the perip! Asma, the C-terminal part of the precursor folds into the outer membrane after truncation of the signal peptide as a porin-like structure, a so-called β-barrel. Through this pore, the N-terminal bound passenger domain is transiently transcribed to the surface (Jose et al., 2002). There, it can be cleaved off - either autoproteolytically or by an additional protease - or can remain anchored to the cell membrane via the transporter domain. Replacement of the natural passenger with a recombinant protein results in its surface translocation. To do this, a non-natural precursor must be constructed using genetic engineering techniques a signal peptide, the recombinant passier, the β-barrel, and a linker region therebetween, which is required for unrestricted access to the surface. In this way, the AIDA-I car transporter has been used successfully for efficient surface presentation of various passenger domains (Henderson et al., 2004). In the autodisplay system, self-association of subunits to an active enzyme was observed, for example, in the dimeric enzyme sorbitol dehydrogenase (Jose, 2002, Jose and von Schwichow, 2004).

In particular, auto-display technology is an expression method for certain proteins on the outer membrane surface of E. coli and other Gram-negative bacteria, the auto-display system being based on the natural secretory mechanism of auto transporter proteins (A. Banerjee et al. (2002)) , In this case, the transport of the recombinant passenger protein can be carried out simply by introducing, in reading frame, its coding sequence between the signal peptide and the translocating domain of the autodisplay vector using standard genetic engineering techniques. The signal peptide may be derived from a subunit of the cholera toxin, and it may be combined with a non-natural promoter. Thus, the passenger protein that is to undergo translocation through the outer membrane is expressed as a recombinant fusion protein with another protein, called an auto transporter, on the outer membrane of E. coli (AIDA-i) (Jose, 2006). The C-terminal part of the car transporter protein forms a porin-like structure (β-barrel) within the outer membrane of E. coli. This porin-like structure allows translocation of the recombinant pass protein to the outer membrane surface of E. coli (Jose, 1995, 2006, 2007).

The concept of immobilization of redoxcofactor-regenerating polypeptides dates back to 1974, when Sarborsky and Ogletree described the immobilization of glucose oxidase via activation of their carbohydrate moieties. Likewise, the concept of the auto-display system, first by Jose ei a /. (1995) has been known for more than 15 years. However, to the best of our knowledge, the prior art neither teaches nor suggests the use of the autotransporter system for immobilizing redox factor regenerating enzymes. The object of the present invention is solved by the subject matters of the independent claims. Preferred embodiments may be taken from the dependent claims.

The object of the present invention, according to a first aspect, which is still the first embodiment of the first aspect, is solved by a nucleic acid molecule comprising (1) a portion encoding a signal peptide, (2) a portion containing a heterologous redox factor regenerating polypeptide or a variant thereof, (3) optionally a portion encoding a protease recognition portion, (4) a portion encoding a transmembrane linker, and (5) a portion encoding a transporter domain of an autotransporter or a variant thereof ,

In a preferred embodiment of the present invention, the nucleic acid molecule is operably linked to a sequence for expression regulation. In a preferred embodiment, as used herein, the term "expression repression sequence" refers to a nucleic acid sequence which can regulate the level of expression of a nucleic acid molecule, preferably downstream of the expression regulation sequence The sequence for expression regulation may be, for example, a promoter. The person skilled in the art is familiar with sequences suitable for expression regulation and methods for the functional connection of these sequences with a nucleic acid molecule.

In a preferred embodiment of the present invention, the nucleic acid molecule is part of a recombinant plasmid. In a preferred embodiment, the nucleic acid molecule comprises SEQ ID NO: 1 or variants thereof.

In a preferred embodiment of the present invention, as used herein, the term "heterologous" refers to a nucleic acid constructed using genetic engineering techniques, for example, by assembling an expression regulation sequence and a sequence to be expressed that does not normally have that sequence for expression regulation subordinate, or by Use a sequence that has a point mutation with respect to the original sequence. Consequently, even if only a section of a construct is called heteroiog, it implies that the entire construct is heteroiogical. The person skilled in the art is familiar with genetic engineering methods. In a preferred weathered embodiment, as used herein, the term "heterogeneous" refers to a polypeptide-encoding nucleic acid assembled with an expression-regulatory sequence and / or a fused sequence derived from an organism other than the expression-regulating sequence and in a further preferred embodiment, as used herein in the context of a redox factor-regenerating polypeptide, the term "heteroiog" means that the portion of the nucleic acid encoding the redox factor-regenerating polypeptide is from at least one other organism another section, such as the transporter domain or the expression regulation sequence or the transmembrane linker, has been obtained or taken. For example, the redox factor-regenerating polypeptide is heterologous when recovered from Lactobacillus brevis but all other sequences were obtained from E. coli. In a further preferred embodiment, as used herein, the term "heteroiog" means that the nucleic acid sequence termed heteroiogue is from an organism other than the host or intended host used to express or amplify that nucleic acid sequence.

In a preferred embodiment of the present invention, as used herein, the term "signal peptide" refers to a sequence of amino acids, preferably at the N-terminus of a polypeptide, which causes the polypeptide, when expressed in the cytosome of a host cell, to become one In a particularly preferred embodiment, the host cell is a Gram-negative bacterial cell and the signal peptide causes the resulting or final polypeptide to translocate into the peripiasma or outer membrane of the Gram-negative bacterial cell.

In a preferred embodiment of the present invention, the term "protease recognition site" as used herein refers to a particular amino acid sequence motif in a polypeptide, this sequence motif of said protease is specifically recognized such that it binds and cleaves the polypeptide.

In a preferred embodiment of the present invention, as used herein, the term "transmembrane linker" refers to a flexible polypeptide portion useful for linking the auto-transporter domain to the redox factor-regenerating polypeptide but flexible enough to independently fold and / or transport the protein redox factor-regenerating polypeptide.

In a preferred embodiment of the present invention, the term "transporter domain of an auto-transporter" refers to a domain that can be used to obtain the expression product of the nucleic acid molecule when synthesized ribosomally in the interior of the cell, preferably in bacterial cytopiasma, and to the outside Membrane of the cell, preferably translocated to the side of the outer membrane exposed to the cell environment In a particularly preferred embodiment, the transporter domain causes said expression product to be on the outer surface of the outer membrane In a preferred embodiment of the present invention For example, the transporter domain of an auto-transporter is a protein located on the outer membrane of the cell, and the NADH oxidase-encoding portion is part of or fused to a domain, loop, or other portion of the transporter domain, so that the di e NADH oxidase is presented on the surface of the cell. In another preferred embodiment of the present invention, the transporter domain of an auto-transporter is a protein of a system that can be used to present polypeptides on the surface of a cell. In a particularly preferred embodiment, the transporter domain is a transporter domain of the autodisplay system, also referred to as the autotransporter pathway, of the AIDA-I type Gram-negative bacterial tern.

