specificity

The field of the present invention relates to recombinant enzyme modification to modify substrate specificity.

Cellobiose dehydrogenase (EC 1.1.99.18, CDH) was first discovered in 1974 in the extracellular enzyme system of

Phanerochaete chrysosporium and later on in several other basidiomycetous fungi. A special characteristic of this enzyme is its composition: the combination of a catalytically active flavodehydrogenase domain (also called "flavin domain") , hosting a non-covalently bound FAD, and a haem domain, with a haem b as a cofactor. Both domains are connected by a linker. By its catalytic activity the natural substrate cellobiose is oxidised in a reaction which reduces the FAD of the flavin domain.

CDH or its flavodehydrogenase domain oxidises carbohydrates like its natural substrates cellobiose and cello- oligosaccharides and others like lactose and maltose. CDHs have been discovered and shown previously to be capable of converting glucose efficiently (Harreither et al . , 2011; WO 2010/097462 A). Modified CDHs exist that have reduced activity on maltose

(WO2013/131942) . Other CDHS are known from Metarhizium

anisopliae (Database Uniprot, acc. No. E9DZA5) or Stemphylium lycopersici (Database Uniprot, acc. No. A0A0L1HDV7) . Gao et al . (Plos Genetics, 7 (1), (2001): el001264) describe genomic sequencing of the fungi Metarhizium anisopliae and M. acridum. EP 2 636 733 Al describes artificially mutated CDHs to reduce its maltose oxidation activity.

The problem with CDHs lies in the versatility which

diminishes its use in sensors for detecting single analyte in mixtures of several potential substrates.

It is a goal of the present invention to provide a

cellobiose dehydrogenase or its catalytically active

flavodehydrogenase domain, which is capable to selectively detect lactose in the presence of galacto-oligosaccharides especially with direct electron transfer based electrodes or mediated electron transfer-based electrodes to provide suitable analytically useful sensors.

The present invention relates to recombinant modified cello ^¬ biose dehydrogenases (CDH) with reduced galacto-oligosaccharide turnover, especially of 6-galactosyllactose or 4- galactosyllactose turnover. The aim is to reduce the effect of any galacto-oligosaccharide concentration, but preferably at least of 6-galactosyllactose or 4 -galactosyllactose or both, present in a sample matrix on lactose detection. In particular, the invention provides a modified cellobiose dehydrogenase (CDH) or its functional flavodehydrogenase domain having a substitu ^¬ tion at least at one of the amino acids of the galacto- oligosaccharide binding motif corresponding to amino acids 555- 557, 627-629 and 639-641 of SEQ ID NO: 4 (CDH from N. crassa) or corresponding to amino acids 554-556, 626-628 and 638-640 of SEQ ID NO: 5 (CDH from M. thermophilum) by any other acid amino acid.

The amino acids corresponding to amino acids 555-557, 627- 629 and 639-641 of SEQ ID NO: 4 are in case of SEQ ID NO :4 and other ascomycota N555-D557 (amino acid sequence NTD, also re ^¬ ferred to as "NTD-part" or "GOS motif 1"), S627-E629 (amino acid sequence SFE, also referred to as "SFE-part" or "GOS motif 3") and S639-Y641 (amino acid sequence SQY, also referred to as "SQY-part" or "GOS motif 2") of SEQ ID NO: 4. These amino acids may vary in different CDHs, especially in basidiomycota . E.g., the NTD-part of the motif in SEQ ID NO: 1 could be changed to NTE, NTV or NTE and/or the SQY-part of the motif in SEQ ID NO: 4 could be changed to NQY . In the course of the present invention, this GOS motif was identified as being responsible for galacto- oligiosaccharide binding for oxidation by the CDH independent of smaller substrates like lactose. The GOS motif comprises three parts which form a three-dimensional structure with the three parts in vicinity to each other.

The invention may further comprise an amino acid substitu ^¬ tion at the amino acid corresponding to N721 of SEQ ID NO: 5 (CDH from M. thermophilum) or corresponding to N722 of SEQ ID NO: 4 (CDH from N. crassa) by glutamine, isoleucine, threonine or leucine or a substitution of the asparagine in the active center motif having the amino acid sequence NHW by glutamine, isoleucine, threonine or leucine; hence this motif would be QHW, IHW, THW or LHW, as e.g. described in PCT/EP2017/051577 (WO 2017/129637) . Usually, the NHW-motif is found near the C- terminus of a CDH or the flavodehydrogenase domain, e.g. around amino acids 660 to 780 of a CDH or amino acids 430 to 550 of a flavodehydrogenase domain. Such a mutation reduces glucose or galactose oxidation activity as described in PCT/EP2017/051577 (WO 2017/129637) . Glucose and galactose may be sources of fur ^¬ ther unwanted cross-reaction in lactose detection and said cross-reactivity may be reduced with such a substitution corre ^¬ sponding to N721 of SEQ ID NO: 5 or at the NHW motif.

Preferred modified cellobiose dehydrogenase enzymes of the invention are optimized for use in biosensors based on direct electron transfer (3 ^rd generation) or based on mediated electron transfer (2 ^nd generation) .

To increase the performance of CDH as selective electrode catalyst for lactose, the reduction of galacto-oligosaccharide oxidation activity alone or in combination with an increase of lactose oxidation activity was pursued by genetic modification. With the general description of modifying a CDHs

flavodehydrogenase domain, a domain of high homology in all CDHs, it is possible to modify any CDH according to the

principles outlined herein in order to decrease galacto- oligosaccharide sensitivity. Thus, the present invention

provides in particular a modified cellobiose dehydrogenase (CDH) or its functional flavodehydrogenase domain having reduced galacto-oligosaccharide oxidation activity as compared to the unmodified CDH or its functional flavodehydrogenase domain while essentially maintaining a high lactose oxidation activity, e.g. at most a 40% or at most a 20% reduction in lactose oxidation activity, at standard conditions and as shown in the examples. The inventive substitutions may reduce the activity of lactose oxidation as long as the ratio of lactose oxidation to an oxidation of a galacto-oligosaccharide is increased. This increase in ratio or specificity achieves the goal of reduced side-reactivities in the detection of lactose. The invention further provides a method of oxidizing lactose with the CDH of the invention. Such a method is preferably used in the

analytical detection of lactose in a sample. Also possible is the detection of cellobiose or maltose.