In a preferred embodiment, variants of amino acids or nucleic acid sequences which are explicitly referred to in the present application, for example by the name or the accession number or even by the term "variant of", or implicitly, for example by a description of the function, are included of the present invention Embodiment, the term "variant" as used herein includes amino acid or nucleic acid sequences corresponding to 60, 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99% with the reference In a preferred embodiment, the term "variant" with respect to an amino acid sequence includes those amino acid sequences that have a conservative amino acid exchange or multiple conservative amino acid substitutions with respect to the reference sequence. In a preferred embodiment, the terms "variants" of an amino acid sequence or nucleic acid sequence include active portions and / or fragments of the amino acid sequence or nucleic acid sequence In a preferred embodiment, the term "active portion" as used herein refers to an amino acid sequence or nucleic acid sequence shorter than the full length amino acid or nucleic acid sequence, but at least some of its essential biological activity, e.g. As an NADH oxidase. In a preferred embodiment, the term "variant" of a nucleic acid includes the nucleic acids of the complementary strand that hybridize to the reference nucleic acid, preferably under stringent conditions The stringency of hybridization reactions is readily determinable by one of ordinary skill in the art and is usually empirical as a function of In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures Hybridization generally depends on the ability of the denatured DNA to renaturate when complementary strands are present in an environment whose temperature is lower than the melting temperature of the strands, the higher the degree of homology sought between the probe and a hybridizable sequence, the higher the relative Temperature that can be applied. As a result, higher relative temperatures tend to make the reaction conditions more stringent, while lower temperatures reduce this stringency. Further details and an explanation of the stringency of hybridization reactions can be found in Ex. (1995). In a further preferred embodiment, the term "variant" of a nucleic acid or amino acid refers to a nucleic acid or amino acid having, at least to some extent, the same biological activity and / or function as the reference nucleic acid or amino acid In a preferred embodiment, the term "variant" refers to a Nucleic acid sequence, as used herein, to another nucleic acid sequence encoding an amino acid sequence that is similar to the reference amino acid sequence. In a preferred embodiment of the present invention, as used herein, the term "redox factor" refers to an organic compound that is redox-reactive, ie, that can be oxidized or reduced under physiological conditions. In a preferred embodiment, the redox factor does not comprise any redox-reactive amino acid side chains In a preferred embodiment, the redox factor is a free redox factor, that is, it is not covalently bound to a peptide backbone or peptide.

In a preferred embodiment, the redox factor has a standard reduction potential of not more than 0.85 (ie, for high 0.6V), 0.5, 0.4, 0.2, 0, -0.1, -0.15, -0.2, -0.25, -0.3, or -0.4V up. In a particularly preferred embodiment, the redox factor has a standard reduction potential of -0.325 to -0.2V. In a most preferred embodiment, the redox factor has a standard reduction potential of -0.35 to -0.3.

In a preferred embodiment, the term "redox factor-regenerating polypeptide" as used herein refers to a polypeptide that catalyzes the regeneration of a redox factor. In a preferred embodiment, the term "regenerating a redox factor" as used herein refers to the ability to to reduce an oxidized redox factor that acts as a reducing agent in its reduced form in a chemical synthesis, or the ability to oxidize a reduced redox factor, which in its oxidized form in a chemical synthesis as Oxidationsmitte! acts. In a further preferred embodiment, the term "regeneration of a redox factor" as used herein refers to the restoration of the redox state of a redox factor, preferably the state in which it could be used for a synthesis of interest, most preferably a synthesis by For example, NAD ⁺ consumed in a reaction can be regenerated by oxidizing NADH to NAD ⁺ . In a second embodiment of the first aspect of the present invention, which is also an embodiment of the first embodiment, the redox factor which is regenerated by the redox factor-regenerating polypeptide is selected from the group comprising NADH, NADPH, FADH _2l heme, metal ions, Glutathione, pyrroloquinoline quinone (PQQ), pyridoxal phosphate, thiamine pyrophosphate and ascorbate. Metal ions include, but are not limited to, Fe, Cu, Zn, Ni, Co, Mn, Cr, and Mg ions.

In a preferred embodiment, each of the aforementioned redox factors has both the oxidized and reduced forms or forms of the corresponding conjugated redox couple, for example, both NADH and NAD ^{1 "} in the case of the redox factor NADH, although only one form is expiicitly mentioned.

In a third embodiment of the first aspect of the present invention, which is also an embodiment of the first and second embodiments of the present invention, the redox factor-regenerating polypeptide comprises one or more flavin cofactors.

In a preferred embodiment, the term "flavin cofactor" as used herein refers to a redox-reactive cofactor based on the isoafoxazine ring system In a preferred embodiment, the flavin cofactor is selected from the group comprising riboflavin, flavin mononucleotide (FMN) and fiavin adenine dinucleotide (FAD), both in their oxidized and reduced forms.

In a fourth embodiment of the first aspect of the present invention, which is also an embodiment of the first to third embodiments, the redox factor-regenerating polypeptide is selected from the group comprising NADH oxidase, formate dehydrogenase and glucose dehydrogenase.

In a fifth embodiment of the first aspect of the present invention, which is also an embodiment of the first to fourth embodiments, the redox factor-regenerating polypeptide is selected from the group comprising the NADH oxidases from the genera Lactobacillus, T ermus, Brevibacterium and Streptococcus, preferably the NADH oxidase from Lactobacillus brevis, and variants of it. In a sixth embodiment of the first aspect of the present invention, which is also an embodiment of the first to fifth embodiments, the transporter domain of an autotransporter is selected from the group comprising Ssp, Ssp-h1, Ssp-h2, PspA, PspB, Ssa1, SphB1 , AspA / NalP, VacA, AIDA-I, IcsA, MisL, TibA, Ag43, ShdA, AutA, Tsh, SepA, EspC, EspP, Pet, Pic, SigA, Sat, Vat, EpeA, EatA, Espi, EaaA, EaaC , Pertactin, BrkA, Tef, Vag8, PmpD, Pmp20, Pmp21, AgA1 protease, App, Hap, rOmpA, rmpmp, ApeE, EstA, Lip-1, McaP, BabA, SabA, AlpA, Aae, NanB and variants thereof.

In another preferred embodiment of the present invention, the term "transporter domain of an auto-transporter" as used herein includes a domain presenting a polypeptide other than the transporter domain, particularly a redox factor-regenerating polypeptide, on the surface of a cell when said protein has been inserted into or fused to the amino acid sequence of the transporter domain.

Preferably, the redox factor-regenerating polypeptide is fused to a transporter domain of an auto-transporter. The transporter domain of the autotransporter according to the invention may be any transporter domain of an auto-transporter and is preferably capable of forming a β-barrel structure. A detailed description of the β-barrel structure and preferred examples of β-barrel auto-transporters are disclosed in WO 97/35 022 and incorporated herein by reference. Henderson et al. (2004) describe auto-transporter proteins with suitable auto-transporter domains (a summary can be found in Table 1 of Henderson et al., 2004). The disclosure of Henderson et al. (2004) is incorporated herein by reference. For example, the transporter domain of the autotransporter can be selected from: Ssp (P09489, S. marcescens), Ssp-h1 (BAA33455, S. marcescens), Ssp-h2 (BAA 11383, S. marcescens), PspA (BAA 36466, P. fluorescens), PspB (BAA36467, P. fluorescens), Ssa1 (AAA80490, P. haemolytica), SphB1 (CAC44081, pertussis), AspA / NalP (AAN71715, N. meningitidis), VacA (Q48247, H. pylori), AIDA-I (Q03155, E. coli), IcsA (AAA26547, S. flexneri), MisL (AAD16954, S. enterica), TibA (AAD41751, E. coli), Ag43 (P39180, E, coli), ShdA (AAD251 10, S. enterica), AutA (CAB89117, N.A. meningitidis), Tsh (I54632, E. coli), SepA (CAC05786, S. flexneri), EspC (AAC44731, E. coli), EspP (CAA66144, E. coli), Pet (AAC26634, E. coli), Pic (AAD23953, E. coli), SigA (AAF67320, S. flexneri), Sat (AAG30168, E. coli), Vat (AAO21903, E. coli), EpeA (AAL18821, E. coli), EatA (AA017297, E. coli), Espl (CAC39286 , Co co //), EaaA (AAF63237, co co //), EaaC (AAF63038, co co //), pertactin (P14283, B. pertussis), BrkA (AAA51646, β-pertussis), Tef (AAQ82668, β pertussis), Vag8 (AAC31247, pertussis), PmpD (084818, C. trachomatis), Pmp20 (Q9Z812, C. pneumoniae), Pmp21 (Q9Z6U5, C pneumoniae), IgA1 protease (NP_283693, Λ /. meningitidis) , App (CAC 14670, / v. Meningitidis), IgA1 protease (P45386, H. influenzae), Hap (P45387, H. influenzae), rOmpA (P15921, R. rickettsii), rOmpB (Q53047, P. rickettsii), ApeE (AAC38796, S. enterica) , EstA (AAB61674, P. aeruginosa), Lip-1 (P40601, X. luminescens), McaP (AAP97134,. catarrhalis), BabA (AAC38081, py / o / 7), SabA (AAD06240, H. py / π), AlpA (CAB05386, H. py / on), Aae (AAP21063, \. actinomycetemcomitans), NanB (AAG35309, P haemolytica) and variants of these car transporters. For each of the exemplary autotransporter proteins, examples of suitable Genbank accession numbers and species from which the autotransporter can be obtained are given in the form of a killer. Preferably, the transporter domain of the autotransporter is the AIDA-i protein of E. coli or a variant thereof, such as those described by Niewert et al. (2001). The AIDA autotransporter system has been associated with F18 and Stx2e in pig / o-isolates from pigs diagnosed with edema and so-called post-weaning.

Variants of the above-described auto transporter sequences can be obtained, for example, by altering the amino acid sequence in the loop structures of the β-barrir which do not belong to the transmembrane sections. Optionally, the nucleic acids encoding the surface shingles can be completely deleted. In addition, within the amphipathic β-sheet structures, conservative amino acid substitutions, ie, the replacement of one hydrophilic with another hydrophilic amino acid and / or the replacement of one hydrophobic with another hydrophobic amino acid, can be made. Preferably, a variant at amino acid level has a sequence identity of at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% with the corresponding naturally occurring sequence of the autotransporter domain, especially in the area of beta-sheet structures.

In a seventh embodiment of the first aspect of the present invention, which is also an embodiment of the first to sixth embodiments, the redox factor-regenerating polypeptide is oxygen sensitive. In a preferred embodiment, as used herein, the term "oxygen sensitive," as used herein, means that the corresponding polypeptide undergoes rapid deactivation in the presence of oxygen, preferably at normal, ie, atmospheric, concentrations. In another preferred embodiment, the term " oxygen sensitive, "as used herein, that the corresponding polypeptide loses at least 10, 20, 30, 40, preferably 50, most preferably 80% of its activity, preferably with respect to its kcat, when exposed to oxygen in concentrations or below for at least 30 minutes Conditions in the atmosphere, both in solutions and in other phases.

In another embodiment, the redox factor-regenerating polypeptide is a soluble polypeptide. In a preferred embodiment, the term "soluble polypeptide" as used herein refers to a polypeptide that is not bound to a membrane in its natural environment For example, the Lactobacillus brevis NADH oxidase, a cytosolic protein, is a soluble polypeptide In another preferred embodiment, the redox factor-regenerating polypeptide is a membrane-bound polypeptide, ie, a polypeptide that is covalently or non-covalently bound or attached to a cell membrane or an integral membrane protein For example, the complex IV of the respiratory chain is a membrane-bound polypeptide.

In a second aspect, the object of the present invention is achieved by a polypeptide encoded by a nucleic acid molecule according to an embodiment of the first aspect of the present invention. In a preferred embodiment, the polypeptide comprises SEQ ID NO: 2 or variants thereof. According to a third aspect, the object of the present invention is achieved by a cell which has on its surface a polypeptide according to the second aspect expressed or transformed using a nucleic acid molecule according to an embodiment of the first aspect of the present invention.

In a preferred embodiment, the term "cell", which is used interchangeably with the term "host cell" as used herein, refers to a cell capable of expressing a polypeptide. Such a cell may also be referred to as a "whole-cell catalyst or biocatalyst." In another preferred embodiment, the cell or host cell is a prokaryotic cell, preferably a Gram-negative bacterial cell, most preferably an E. coli cell In another embodiment, the cell is a eukaryotic cell In another preferred embodiment, the cell or host cell is a spore of a prokaryote.

According to a fourth aspect, the object of the present invention is achieved by a membrane fraction which can be obtained from the cell according to the third aspect of the present invention.