Most CDHs are capable of oxidizing lactose. The oxidation reaction can be detected e.g. by monitoring electron acceptors for the redox reaction such quinones, like as DCIP (2,6- dichloroindophenol ) o- or p-benzoquinone or derivatives thereof, methylene blue, methylene green, Meldola' s blue, potassium ferricyanide, ferricenium hexafluorophosphate, FeCl3 or cytochrome c (the latter being a haem domain cofactor) or simply by determining electric current or voltages on an electrode, the electron acceptor being the electrode surface.

It is not necessary to use an entire CDH; the flavin domain, even without the haem domain, is sufficient for catalytical activity. The domain is therefore referred to as "functional domain" as it has the function of oxidizing lactose with a suitable electron acceptor. The activity is exerted by either the whole enzyme cellobiose dehydrogenase or the catalytically active flavodehydrogenase domain.

Wild type CDHs have an undesirable activity to oxidize galacto-oligosaccharides especially when they are structurally similar to lactose (e.g.: 6-galactosyllactose, 4- glactosyllactose) . The modification according to the present invention should now be understood in that the inventive CDHs deviate from the wild-type CDHs by this substantially decreased galacto-oligosaccharide oxidation activity. This substantially decreased galacto-oligosaccharide oxidation activity may be in combination with an increased or even a decrease in lactose oxidation activity, as mentioned above. The invention increases the ratio of the lactose oxidation activity to the galacto- oligosaccharide oxidation activity. Preferably, the ratio of lactose oxidation activity to the oxidation activity of 6- galactosyllactose is at least 2:1, preferably at least 3:1, at comparable conditions, as e.g. described in the examples, in particular at 23°C and atmospheric pressure in aqueous solution.

Preferred modified CDHs or their functional flavodehydrogen ^¬ ase domain are of a CDH of the subkingdom dikarya. Preferably, the CDH is from Ascomycota or Basidiomycota . Preferred ascomyco ^¬ ta are selected from Myriococcum thermophilum, Corynascus ther- mophilus, Chaetomium atrobrunneum (Myceliophthora fergusii) , Hy- poxylon haematostroma , Neurospora crassa, Stachybotrys bisby or Humicola insolens . Preferred Basidiomycota are Athelia rolfsii, Gelatoporia subvermispora , Phanerochaete chrysosporium, Trametes versicolor. Ascomycota are particularly preferred according to the invention. Such unmodified CDHs are described in WO

2010/097462 A and in WO2013/131942, including encoding nucleotide sequences, and in sequences of SEQ ID NO: 1-10 herein.

Suitable sequences are also available in sequence databases, in particular the NCBI database, e.g. accession numbers ADT70773.1, ADT70775.1, ADT70772.1, XP_956591.1, ABS45567.2, ADT70777.1, AA064483.1, ACF60617.1, AAB61455.1, XP_008041466.1 , AAF69005. "Unmodified" as used herein is a CDH without the inventive modi ^¬ fication that decreases galacto-oligosaccharide oxidation activ ^¬ ity. There may be further modifications that increase interac ^¬ tion between the flavin and the haem domain if an entire CDH is used (such as described in WO 2010/097462) or a modification that reduce glucose oxidation activity in relation with lactose oxidation activity as described in PCT/EP2017/051577 (WO

2017/129637) .

The inventive CDH or flavodehydrogenase domain has a substi ^¬ tution at least at one of the amino acids of the galacto- oligosaccharide binding motif corresponding to amino acids 555, 556, 557, 627, 628, 629, 639, 640 and 641 of SEQ ID NO: 4 (CDH from N. crassa) . Regarding other CDH sequences provided herein, the galacto-oligosaccharide binding motif (GOS motif) corre ^¬ sponding to amino acids 555-557, 627-629 and 639-641 of SEQ ID NO: 4 may be found at amino acids 556-558, 628-630 and 640-642 of SEQ ID NO: 1, amino acids 571-573, 643-645 and 655-657 of SEQ ID NO: 2, amino acids 550-552, 622-624 and 635-637 of SEQ ID NO: 3, amino acids 554-556, 626-628 and 638-640 of SEQ ID NO: 5, amino acids 554-556, 626-628 and 638-640 of SEQ ID NO: 6, amino acids 535-537, 606-608 and 623-625 of SEQ ID NO: 7, amino acids 538-540, 609-611 and 626-628 of SEQ ID NO: 8, amino acids 537- 539, 608-610 and 625-627 of SEQ ID NO: 9, amino acids 542-544, 613-615 and 630-632 of SEQ ID NO: 10, amino acids 548-550, 620- 622 and 633-635 of SEQ ID NO: 17 (see alignments at Fig. 1-4, note Fig. 1 only shows the flavodehydrogenase domain and is num ^¬ bered accordingly) .

A preferred selection of amino acids to be substituted are those at an amino acid corresponding to N555, D557, S627 and S639 of the CDH of SEQ ID NO: 4. Substitutions at these sites are particularly effective.

The invention has shown that any substitution at these positions, introducing a change to the wild type sequence as e.g. found in SEQ ID NO: 1-10, reduces galacto-oligiosaccharide oxi ^¬ dation activity, as such or in relation to lactose. Therefore, any substitution is possible to achieve the inventive goal of a robust lactose oxidation with less interference from galacto- oloigiosaccharides . Some substitutions are of course more effec ^¬ tive than others. Preferred substations are by at least one ami ^¬ no acid change corresponding to a substitution selected from D557V, D557L, S627D, S639V, S639N, N555M, S639I, D557R, S639G, D557T, D557I, D557E, S627K, S627T, S627P, N555L, D557P, S639M, S639R, D557G, S639E, D557A, S639Q, S639K, S639C, D557S, S639D, D557M, D557H, S639W, S627Q, D557W, S639F, N555D, S627Y, D557Q, S639Y, S639L, N555K, S639H, S627W, D557C of the CDH of SEQ ID NO: 4.