A bacterial cell comprises a series of compartments separated by hydrophobic membranes. A Gram-positive bacterial cell has a plasma membrane that confines the cytosol, the interior of the cell. The plasma membrane is surrounded by a peptidoglycan layer. In contrast, Gram-negative bacteria have, in addition to the plasma membrane, another membrane called the outer membrane. The term "surface" as used herein preferably refers to a layer of the microorganism which is exposed to the environment, the environment being, for example, the liquid culture medium used to grow the cell of interest In a most preferred embodiment a polypeptide according to the present invention is expressed on the outside of the outer membrane of a Gram-negative bacterial cell In a preferred embodiment, a polypeptide according to the present invention on the inside of the outer membrane of a Gram-negative Bacterial cell expressed. In a further preferred embodiment, the polypeptide of the invention is expressed on the outside of a spheroplast, which is a Gram-negative bacterial cell in which the outer membrane has been removed. Those skilled in the art will be familiar with methods that can be used to make spheroplasts. In a further preferred embodiment, the polypeptide according to the invention is expressed on the outside of a gram-positive bacterial cell. Just as the terms "presented on the surface" and "expressed on the surface" are used here, they are synonymous.

In a preferred embodiment of the present invention, the membrane fraction or the membrane preparation, wherein the two terms are used interchangeably, comprises a redox factor-regenerating polypeptide according to the first aspect of the invention, preferably in a catalytically active state. The terms "membrane fraction" and "membrane preparation" as used herein preferably refer to a product that accumulates in membrane constituents, preferably constituents of the outer membrane of a Gram-negative bacterium. One skilled in the art will be familiar with methodology and procedures that can be used to prepare membrane preparations. For example, bacterial cells can be harvested from a culture and lysed, for example by freeze-thaw cycles, sonication, resuspension in lysis buffer or the like, followed by differential centrifugation to isolate membrane fractions of the cells. In a preferred embodiment of the present invention, the membrane preparation is an outer membrane preparation, ie a preparation in which the outer membrane constituents are enriched compared to the constituents of other membranes and compartments, such as the cytosol, inner membrane and periplasm are. One of ordinary skill in the art will be familiar with methodology and procedures that may be used to isolate or enrich the outer membrane or components thereof, such as lysozyme treatment of bacterial cells and subsequent centrifugation steps. In a preferred embodiment, the membrane fraction may be a treated membrane fraction, ie, the content or properties of the membrane fraction have been altered, for example by purifying a protein component of the membrane fraction or by solubilizing the membrane fractions and / or incorporation of components the membrane fraction in vesicles. In a preferred embodiment of the present invention, the membrane fraction may be immobilized - for example on the surface of a vessel or column.

According to a fifth aspect, the object of the present invention is achieved by a method for regenerating a redox factor, comprising the following steps: a) providing the polypeptide according to the second aspect, the membrane preparation according to the fourth aspect or the cell according to the third aspect of the present invention and b) contacting the polypeptide according to the second aspect, the membrane preparation according to the fourth aspect or the cell according to the third aspect with at least one substrate of the redox factor-regenerating polypeptide. In a preferred embodiment, step a) and / or step b) take place in the absence of a reducing agent and / or in an oxidative environment.

According to a sixth aspect, the object of the present invention is achieved by using the cell according to the third aspect or the membrane fraction according to the fourth aspect or the polypeptide according to the second aspect for regenerating a redox factor.

According to a seventh aspect, the object of the present invention is achieved by the use of the cell according to the third aspect, the membrane preparation according to the fourth aspect and the polypeptide according to the second aspect of the present invention for adjusting the redox environment of an aqueous solution, preferably by deoxidizing the aqueous Solution.

In a preferred embodiment of the present invention, the term "adjusting the redox environment of an aqueous solution" as used herein refers to any action directly related to the redox conditions in an aqueous solution, ie, the ability of the solution to oxidize or bind a compound In a preferred embodiment of the present invention, the term "deoxygenating an aqueous solution" as used herein refers to any action that can be taken to reduce the concentration of oxygen in that solution. The person skilled in the art is familiar with methods for measuring the oxygen concentration in an aqueous solution can be used, such as the use of oxygen electrodes.

According to an eighth aspect, the object of the present invention is achieved by a method of producing a cell presenting on its surface a redox factor-regenerating polypeptide, comprising: (a) introducing the nucleic acid into a cell according to an embodiment of the first aspect of the present invention and b) optionally contacting the cell with one or more redox reactive prosthetic groups.

According to a ninth aspect, the object of the present invention is achieved by a method for producing the product of a redox factor-dependent polypeptide, comprising the following steps: a) providing a redox factor-dependent enzyme and (b) contacting the redox-factor-dependent polypeptide with one or more of its substrates in the presence of the line according to the third aspect, the membrane preparation according to the fourth aspect and the polypeptide according to the second aspect of the present invention, whereby the polypeptide according to the second aspect regenerates the redox factor on which the redox factor-dependent polypeptide depends , In a preferred embodiment, the term "redox factor-dependent polypeptide" as used herein refers to a polypeptide having a biological activity, preferably an enzyme activity, which requires a redox factor as cofactor, prosthetic group or, preferably, substrate.

In another preferred embodiment, the object of the present invention is achieved by a composition comprising the cell according to the third aspect, the membrane preparation according to the fourth aspect and the polypeptide according to the second aspect of the present invention, and a redox factor-dependent polypeptide and the redox factor. In one embodiment, the redox factor-dependent polypeptide is presented using the autotransporter system, ie, its expression is effected by a nucleic acid comprising: (1) a portion encoding a signal peptide, (2) a portion that is a preferably heterologous one redox factor-regenerating polypeptide or a variant thereof, (3) optionally one Section encoding a protease recognition site; (4) a section encoding a transmembrane linker; and (5) a section encoding a transporter domain of an autotransporter or a variant thereof.

The present invention is further illustrated by the following figures and non-limitative examples to which further features, embodiments, aspects, and advantages of the invention may be derived.

Fig. 1 shows the restriction map of the plasmid pAT-NOx.

Figure 2 shows an SDS gel showing NADH oxidase in the outer membrane of E. coli as well as orientation by whole cell digestion. M) molecular weight standard; 1) E. coli UT 5600 {DE3); 2) E. coli UT5600 (DE3) pAT-NOx, not induced; 3) E. coli UT5600 (DE3) pAT-NOx induced; 4) E. coli UT5600 (DE3) pAT-NOx induced, digested with proteinase K; 5) E. coli UT5600 (DE3) pAT-NOx induced, digested with trypsin.

Fig. 3 shows absorbance spectra of the supernatant following an NADH oxidase assay using A) E. coli UT5600 (DE3) pAT-NOx and β) E. coli BL21 (DE3) paT-NOx, each after 1 h of induction ,

Fig. 4 shows the absorption spectra of the supernatant following an NADH oxidase assay using A) E. coli UT5600 (DE3) pAT-NOx and B) E. coli BL21 (DE3) paT-NOx, each after 4 hours Induction.

Figure 5 shows the absorption spectra of the supernatant following an NADH oxidase assay using A) E. coli UT5600 (DE3) pAT-NOx and B) E, coli BL21 (DE3) paT-NOx, each after 20 h Induction.

Fig. 6 shows the results of the NADH oxidase assay performed on microtiter plates using E. Coli BL21 (DE3) pAT-NOx after growth in M9 medium. The figure shows the decrease in absorbance at 340 nm over time.

Figure 7 shows the results for the NOX whole cell biocatalyst stored at + 8 ° C for more than 7 weeks. The activity test was performed with cells pre-stored [A] and cells given one week [B], two weeks [C], three weeks [D], four weeks [E], five weeks [F], six weeks [G] and seven weeks [H] had been stored.

Figure 8 shows the results for the NOX whole-cell biocatalyst stored at -18 ° C for more than 7 weeks. The activity test was carried out with cells before storage [A] and lines lasting one week [B], two weeks [C], three weeks [D], four weeks [E], five weeks [F], six weeks [G ] and seven weeks [H] had been stored.

Figure 9 shows the results for the NOX whole cell biocatalyst stored at -70 ° C for more than 7 weeks. The activity test was carried out with cells before storage [A] and cells that lasted one week [B], two weeks [C], three weeks [D], four weeks [E], five weeks [F], six weeks [G ] and seven weeks [H] had been stored.

Fig. 10 shows the determination of the number of cells in samples stored for stability tests for more than seven weeks. It is the decrease in the number of cells in samples stored at -18 ° C (■), as well as the decrease in the number of cells in samples stored at + 8 ° C (·) and in samples stored at -70 ° C (A). To determine the number of cells, 50 μΙ were taken from a stored sample and used to prepare a series of dilutions. 50 ml of each of the 10 ^{~ 9} - and 10 ^"0 dilutions was plated on 30 mg / i kanamycin LB agar plates The plates were incubated at 37 ° C overnight, and the next day, the colonies were counted the number.. of cells / ml was calculated based on the number of co-ionizing units.

Fig. 11 shows the recyclability of the NOX whole-cell biocatalyst in a test which was repeated five times in a cyclic manner: ♦ = host strain E. coli BL21 (DE3), ~~ E. coli BL21 (DE3) pAT-NOX. In the narrower sense, the photometric determination of the decrease in NADH concentration in the first [A], second [B], third [C], fourth [D] and fifth [E] cycle is shown.

12 shows the regeneration of NAD * using the biocatalyst E. coli BL21 {DE3) pAT-NOx, in the narrower sense the NADH conversion by the whole-cell biocatalyst E. coli BL21 (DE3) pAT-NOx (■), and in the regeneration system (·). As a control, E. coli BL21 {DE3) pAT-NifAf (Δ) was used. It became the extinction of NADH at 340 nm. The absorbance of the cells was monitored at 590 nm. The evaluation was carried out by calculating the difference E ₃₄₀ -E ₅ g ₀ . A1DH 60 mU, lines OD 1, reaction started by 200 μ NADH.

Figure 13 shows the regeneration of NAD ⁺ , more specifically the concentration of NADH as a function of absorbance at 340 nm over time, especially the NADH concentration during the AIDH reaction and during the AIDH reactions using E. coli BL21 (DE3) pAT-NOx. The NADH concentration was normalized by calculating the quotient E _{3 (} , ₀ / E59o relative to the number of cells: AIDH 30 mU, E. coli BL21 (DE3) pAT-NOx OD 5, E. coli BL21 (DE3) OD 1, reaction started by 400 μΜ NAD ⁺ .

Examples

It is possible to detect the surface finish of a passenger, in this case NADH oxidase, by incubating the bacteria in the presence of serine protease, trypsin or protein kinase K. Because trypsin and proteinase K are unable to pass through the outer membrane of E. coli because of their size, incubation of whole cells in the presence of these proteases results in the digestion of only the proteins that are on the surface of the outer membrane. Thus, a surface passenger is digested while protein components integrated into the membrane are protected from enzyme attack and therefore remain intact. This can be confirmed by SDS-PAGE. The orientation of the enzyme was assessed by whole cell digestion. Figure 2 depicts the isolated outer membranes of E. coli strain UT5600 (DE3) pAT-NOx after expression induction with IPTG and also isolated outer membranes after whole cell digestion. Upon induction with IPTG, the expected fusion protein of about 100 kDa be detected in the outer membrane (lane 3). Digestion of whole cells with trypsin resulted in a decrease in the intensity of the corresponding band as compared to the OmpF / C and OmpA representing bands of initially approximately equal intensity, thus confirming that the protein is oriented towards the outside of the membrane (lanes 4 and 5) ). It has been demonstrated that NADH oxidase is presented on the surface of E. coli using the auto-display system. Example 2: Establishment of Growth and Test Conditions for Tracking NADH Oxidase Activity

The activity of NADH oxidase was detected by a photometric assay. NADH shows two extinction maxima, namely at the wavelengths 260 nm and 340 nm, while NAD ^{+ shows} only one maximum, at 260 nm. This difference can be exploited to follow the oxidation of NADH to NAD * photometrically. FAD was added to the nutrient media or buffers used to allow proper folding of the enzyme as well as uptake of FAD.

Breeding the NOX biocatalyst

20 ml of LB medium (15 μg ml of kanamycin) were inoculated with 10 .mu.l of Gyclerin culture of the corresponding strain and incubated at 37.degree. C. and 200 revolutions per minute overnight. To prepare the main culture, 100 ml of LB medium (15 μg ml kanamycin, 10 μM EDTA, 10 mM mercaptoethanol) were inoculated with 2.5 ml of the overnight culture. At OD ₅₇₈ = 0.6, 50 ml of the culture was induced with 1 mM IPTG. In addition, 10 μΜ FAD were added in the case of NADH oxidase. The cultures were induced for 1 h, 4 h or 20 h at 30 ° C and 200 revolutions per minute. The other half of the culture was not induced but otherwise treated exactly as the induced culture.