In further preferred embodiments of the invention, an amino acid corresponding to amino acid 557 of SEQID NO: 4 is prefera ^¬ bly substituted by any non-aspartic acid natural amino acid, such as by glutamic acid, valine, or asparagine. An amino acid corresponding to amino acid 639 of SEQ ID NO: 4 (CDH from N.

crassa) or corresponding amino acid 638 of SEQ ID NO: 5 (CDH from M. thermophilum) is preferably substituted by any natural non-serine amino acid, such as by glutamic acid, valine, or as ^¬ paragine. Of course, such changes apply to all CDHs, preferably those related to SEQ ID NO: 1-10 and 17.

As said a preferred further substitution increases the ratio of lactose oxidation to glucose or galactose oxidation. Prefera ^¬ bly the modified CDH or its functional flavodehydrogenase domain is based on an unmodified CDH or flavodehydrogenase domain with an active center motif having the sequence X ₁ X ₂ NHWX ₃ X ₄ X ₅ , wherein Xi is any amino acid, preferably selected from N, C and R; X ₂ is selected from S and A; X ₃ is selected from V, M and I; X ₄ is any amino acid, preferably selected from G and S; and X5 is any amino acid, preferably selected from S, A and T; especially preferred X ₄ and X5 are not both S. According to this additional substitu ^¬ tion, the N in this motif is preferably changed to Q, I, T or L. This change facilitates the activity change reducing activity on glucose and/or galactose. With the information of the motif, the position for the substitution can be determined. Preferably, al ^¬ so the modified CDH or the domain comprises the sequence

X1X2 Z HWX3X4X5 , with Z, being Q, I, T or L and X1-X5 being defined as above. Preferably, the amino acid corresponding to N721 of SEQ ID NO: 5 or asparagine at the NHW motif is amino acid N723 of SEQ ID NO: 1, N738 of SEQ ID NO: 2, N718 of SEQ ID NO: 3, N722 of SEQ ID NO: 4, N721 of SEQ ID NO: 5, N721 of SEQ ID NO: 6, N704 of SEQ ID NO: 7, N707 of SEQ ID NO: 8, N706 of SEQ ID NO: 9, N701 of SEQ ID NO: 10, N716 of SEQ ID NO: 17.

The modified CDH or its functional flavodehydrogenase domain preferably comprises a modified flavodehydrogenase domain based on one of the unmodified flavodehydrogenase domains according to amino acids 253-831 of SEQ ID NO: 1, amino acids 269-845 of SEQ ID NO: 2, amino acids 249-787 of SEQ ID NO: 3, amino acids 253- 829 of SEQ ID NO: 4, amino acids 251-828 of SEQ ID NO: 5, amino acids 251-829 of SEQ ID NO: 6, amino acids 233-771 of SEQ ID NO: 7, amino acids 236-774 of SEQ ID NO: 8, amino acids 235-773 of SEQ ID NO: 9, amino acids 230-768 of SEQ ID NO: 10, amino acids 246-785 of SEQ ID NO: 17. Preferably the inventive modified fla ^¬ vodehydrogenase domain has a sequence with at least 50 %, pref ^¬ erably at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, in particular preferred at least 99% , se quence identity with one of said unmodified flavodehydrogenase domains and further comprises at least one amino acid substitu ^¬ tion at the amino acid corresponding to amino acids 555-557, 627-629 and 639-641 of SEQ ID NO: 4 or at the GOS motif, suita ^¬ ble for reducing the galacto-oligosaccharide oxidation activity.

Homologous CDHs or flavodehydrogenase domains within these sequence requirements can be readily identified by sequence com ^¬ parisons such as by sequence alignment using publicly available tools, such as BLASTP, ClustalO, ClustalW or FastDB. Preferably, a homologous or modified CDH or the domain thereof has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, at least 11, at last 13, at least 15, at least 17, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 80, at least 100 and/or up to 100, up to 80, up to 60, up to 50, up to 40, up to 30, up to 30, up to 20, up to 15 further amino acid substitutions, deletions, insertions or modifications, and any ranges between these val ^¬ ues, as compared to any one of the CDHs of SEQ ID NOs 1-10 or any of their flavodehydrogenase domains. A particular preferred base CDH is of N. crassa, SEQ ID NO: 4. A prediction of such modifications can be made by computational methods using e.g. molecular docking into crystal structures such as of PDB data ^¬ base entry "lkdg" or "4qi5". Preferably, the inventive CDH or flavodehydrogenase domain has 1 substitution at the GOS motif, preferably at its parts GOS motif 1 or GOS motif 2; but further mutations in this motif and its -1 or -2 parts are possible, such as 2, 3, 4, 5, or more substitutions. Such combinations are e.g. one substitution in each of the GOS motif-1 and GOS motif-2 parts or one substitution in each of the three parts.

Further modifications outside the GOS motif are possible. The effect of such further modified amino acids in the active site and the substrate binding site can be determined for homogeneous catalysis by photometric methods and for

heterogeneous catalysis by electrochemical measurements using enzyme electrodes as described herein.

The methods for further modification may be any known in the art such as amino acid mutations, including amino acid substitu ^¬ tions, deletions or additions but also chemical modifica- tion/derivatisation of amino acid side chains.

The inventive enzyme or domain is usually recombinantly expressed. Also provided are preparations comprising the

modified CDH or domain. The term "enzyme" or "enzyme

preparation" as used herein refers to a cellobiose dehydrogenase or its flavodehydrogenase domain from a specified organism which is at least about 20% pure, preferably at least about 40% pure, even more preferably at least about 60% pure, even more

preferably at least 80% pure and most preferably at least 90% pure, as determined by polyacrylamide gel electrophoresis (SDS- PAGE) .