Prepare the cells for the NADH oxidase assay

Following induction, the cells were first stored on ice for 15 minutes and then pelleted by sedimentation at 5000 rpm for 10 minutes. The sediment was washed twice in 20 ml of 0.1 M potassium phosphate buffer, pH 7.5, 10 μΜ FAD. The OD of the cells was then adjusted to 50 with the same buffer.

NADH oxidase tests

The test for detecting NADH oxidase activity was carried out with 1 m! Samples containing 0.1 M potassium sulfate buffer, pH 7.5, 1 mM DTT and 100 μM NADH as substrate. Cells were assayed at a final OD ₅₇₈ of 1 in the test After one hour of incubation at room temperature, the cells were removed from the samples by centrifugation. The supernatant spectra were recorded at wavelengths between 200 and 400 nm.

First, expression of the NADH oxidase gene was induced for 1 h in both strains. The UV spectra in Fig. 3 show that it was impossible to detect enzyme activity under these conditions because the NADH had already been oxidized by host cells lacking the plasmid, resulting in the disappearance of the absorbance maximum at 340 nm. The added enzyme is either taken up by the cells or reacted by enzymes secreted by the cells. Both effects can be explained by the high metabolic rate that the cells exhibit in the exponential growth phase, as shown by the cells here.

In order to put the cells in the stationary growth phase and thus to minimize their primary metabolism, the expression induction was initially extended to 4 h. It turned out that the added NADH had again been oxidized by the host cells (FIG. 4). However, the NADH oxidizing activity of cells containing pAT-Nox exceeded the activity of host cells lacking the plasmid. In addition, a quantitative difference between uninduced and induced cells could be detected.

After an extension of the induction time to 20 h, NADH oxidation by host cells lacking the plasmid could no longer be detected, as shown by the fact that in the case of negative control without cells, the extinction maximum of NADH is still present, as in FIG 5 is shown. Now it was possible to detect the oxidation of the added NADH by the surface NADH oxidase in these so-called "quiescent cells" (cells in the stationary phase) .Different differences between E. coli BL21 (DE3) and E. coli UT5600 (DE3) are obvious, in the case of E. coli BL21 (DE) pAT-NOx, uninduced cells convert about half of the NADH offered into NAD ⁺ , while induced cells transduce all NADH present thus explain that the T7 system used for the expression of NADH oxidase is not "dense", ie that the repression of the T7 promoter is incomplete, which leads to low expression of the gene by T7 polymerase even in the uninduced state , In contrast, E. coli UT5600 (DE3) shows no activity in the uninduced state, however, induced cells convert only about half of the NADH.

These results demonstrate that the Lactobacillus brevis flavoprotein NADH oxidase can be expressed on the surface of E. coli in its active form and using the autotransporter system. The subsequent incorporation of FAD into the apoenzyme appears to proceed properly and requires only the addition of FAD. All measurements so far have been of a qualitative nature to set up the test and to find conditions to track the activity of the enzyme.

For quantitative tracking of enzyme activity by measuring NADH oxidation over time, the optical assay was transferred to microtiter plates to allow parallel observation of as many samples as possible.

Growing the cells in minimal medium (M9)

10 ml of M9 medium (15 μg ml of kanamycin) were inoculated with 20 .mu.l of glycerol culture of the corresponding strain and incubated at 37.degree. C. and 200 revolutions per minute overnight. For inoculating the major culture, 160 ml M9 (15 g ml kanamycin), the entire overnight culture was used. After culturing the cultures for 7 hours, 1 mM IPTG and 0.2% lactose as a carbon source were added to half of the culture. In addition, 10 μΜ FAD were added for NADH oxidase. The induction was carried out for 16 h at 30 ° C and 200 revolutions per minute. No IPTG was added to the other half of the cultures, otherwise these cultures were treated in exactly the same way.

Prepare the cells for activity testing

After induction, the cells were first stored on ice for 15 minutes and then pelleted by sedimentation at 5000 rpm for 10 minutes. The sediments were washed twice in 20 ml 0.1 M potassium phosphate buffer, pH 7.5, 10 μΜ FAD. Then the cells were adjusted to OD ₅₇₈ = 10 with the same buffer,

NADH oxidase assay on microtiter plates The NADH oxidase assay for 96-well microtiter plates was performed in samples of 240 μΙ per well in 0.1 M potassium phosphate buffer. The cells were used in amounts such that an OD578 of 1 was achieved in the assay. 200 μΜ NADH was added to start the reactions. After a 0, 3, 7, 15, 30, 45, or 60-minute incubation at room temperature, the assay was monitored in a microtiter plate at 340 nm (Mithras, Berthold Technologies). In order to avoid sedimentation of the cells, the plate was shaken before the absorbance measurement. As a reference, a cell suspension of the corresponding OD in buffer was used.

Based on a series of experiments, the product tint of the NADH oxidase whole cell catalyst could be optimized to have i) higher activities, ii) increase the difference between induced and uninduced cells, and iii) only one in the nonsense controls low activity occurred. In particular, it has been found that the culture used to measure NADH oxidase must be in an optimal state. Figure 6 shows the results of an NADH oxidase assay performed under standard conditions as described in the section "Methods".

Several factors related to the growth and test conditions are responsible for this improved enzyme activity. Already with a view to a larger scale use, the growth of the cells was modified such that M9 medium defined instead of the complex LB medium was used. During induction, lactose was added as a carbon source and the cells were induced earlier, at an OD ₅₇ 8 of about 0.35. Subsequent induction, usually 0.6, results in the need for too long growth in the M9 medium, resulting in spontaneous cell lysis, with the effect that both the nonsense controls and the uninduced controls do oxidized as a substrate supplied NADH. This is most likely due to enzymes released as a result of cell lysis.

The NADH oxidase assay was then performed directly in the cell suspension as described above in the microtiter plate and observed. This makes handling much easier and the data is easier to reproduce. A determination of absolute values of the enzyme activity is not possible. The activity of enzymes determined by a photometric method is calculated according to Lambert-Beer's law. In order to apply this equation, the layer thickness of the cuvette or the 96-weli plate must be known. In the present experimental approach, this parameter can not be determined. Therefore, the activity of NADH oxidase was determined with a photometer, using cuvettes with a defined layer thickness of 1 cm. Consequently, the Lambert-Beer law can be applied. At a temperature of 30 ° C., the activity in the LB medium was determined to be 12.23 mU / ml. The activity at 30 ° C in the M9 medium was slightly higher, namely 16.38 ml / ml. In the publication by Hummel et al. (2003) reported the activity of a readily purified enzyme at 10-15 U / mg.