The present invention relates to modified/genetically engineered cellobiose dehydrogenases or flavodehydrogenase domain from existing protein scaffolds, which oxidise galacto- oligosaccharides less efficiently with or without a more

efficient lactose turnover than the currently known cellobiose dehydrogenases, especially those that is closest related to the modified CDH or the flavodehydrogenase domain, such as a CDH or flavodehydrogenase domain of sequences SEQ ID NO: 1-10 and 17 (the portions for the flavodehydrogenase domain are given below in the description of Fig. 1) .

Of course, the modified CDH or the flavin domain (aka fla- vodehydrogenase domain) may still have activity for an electro- catalytic oxidation of galacto-oligosaccharides . It is suffi ^¬ cient that the concentration dependence of the signal from galacto-oligosaccharides is reduced. Essentially constant back ^¬ ground signals dependent on galacto-oligosaccharides can be re ^¬ moved from the lactose detection by normalization. Especially preferred the CDH or flavin domain has the property that a sig ^¬ nal of galacto-oligosaccharides during lactose detection or lac ^¬ tose concentration determination is below 40%, preferably below 20%; particularly the signal, e.g. electrode current or electro ^¬ chemical reduction of an electron acceptor, of 0.25 g/L 6- galactosyllactose or 4-galactosyllactose in a solution compared to the signal of a 0.25 g/L lactose solution is below 40%, pref ^¬ erably below 20%. In preferred embodiments of the invention the 6-galactosyllactose or 4-galactosyllactose oxidation activity of the CDH or the domain is reduced in relation to lactose oxida ^¬ tion activity. Especially preferred the concentration dependency of the 6-galactosyllactose or 4-galactosyllactose oxidation is reduced so that - if 6-galactosyllactose or 4-galactosyllactose is present - only a substantially constant contribution of 6- galactosyllactose or 4-galactosyllactose to the signal is de ^¬ tected in relation to a lactose signal.

It is understood that one of skill in the art may engineer the mentioned or other cellobiose dehydrogenases to obtain the modified CDH or the active flavodehydrogenase domain using the principles outlined herein or in the prior art (WO 2010/097462 A, WO2013/131942) like the rational enzyme engineering via site- directed mutagenesis or directed evolution approaches (e.g., gene shuffling, error-prone PCR, etc.) and subsequent screening of the generated diversity. The techniques to introduce a further mutation into the nucleic acid sequence to exchange one nucleotide for another nucleotide with the aim to exchange one amino acid for another in the resulting protein may be

accomplished by site-directed mutagenesis using any of the methods known in the art. The amino acid positions and

modification to modify a CDH can be obtained by homology

modelling using the crystal structure of Phanerochaete

chrysosporium CDH (PDB database entry lkdg) as template and superimposition of the obtained models as well as docking studies. It is possible to use a CDH as template and by sequence comparison to obtain corresponding amino acids for modification to reduce galacto-oligosaccharide oxidation activity.

The modified CDH or its functional flavodehydrogenase domain may be isolated by diafiltration, ion exchange chromatography and preferably being further purified by hydrophobic interaction chromatography. Preferably the CDH or the domain is recombinant- ly produced by Pichia pastoris .

The invention further provides a nucleic acid molecule en ^¬ coding a modified CDH or its functional flavodehydrogenase do ^¬ main as described above. Unmodified CDH sequences are known in the art, e.g. in the NCBI sequence database and disclosed in WO 2010/097462 A and WO2013/131942 (incorporated herein by reference) . The nucleic acid can be modified to encode the inventive CDH or flavin domain with the substitution or further mutation. The nucleic acids, CDH or domains described herein may be iso ^¬ lated and/or purified.

Further provided is a method of producing a modified CDH or its functional flavodehydrogenase domain of the invention, com ^¬ prising recombinantly expressing a nucleic acid molecule encod ^¬ ing said modified CDH or its functional flavodehydrogenase do ^¬ main in a host cell.

In a further aspect, the invention further provides an electrode comprising an immobilised cellobiose dehydrogenase or its functional flavodehydrogenase domain of the invention.

Preferably the electrode comprises an immobilised CDH in di ^¬ rect- or mediated electron transfer mode (Tasca et al . 2011a, Tasca et al . 2011b, Ludwig et al. 2010, Safina et al. 2010, Tas ^¬ ca et al 2010a, Tasca et al . 2010b) or an immobilised flavodehy ^¬ drogenase domain in mediated electron transfer mode. As elec ^¬ trode, any suitable surface for collecting electrons from CDH is understood. The electrode may be of any material suitable to im ^¬ mobilise the CDH, e.g. carbon such as graphite, pyrolytic graph ^¬ ite, glassy carbon, carbon nanotubes (single or multi-walled) , carbon fibres, boron doped diamond, gold electrodes modified with promoters e.g., thiols or screen-printed electrodes. This is a non-exhaustive list of possible electrodes, which may e.g. contain other nanoparticles (gold,...) to increase the specific surface area. Particular uses of the inventive electrodes are in the provision of biosensors, more specifically to lactose bio ^¬ sensors using the direct electron transfer properties (DET) of cellobiose dehydrogenase (CDH) or using mediated electron trans ^¬ fer properties (MET) to measure the lactose concentration at acidic, neutral, alkaline pH.

On the electrode, the CDH or the flavodehydrogenase domain may be immobilised by adsorption, preferably also physical en ^¬ trapment in a polymer, complex formation, preferably via an ad- ditional complexing linker, covalent binding, in particular cross-linking, or ionic binding and/or the immobilized cellobiose dehydrogenase can be cross-linked, in particular by bifunc- tional agents, to increase stability or activity. It has been shown that cross-linking with bifunctional agents, such as agents with two reactive groups making a connection with the CDH, can stabilize the CDH and even increase its activity on graphite electrodes measurable by amperometric methods described herein. This advantage can lead to an increased sensitivity and lowering the detection limit for lactose. Such a cross-linking agent is e.g. glutaraldehyde or any other dialdehydes. Further methods for immobilization are described in e.g. WO 2010/097462 and WO2013/131942, which can be used according to the invention.