Step Example! 3: Stability and storage stability test of the NADH oxidase whole cell catalyst

For the study of shelf life and stability of the NADH oxidase whole cell catalyst, induced cells were stored at 8 ° C in the refrigerator or at -18 ° C or at -70 ° C in the freezer. These cells were checked weekly by triplicate with the NADH oxidase assay.

The cells intended for storage were washed twice with potassium phosphate buffer (0.1 M, pH 7.5) following the induction of gene expression, adjusted to OD ₅₇ 8 = 10 in this buffer, divided into partial samples and grown at + for eight weeks Stored at 8 ° C. When cells were to be stored at -18 ° C and -70 ° C, respectively, 20% glycerol was added to the freeze buffer. For activity assay, the cells were diluted to OD ₅₇₃ -1 in 200μl samples with the above-mentioned buffer and then 0.2mM NADH was added to start the reaction. The oxidation of NADH to NAD was tracked in triplicate by measuring the absorbance decrease at λ _max = 340 nm. To account for the effect of cell lysis occurring during storage, the host strain E. coli BL21 (DE3) was similarly stored and subjected to the same type of activity assay as the whole cell biocatalyst BL21 (DE3) pAT-NOX. The panels in Figure 7 show the decrease in NADH concentration in cell suspensions of different ages stored at 8 ° C. As expected, the host strain E. coli BL21 (DE3) used as a control does not show any NADH activity before storage. However, after a one-week storage period, a marked decrease in the NADH concentration in the test can be detected in these host cells, the decrease being more pronounced the longer the storage takes. This putative NADH oxidase activity can be explained by the lysis of bacterial cells, which experience has shown to be relatively rapid on storage of cells at 8 ° C. Cells stored at this temperature in the refrigerator show an increased viscosity in solution after just a few days and can not be completely resuspended. This increased viscosity is due to release of chromosomal DNA in the cell. In addition, as a result of lysis, enzymes are released from the cells which apparently oxidize the NADH used as a substrate in the assay. This effect can also be observed in the case of storage of the NADH oxidase whole cell catalyst E. coli BL21 (DE3) pAT-NOX and a subsequent activity test on it. As the duration of storage increases, NADH oxidation, which is caused by the lysis of the cells in the refrigerator, exceeds the activity of the catalyst measured before storage. Consequently, it is not recommended to store the NADH oxidase whole cell catalyst in the refrigerator.

When cells are stored at -18 ° C, the situation is different (Figure 8). The host lines used as a negative control after storage in a freezer at -18 ° C no oxidation of NADH result, whereas the NADH oxidase whole-cell Kataiysator even after 8 weeks still shows an NADH oxidase activity, which - in a Normal distribution - comparable to the initial activity.

In a 7-week storage of the total Zelf biocatalyst at -70 ° C, no significant decrease in activity is observed (Figure 9). Lysis of host lines can also be excluded in this case. Consequently, it is recommended to store the whole-cell biocatalyst at -70 ^D C.

In the meantime, the number of living cells has been determined parallel to the activity of the biocatalyst. The results are shown in FIG. When stored at +8 ° C, the number of live cells decreases by three within the first four weeks Orders of magnitude. In a further three weeks of storage then the number of living cells remains relatively constant. This curve can be observed even at storage of the biocatalyst at -18 ° C and -70 ° C, respectively. However, in this case, the decrease in the number of live lines during the first four weeks is only two orders of magnitude. The decrease in the number of live cells does not correlate with the activity of the biocatalyst over a period of seven weeks. While the number of living cells decreases when stored at -18 ° C and -70 ° C, the activity remains approximately constant. However, this experiment shows that storage in the freezer (-18 ° C or -70 ° C) is preferable to storage at + 8 ° C in the refrigerator.

Example 4 Investigation of the Recyclability of the Whole Cell Biocatalyst

The recyclability of whole-cell Catalyst E. coli BL21 (DE3) pAT-NOx was determined for a 5-minute reaction of NADH over a period of 30 minutes. For this assay, following induction of gene expression, cells were washed twice with potassium phosphate buffer (0 , 1 M, pH 7.5) and resuspended in this buffer to a final OD ₅ 7 ₈ of 10. For the determination of activity, cells in 200 μΙ samples were diluted with the above-mentioned buffer in the microtiter plate to an OD ₅₇ 8 of 1 and then 0.2 mM NADH was added to start the reaction. The oxidation of NADH to NAD was tracked in triplicate by measuring the absorbance decrease at λ _max = 340 nm. For recycling, after completion of the reaction, the tents were spun down and resuspended in 160 μM potassium phosphate buffer. Again, 0.2 mM NADH was added to start the reaction and the oxidation was followed photometrically. This procedure was repeated for a total of 5 cycles. In order to take into account the influence of the zetllyis which may occur during the treatment, the host strain E. coli BL21 (DE3) was subjected to the same test as the whole-cell biocatalyst BL21 (DE3) as a negative contrast.

As control, host cells were used which lacked the biocatalyst. As can be seen from Figure 11, the host cells do not convert NADH. In the first cycle (A), the whole-cell biocatalyst converts almost 100% of the existing NADH within 30 minutes. In the further cycles, the turnover and the turnover speed decrease. In the last cycle (5th cycle, D) within the Incubation time 30% less NADH implemented. The decrease in activity in subsequent cycles could be due to the centrifugation of the bacterial suspension. The centrifugal force is a mechanical stress for the cell and in particular for the molecules expressed on its surface.

Example 5: Regeneration of NADH to NAD ⁺ using the whole-cell biocatalyst E. coli BL21 (DE3) pAT-NOx

This experiment was carried out to investigate whether the whole-cell biocatalyst E. coli BL21 (DE3) pAT-NOx can be used as a regeneration system for NAD ⁺ -reducing enzymes. This was attempted using a system comprising the whole-cell biocatalyst and an NAD ⁺ -reducing enzyme, aldehyde dehydrogenase (AIDH). AIDH belongs to a group of enzymes that oxidize aldehydes to the corresponding carboxylic acids by reducing NAD ⁺ to NADH. The concentration of NADH was traced photometrically.

As can be seen from Fig. 12, as expected, the NADH concentration in the sample containing the whole-cell biocatalyst E. coli BL21 {DE3) pAT-Nox decreases continuously. In contrast, equilibrium between NAD ⁺ and NADH is achieved when the recombinant enzyme AIDH is added to the biocatalyst because NAD +, which is continuously reduced to NADH by the AIDH, is regenerated by the biocatalyst E. coli BL22 (DE3) pAT-Nox. Consequently, this system is in equilibrium. The so-called nonsense control was E. coli BL21 (DE3) -NitAf. These cells present on the surface the nitrilase of A. faecalis, which is unable to oxidize NADH, as can also be seen in FIG.