The electrodes might be used in form of a single electrode or electrode stacks, e.g. of 2, 3, 4, or more electrodes.

Electrode configurations and uses, including measurement pa ^¬ rameters are described in e.g. WO 2010/097462 and WO2013/131942, which can be used according to the invention.

The invention further provides a method of oxidizing lactose with the inventive CDH or flavin domain, especially in a method of detecting or quantifying lactose in a sample comprising the step of oxidizing lactose in said sample with a modified CDH or its functional flavodehydrogenase domain or an electrode as de ^¬ scribed herein and detecting or quantifying said oxidation, preferably wherein said sample comprises or is suspected of com ^¬ prising a galacto-oligosaccharide, such as 6-galactosyllactose or 4-galactosyllactose . The fluid sample may be any fluid which potentially comprises lactose, including milk or milk or dairy products, such as whey or cheese. If solid products are ana ^¬ lysed, such as cheese, the carbohydrates, including lactose, may be extracted or the solid product may be fluidized, such as by dissolving .

In a further aspect, the present invention provides a lac ^¬ tose assay kit comprising the modified cellobiose dehydrogenase or its functional flavodehydrogenase domain or an electrode as described herein. The kit may in preferred embodiments also com ^¬ prise auxiliary substances, like buffers, and containers such as a sample holding means and/or lactose standards. Lactose stand ^¬ ards may be used to calibrate the assay. The kit may also com ^¬ prise a reader for a signal, especially an electrochemical sig- nal such as a potentiostat , a computer readable memory device with software for calibration and/or measurement calculations.

In preferred embodiments, the invention is defined as fol ^¬ lows :

1. A modified cellobiose dehydrogenase (CDH) or its func ^¬ tional flavodehydrogenase domain having a substitution at least at one of the amino acids of the galacto-oligosaccharide binding motif corresponding to amino acids 555-557, 627-629 and 639-641 of SEQ ID NO: 4 (CDH from N. crassa) .

2. The modified CDH or its functional flavodehydrogenase domain according to 1, further comprising a substitution at the amino acid corresponding to N721 of SEQ ID NO: 5 (CDH from M. thermophilum) , preferably by glutamine, isoleucine, threonine or leucine or a substitution of the asparagine in the active center motif having the amino acid sequence NHW, preferably by gluta ^¬ mine, isoleucine, threonine or leucine.

3. The modified CDH or its functional flavodehydrogenase domain according to 2, wherein active center motif has the sequence X1X2NHWX3X4X5 , wherein Xi is any amino acid, preferably se ^¬ lected from N, C and R; X2 is selected from S and A; X ₃ is se ^¬ lected from V, M and I; X4 is any amino acid, preferably selected from G and S; and X5 is any amino acid, preferably selected from

5, A and T; especially preferred X ₄ and X ₅ are not both S.

4. The modified CDH or its functional flavodehydrogenase domain according to any one of 1 to 3, characterised in that the CDH is of dikaryota, preferably selected from ascomycota or ba- sidiomycota , preferably wherein the CDH is a modified CDH of a CDH from Myriococcum thermophilum, Corynascus thermophilus, Chaetomium atrobrunneum (Myceliophthora fergusii) , Hypoxylon haematostroma , Neurospora crassa, Stachybotrys bisby, Athelia rolfsii, Gelatoporia subvermispora , Phanerochaete chrysosporium, Trametes versicolor or Humicola insolens .

5. The modified CDH or its functional flavodehydrogenase domain according to any one of 1 to 4, comprising a modified flavodehydrogenase domain based on one of the unmodified fla- vodehydrogenase domains according to amino acids 253-831 of SEQ ID NO: 1, amino acids 269-845 of SEQ ID NO: 2, amino acids 249- 787 of SEQ ID NO: 3, amino acids 253-829 of SEQ ID NO: 4, amino acids 251-828 of SEQ ID NO: 5, amino acids 251-829 of SEQ ID NO:

6, amino acids 233-771 of SEQ ID NO: 7, amino acids 236-774 of SEQ ID NO: 8, amino acids 235-773 of SEQ ID NO: 9, amino acids 230-768 of SEQ ID NO: 10, amino acids 246-785 of SEQ ID NO: 17; said modified flavodehydrogenase domain has a sequence with at least 50%, preferably at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, in particular pre ^¬ ferred at least 99%, sequence identity with one of said unmodi ^¬ fied flavodehydrogenase domains and further comprises the sub ^¬ stitution mutation at least at an amino acid corresponding to amino acids 555-557, 627-629 and 639-641 of SEQ ID NO: 4.

6. The modified CDH or its functional flavodehydrogenase domain according to any one of 1 to 5, wherein the galacto- oligosaccharide binding motif corresponding to amino acids 555-

557, 627-629 and 639-641 of SEQ ID NO: 4 is at amino acids 556-

558, 628-630 and 640-642 of SEQ ID NO: 1, amino acids 571-573, 643-645 and 655-657 of SEQ ID NO: 2, amino acids 550-552, 622- 624 and 635-637 of SEQ ID NO: 3, 557, 627-629 and 639-641 of SEQ ID NO: 4, amino acids 554-556, 626-628 and 638-640 of SEQ ID NO: 5, amino acids 554-556, 626-628 and 638-640 of SEQ ID NO: 6, amino acids 535-537, 606-608 and 623-625 of SEQ ID NO: 7, amino acids 538-540, 609-611 and 626-628 of SEQ ID NO: 8, amino acids 537-539, 608-610 and 625-627 of SEQ ID NO: 9, amino acids 542- 544, 613-615 and 630-632 of SEQ ID NO: 10, amino acids 548-550, 620-622 and 633-635 of SEQ ID NO: 17.

7. The modified CDH or its functional flavodehydrogenase domain according to any one of 1 to 6, being recombinantly pro ^¬ duced by Pichia pastoris, isolated by diafiltration, ion ex ^¬ change chromatography and preferably being further purified by hydrophobic interaction chromatography.

8. The modified CDH or its functional flavodehydrogenase domain according to any one of 1 to 7, wherein the substitution is at an amino acid corresponding to N555, D557, S627 and S639 of the CDH of SEQ ID NO: 4.

9. The modified CDH or its functional flavodehydrogenase domain according to 8, wherein the substitution is by an amino acid corresponding to a substitution selected from D557V, D557L, S627D, S639V, S639N, N555M, S639I, D557R, S639G, D557T, D557I, D557E, S627K, S627T, S627P, N555L, D557P, S639M, S639R, D557G, S639E, D557A, S639Q, S639K, S639C, D557S, S639D, D557M, D557H, S639W, S627Q, D557W, S639F, N555D, S627Y, D557Q, S639Y, S639L, N555K, S639H, S627W, D557C of the CDH of SEQ ID NO: 4. 10. The modified CDH or its functional flavodehydrogenase domain according to any one of 1 to 9, wherein the ratio of lac ^¬ tose oxidation activity to the oxidation activity of 6- galactosyllactose is at least 2:1, preferably at least 3:1.

11. A nucleic acid molecule encoding a modified CDH or its functional flavodehydrogenase domain according to any one of 1 to 10.

12. A method of producing a modified CDH or its functional flavodehydrogenase domain according to any one of 1 to 10, com ^¬ prising recombinantly expressing a nucleic acid molecule accord ^¬ ing to 11 in a host cell.

13. An electrode comprising an immobilised cellobiose dehy ^¬ drogenase or its functional flavodehydrogenase domain according to any one of 1 to 10; preferably wherein the cellobiose dehy ^¬ drogenase is immobilised by adsorption, complex formation, espe ^¬ cially preferred via an additional complexing linker, covalent or ionic linkage, and/or wherein preferably the immobilized cel ^¬ lobiose dehydrogenase is cross-linked, in particular by bifunc- tional agents, to increase stability or activity.

14. Method of detecting or quantifying lactose in a sample comprising the step of oxidizing lactose in said sample with a modified CDH or its functional flavodehydrogenase domain accord ^¬ ing to any one of 1 to 10, or an electrode according to 13 and detecting or quantifying said oxidation, preferably wherein said sample comprises or is suspected of comprising a galacto- oligosaccharide, especially preferred a milk or milk product containing sample.

15. A lactose assay kit comprising the modified Cellobiose dehydrogenase or its functional flavodehydrogenase domain ac ^¬ cording to any of the 1 to 10 or an electrode according to 13 and a sample holding means and/or lactose standards.

The present invention is further illustrated by the

following figures and examples without being restricted thereto.

F i g u r e s:

Figure 1 is a sequence alignment of amino acid sequences of the flavodehydrogenase domains ("flavin domains") of the wild- type CDHs from Chaetomium atrobrunneum (aa 253-831 of SEQ ID NO: 1), Hypoxylon haematostroma (aa 269-845 of SEQ ID NO: 2),

Corynascus thermophilus (aa 249-787 of SEQ ID NO: 3), Neurospora crassa (aa 253-829 of SEQ ID NO: 4), Myriococcum thermophilum (aa 251-828 of SEQ ID NO: 5), Stachybotrys bisby (aa 251-829 of SEQ ID NO: 6), Athelia rolfsii (aa 233-771 of SEQ ID NO: 7), Gelatoporia subvermispora (aa 236-774 of SEQ ID NO: 8),

Phanerochaete chrysosporium (aa 235-773 of SEQ ID NO: 9),

Trametes versicolor (aa 230-768 of SEQ ID NO: 10), Humicola insolens (aa 246-785 of SEQ ID NO: 17) . The mutation site at the galacto-oligosaccharide (GOS) binding motif corresponding to amino acids 555-557, 627-629 and 639-641 of SEQ ID NO: 4 are marked by "*". An optional additional mutation site relating to glucose/lactose sensitivity at the amino acid corresponding to N721 of SEQ ID NO: 5 is highlighted and marked by (position 483 of the shown consensus numbering of the shown flavin

domains) .

Figure 2 shows a sequence alignment of the CDHs at the position of the GOS motif 1. Figure 3 shows a sequence alignment of the CDHs at the position of the GOS motif 2. Figure 4 shows a sequence alignment of the CDHs at the position of the GOS motif 3. Numbering corresponding SEQ ID NO : 4 was used.

Figure 5 shows activities of the supernatants of the cultures NcCDH, NcCDH D557V, NcCDH N722Q D557V, NcCDH N722Q D557E, NcCDH N722Q S639N, and NcCDH N722Q measured with the cyt c assay with 5 g L ^_1 glucose, 0.25 g ^~L lactose or 0.25 g ^~L 6-gal- lac. The activities are normalized to the average activity with lactose of each enzyme variant.

Figure 6 shows the sensor output of the enzyme variant NcCDH N722Q D557E and its parent NcCDH N722Q with solutions of 0.25 g L ^-1 lactose, 0.25 g L ^-1 6-gal-lac, 5 g L-l glucose and of a combination of 0.25 g L ^-1 lactose and 0.25 g L ^-1 6-gal-lac. The activities are normalized to the average activity with lactose of each enzyme variant.

E x a m p l e s :

Example 1 : Materials

Chemicals used in buffers and fermentation media were commercial products and at least of analytical grade if not otherwise stated. Substrates for kinetic studies were lactose, glucose and cytochrome c (cyt c) from Sigma-Aldrich and 6' -galactosyllactose (6-gal-lac) from Carbosynth in the highest grade of purity available. Buffers were prepared using water purified and deionised (18 ΜΩ) with a Milli-Q system (Millipore, Bedford, MA, USA) .