Figure 13 shows in the upper half of the panel the AIDH mediated NADH increase. As for the addition of the biocatalyst E. Coli BL21 (DE3) pAT-NOx to the regeneration of NAD ⁺ , the host strain E. coli BL21 (DE3) had already been added to this reaction to increase the NADH concentration {as absorbance at 340 nm) with respect to the number of cells. After addition of the biocatalyst E. coli BL21 (DE3) pAT-NOx to the AIDH reaction, no increase in NADH could be detected over time, because of AIDH formed NADH was continuously reoxidized to NAD ⁺ . Consequently, this system is in equilibrium.

Sources

As far as reference is made here to various documents of the prior art, these documents are incorporated by reference in their entirety part of the present description. The following is the complete bibliographic information for the cited prior art documents:

Sanjust e. (1997), Biochem Mol Biol Int (3), 555-62.

Hummel and Riebe! (2003), Biotechnol Lett. (1), 51-4.

Ahmed and Claiborne (1992), J. Biol. Chem. 267 (36): 25822-9.

El-Zahab et al. (2004), Appl Biochem Biotechnol., 17 (3), 165-74.

Lee and Ping (2007), Biotechnology Adv 25, 369-384.

Jose and Meyer (2007), Microbio! Mol Biol Rev 71 (4), 600-9.

Jose (2006), Appl. Microbiol. Biotechnol. 2006, 69, 607-614.

Jose ei al. (2002), J. Biotechnol. 2002, 95, 257-268.

Jose ei al. (1995), Mol. Microbiol., (2), 378-80.

Ausubel et al. (1995), Current Protocols in Molecular Biology, Wiley Abstracts

Publishers.

Henderson et al. (2004), Microbiol. and Molecular Biology Reviews, 68 (4), 692-744. Jose and von Schwichow (2004), ChemBioChem, 5, 491-499.

Banerjee et al. (2002) Appl. Microbiol. Biotechnol. 2002, 60, 33-44.

Sarborsky and Ogletree (1974), Biochem Biophys Res Commun., 61 (1): 210-6.

"Export Systems for recombinant proteins", international patent application WO 97/35 022.

Niewert et al. (2001) Diarrhea. Clin. Diagn. Lab. Immunol. (1): 143-149; 9.

The features of the present invention disclosed in the specification, the sequence listing, the claims and / or the drawings may, both alone and in any combination, be material to the realization of the invention in its various forms.

Claims

claims

1 . Nucleic acid molecule comprising the following components:

(1) a portion encoding a signal peptide

(2) a section comprising a hetero-redox factor-regenerating polypeptide,

(3) optionally a portion encoding a protease recognition site,

(4) a portion encoding a transmembrane linker, and

(5) a portion encoding a transporter domain of an autotransporter or a variant thereof.

The nucleic acid molecule of claim 1, wherein the redox factor regenerated by the redox factor-regenerating polypeptide is selected from the group comprising NADH, NADPH, FADH ₂ , heme, metal ions, glutathione, pyrroloquinoline quinone, pyridoxaiphosphate, thiamine pyrophosphate and ascorbate ,

3. Nucleic acid molecule according to one of claims 1 to 2, wherein the redox factor-regenerating polypeptide comprises one or more flavin cofactors.

4. Nucleic acid molecule according to one of claims 1 to 3, wherein the redox factor-regenerating polypeptide is selected from the group comprising NADH oxidase, formate dehydrogenase, glucose dehydrogenase.

The nucleic acid molecule of claim 4, wherein the redox factor-regenerating polypeptide is selected from the group comprising the NADH oxidases of Lactobacillus brevis, Thermus thermophilus, Thermus aquaticus, Brevibacterium sp. KU139 and Streptococcus faecalis, preferably the Lactobacillus brevis NADH oxidase, and variants thereof.

6. A nucleic acid molecule according to any one of claims 1 to 5, wherein the transporter domain of an autotransporter is selected from the group comprising Ssp, Ssp-h1, Ssp-h2, PspA, PspB, Ssa1, SphB1, AspA / NalP, VacA, AIDA-I , IcsA, MisL, TibA, Ag43, ShdA, AutA, Tsh, SepA, EspC, EspP, Pet, Pic, SigA, Sat, Vat, EpeA, EatA, Espl, EaaA, EaaC, Pertactin, BrkA, Tef, Vag8, PmpD . Pmp20, Pmp21, AgA1 Protease, App, Hap, rOmpA, rmpmp, ApeE, EstA, Lip-1, McaP, BabA, SabA, AlpA, Aae, NanB, and variants thereof.

7. Nucleic acid molecule according to any one of claims 1 to 6, wherein the redox factor-regenerating polypeptide is sensitive to oxygen.

8. polypeptide encoded by a nucleic acid molecule according to any one of claims 1 to 7.

A cell having on its surface a polypeptide according to claim 8 expressed or transformed using a Nukleinsäuremoieküls according to any one of claims 1 to 7.

10. Membrane fraction obtainable from the cell according to claim 9.

11. A method for regenerating a redox factor, comprising the following steps:

(a) providing the polypeptide of claim 8, the membrane preparation of claim 10 or the cell of claim 9,

(b) contacting the polypeptide of claim 8, the membrane preparation of claim 10, or the cell of claim 9 with one or more substrates of the redox factor-regenerating polypeptide, wherein the one or more substrates comprises the redox factor.

Use of the cell of claim 9 or the membrane preparation of claim 10 or the polypeptide of claim 8 for regenerating a redox factor.

Use of the cell according to claim 9, the membrane preparation according to claim 10 and the polypeptide according to claim 8 for incorporating the redox environment of an aqueous solution, preferably by deoxidizing the aqueous solution.

14. A method for producing a line presenting on its surface a redox factor-regenerating polypeptide, comprising the following steps:

(a) introducing the nucleic acid according to any one of claims 1 to 7 into a cell, (b) optionally contacting the cell with one or more redox-reactive prosthetic groups.

15. A method for producing the product of a redox factor-dependent enzyme, comprising the following steps:

(a) providing a redox factor dependent enzyme,

(b) providing the cell of claim 9, the membrane preparation of claim 10 or the polypeptide of claim 8 and

(c) contacting the redox factor-dependent enzyme with one or more of its substrates in the presence of the line of claim 9, the membrane preparation of claim 10 or the polypeptide of claim 8, wherein the cell of claim 9, the membrane preparation of claim 10 or the polypeptide according to claim 8 comprising a polypeptide which regenerates the redox factor on which the redox factor-dependent polypeptide depends.