Example 2: Enzymatic activity assays and steady-state kinetics

Enzyme activity was assayed at 30°C using cyt c (Canevascini et al.,1991) as electron acceptor. Stock solutions of carbohydrates used for kinetic measurements were prepared in the respective buffer and allowed to stand overnight for mutarotation, while stock solutions of electron acceptors were prepared in water and immediately used. The protein concentration was determined with the Bradford assay.

Example 3 : Protein characterisation

The protein concentration was determined by the dye-staining method of Bradford using a pre-fabricated assay from Bio-Rad Laboratories Hercules, CA, USA) and bovine serum albumin as standard according to the manufacturers recommendations.

For electrophoretic characterisation SDS-PAGE was carried out on a Hoefer SE 260 Mighty Small II vertical electrophoresis unit. Gels (10.5x10 cm; 10% T, 2.7% C) were cast and run according to the manufacturers' modifications of the Laemmli system.

Example 4 : Docking study

A 6-galactosyl-lactose tri-saccharide structure was docked to the structure of CDH form Myriococcum thermophilium (PDB

database entry 4qi5) . Docking studies were performed using

Molecular Operating Environment (MOE) software (Chemical

Computing Group, Quebec, Canada) . Quality was assessed by comparison of the docking position of the 6-galactosly-lactose molecule to the cellobionolactam molecule co-crystalized in the 4qi5 structure. The best fit was chosen for further

investigation. Three regions (GOS motif 1 (N. crassa_N555-D557 ) , GOS motif 2 (N. crassa_S 639-Y641 ) , GOS motif 3 (N. crassa_S621 - E629)) defined by amino acid residues in close vicinity to the modeled galactosyl residue were selected and in silico site saturation mutagenesis was performed. Best results judged by theoretical binding affinity and stability values calculated by the software were selected for expression studies. Amino acids N555, D557, S627 and S639 were preliminarily closer

investigated. In descending order, the following amino acid substitutions reduce GOS binding to the CDH: D557V, D557L, S627D, S639V, S639N, N555M, S639I, D557R, S639G, D557T, D557I, D557E, S627K, S627T, S627P, N555L, D557P, S639M, S639R, D557G, S639E, D557A, S639Q, S639K, S639C, D557S, S639D, D557M, D557H, S639W, S627Q, D557W, S639F, N555D, S627Y, D557Q, S639Y, S639L, N555K, S639H, S627W, D557C (all amino acid designations with regard to SEQ ID NO: 4, the N. crassa) . Surprisingly, various substitute amino acids with various properties (acidic, basic, aliphatic, aromatic, hydroxylic, sulphurous, amidic, large, small) decrease affinity. This means that the GOS motif is in wild type CDH optimized for GOS binding and any change can reduce or disrupt such binding. Promising candidates were selected for wet-chemical validation.

Example 5 : Generation of Neurospora crassa CDH variants by site- directed mutagenesis

The amino acid exchanges D557E, D557V and S639N were selected for site-directed mutagenesis. The previously reported plasmid pNCIIA, encoding the N. crassa CDH gene (XM_951498, SEQ ID NO: 12 of WO 2010/097462, incorporated herein by reference) with its native signal sequences cloned under the control of the metha ^¬ nol-inducible AOX1 promoter was used as templates for the ampli ^¬ fication of the target gene with primers PRll (5 ^r - CAACACCGAAACCGTCATCCAGC-3 ' , SEQ ID NO: 11) and PR12 (5'- GATGACGGTTTCGGTGTTGGTG-3 ' , SEQ ID NO: 12) for mutation D557E. The plasmid pDS63, encoding the N. crassa CDH gene (XM_951498, SEQ ID NO: 12 of WO 2010/097462, incorporated herein by reference) with its native signal sequences cloned under the control of the methanol-inducible AOX1 promoter, modified with the amino acid exchange N722Q was used as template for the amplification of the target gene with primers PRll (5 ^r -

CAACACCGAAACCGTCATCCAGC-3 ' , SEQ ID NO: 11) and PR12 (5'- GATGACGGTTTCGGTGTTGGTG-3 ' , SEQ ID NO: 12) for mutation D557E; PR9 (5 ' -CAACACCGTTACCGTCATCCAGC-3 ' , SEQ ID NO: 13) and PR10 (5'- GATGACGGTAACGGTGTTGGTG-3 ' , SEQ ID NO: 14) for mutation D557V, and PR7 (5 ' -GCCATGACCATGAACCAGTACCTTGGCCGTGGC-3 ' , SEQ ID NO: 15) and PR8 (5 ' -CATGGTCATGGCGTAGCCGTC-3 ' , SEQ ID NO: 16) for muta- tion S639N. PCR was performed with Phusion high-fidelity DNA polymerase from New England BioLabs, a deoxynucleoside triphos ^¬ phate (dNTP) mix from Fermentas, oligonucleotide primers from VBC Biotech (Vienna, Austria) , and a C-1000 thermocycler from Bio-Rad Laboratories. The resulting PCR fragments were digested with Dpnl and transformed into E.coli. The correct sequences of the resulting plasmids were confirmed by sequencing. Linearized, verified plasmids were used for transformation into electrocom- petent P. pastoris cells and transformants were selected on YPD Zeocin plates (100 mg L ^-1 ) .

Example 6 : Lactose to galacto-oligosaccharide activity

The resulting four P. pastoris clones expressing the enzyme variants NcCDH D557E, NcCDH N722Q D557E, NcCDH N722Q D557V and NcCDH N722Q S639N were produced together with the Wildtype NcCDH and the parent variant NcCDH N722Q in 96-well deep well plates according to methods described previously by Weis et al . (2004) . The activities of the supernatants of each cultivation were measured with the cyt c assay with 5 g L ^-1 glucose, 0.25 g ^~L lac ^¬ tose or 0.25 g ^~L 6-gal-lac. 6-gal-lac was chosen as a representa ^¬ tive of galacto-oligosaccharides (Sako et al . , 1999), because of its strong presence in hydrolyzed low lactose milk, rendering it a significant substance for applications. Fig. 2 shows the re ^¬ sults of the activity measurements and Table 1 shows the ratios of activities lactose : 6-gal-lac and lactose : glucose .

Table 1: Ratios of lactose to glucose or 6-gal-lac ratio lactose : glucose ratio lactose : 6-gal-lac

NcCDH 7.3:1 0.8:1

NcCDH D557E 9.7:1 2.1:1

NcCDH N722Q D557V 20 : 1 8.1:1

NcCDH N722Q D557E 13.6:1 3.9:1

NcCDH N722Q S639N 19.7:1 4.4:1

NcCDH N722Q 40.3:1 0.9:1

The ratios lactose : 6-gal-lac are strongly increased by the mutation; the ratios lactose : glucose are reduced, but still high enough for all variants with a substitution in the GOS motif.

Example 7 : Production of recombinant CDH

Parent enzyme NcCDH N722Q as reference and the variant NcCDH N722Q D557E were produced in 1L baffled flasks. Precultures were grown overnight in 30 mL of YPD medium at 30°C and 120 rpm. Af ^¬ ter approximately 18 hours the precultures were transferred into 1L baffled flasks containing 200 mL BMGY medium without metha ^¬ nol. Induction with methanol was started immediately using a multichannel peristaltic pump (Minipuls Evolution, Gilson, Mid- dleton, WI, USA) . Each flask was supplied eight times a day with methanol yielding a total concentration of 2% (v/v) methanol per day. Increase in activity was monitored using the DCIP and the cytochrome c enzyme assays. Cultivation was stopped at day five of methanol induction, cells removed by centrifugation (4000 rpm, 20 min) and the supernatant set to a final ammonium sulfate concentration of 20%.

Example 8 : Purification of recombinant CDH

Both enzymes were purified to homogeneity in a two-step purifi ^¬ cation. The sample was loaded on a 20 mL PHE Sepharose FF column (HR26/20) equilibrated with 50 mM Na-acetate buffer pH 5.5 con ^¬ taining 20% ammonium sulfate. Proteins were eluted by increasing the concentration of the elution buffer (50 mM Na-acetate buffer pH 5.5) from 0 to 100% in 5 column volumes and fractions con ^¬ taining CDH activity were pooled. After diafiltration with a polyethersulfone flat-stack cross flow module with a cut-off of 10 kDa (Viva Flow 50, Sartorius, Gottingen, Germany) until con ^¬ ductivity of 5 mS cm ^-1 in 20 mM Na-acetate pH 5.5 the samples were loaded on a 20 mL Q-Source column (HR26/20) equilibrated with a 20 mM Na-acetate buffer pH 5.5. CDH was eluted by in ^¬ creasing the concentration of the elution buffer (50 mM Na- acetate buffer pH 5.5 containing 0.5 M NaCl) from 0 to 100% in 50 column volumes. Fractions were tested for CDH activity and pooled according to the highest Reinheitszahl (RZ, calculated from the absorbance ratio 420 nm/280 nm) . Purified enzymes were concentrated and diafiltered in 50 mM citrate buffer, pH 5.5, aliquoted and kept at 4°C for further use. Example 9 : Electrochemical Measurements

A screen-printed electrode with a three-electrode setup

(Dropsens, Oviedo, Spain) was connected to an Autolab potenti- ostat ( PGSTAT204 , Metrohm Autolab B. V., Utrecht, The Nether ^¬ lands) and used for electrochemical measurements. The enzyme- modified, screen-printed electrodes (DropSens, Oviedo, Spain) were mounted using an electrode holder (DropSens, Oviedo,

Spain) . 100yL drops of buffer containing various substrate concentrations were pipetted to cover all three electrodes. The measurements were started immediately. The system was controlled by the NOVA software version 1.11.0 (Metrohm Autolab B. V., Utrecht, The Netherlands) .

Example 10: Mutated CDHs from N. crassa - reduced galacto- oligosaccharide activity on electrodes

CDH from N. crassa oxidises besides lactose also glucose

(Harreither et al . , 2007) and galacto-oligosaccharides , which has negative side effects on the lactose detection accuracy if glucose and galacto-oligosaccharides are present. To reduce the activity with glucose, the mutation N722Q was previously

introduced in N. crassa CDH. The enzyme variant NcCDH N722Q was nevertheless still active with galacto-oligosaccharides. Enzyme variants were designed to reduce this activity as described above. The molecular weights of variant NcCDH N722Q D557E did not differ significantly from the parent enzyme.

To investigate the electrochemical performance of the N. crassa CDH variant, the enzyme was immobilized on carbon

electrodes and the current responses to different concentrations of glucose, 6-gal-lac and lactose were measured.

Figure 3 shows the current response to 0.25 g L ^-1 lactose, 0.25 g L ^-1 6-gal-lac, 5 g L-1 glucose and of a combination of 0.25 g L ^-1 lactose and 0.25 g L ^-1 6-gal-lac. Table 2 shows the ratios of activities lactose : 6-gal-lac, lactose : glucose and

lactose :lactose+6-gal-lac.

Table 2: Ratios of lactose to glucose, 6-gal-lac, or lactose+6- gal-lac

ratio lactose:glucose ratio lactose:6-gal-lac ratio lactose:lactose+6-gal-lac

NcCDH N722Q 19.5:1 3.1:1 0.75:1

NcCDH N722Q D557E 19.7:1 6.4:1 0.93:1 In contrast to the parent enzyme the mutation D557E reduces the influence of 6-gal-lac on lactose measurement dramatically. The ratio lactose : 6-gal-lac more than doubles, while the ratio lactose : glucose stays almost identical. The ratio

lactose : lactose+6-gal-lac shows that there is only a small influence of the signal strength when 6-gal-lac is added to a lactose solution. Therefore, the enzyme variant is an

extraordinarily improved biocatalyst for the measurement of lactose in the presence of the major interfering sugars in milk - glucose and galacto-oligosaccharides .

References

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Safina et al. (2010) Electrochimica Acta 55: 7690-7695.

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Tasca et al . (2011) Anal. Chem. 83:3042-3049.

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