WO2016097324A1

WO2016097324A1 - Alpha-galactosidases and uses thereof

Info

Publication number: WO2016097324A1
Application number: PCT/EP2015/080556
Authority: WO
Inventors: Hanna KONTAKANEN; Kristiina Kruus; Pascale Joseph; Jean-Paul Leonetti
Original assignee: Deinove
Priority date: 2014-12-19
Filing date: 2015-12-18
Publication date: 2016-06-23

Abstract

The present invention relates to the use of novel alpha-galactosidases, in particular in food, feed and medical fields, paper industry and in biomass conversion technologies to produce biofuels or other compounds of interest.

Description

ALPHA-GALAC TO SID ASES AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to novel bacterial enzymes and uses thereof, in particular in food, feed and medical fields, paper industry and in biomass conversion technologies to produce bio fuels or other compounds of interest.

BACKGROUND OF THE INVENTION

Alpha-galactosidases (E.C. 3.2.1.22) are exoglycosidases that catalyze the hydrolysis of the terminal non reducing -l,6-linked galactose residues from a-D-galactosides including raffinose family oligosaccharides (RFO) such as melibiose, raffinose and stachyose. These enzymes are also involved in hemicellulose degradation by removing branched galactose on hemicelluloses polysaccharides such as galactomannan, galactoglucomannan and arabinogalactan. Galactomannans may be found for example in coffee, tobacco, bean, guar gum and locust beam gum. Arabinogalactans and galactoglucomannan may be found for example in softwood. Alpha-galactosidases are also known to hydrolyze the terminal alpha-galactosyl moieties from glycolipids and glycoproteins.

These enzymes have potential uses in various applications, in particular in the food, feed, medical, and many other industries such as biomass conversion technologies to produce bio fuels, biochemical and /or biomaterials (Katrolia et al., 2013, Critical Reviews in Biotechnology, 8551, 1-11).

In food and feed industries, -galactosidases can be used to hydrolyze RFOs in legume derived food products that contain high amounts of these sugars, in particular in soy milk, thereby reducing their undesirable digestive side-effects on humans and monogastric animals. They are thus particularly useful in the pre-treatment of animal feed to improve the nutritive value of feedstuff by removing non-nutritive RFOs. Alpha-galactosidases can also be used in sugar industry to hydrolyze raffinose from beet sugar syrup and thus facilitating the sugar crystallization from molasses.

In medical area, these enzymes have potential use in transfusion therapy by converting blood group type B to universal donor type O, and in the treatment of Fabry disease caused by a mutational deficient activity of a-galactosidase A and resulting in accumulation of a glyco lipid within the blood vessels, other tissues and organs which leads to painful neuropathy and renal, cardiovascular and cerebrovascular dysfunction. α-galactosidases are also useful in biorefinery industry. Indeed, biomass is a very promising resource for replacing fossil raw materials in applications in which carbon is indispensable, such as liquid fuels, materials and chemicals. However, technologies based on the fermentation of sugars derived from starch and sugar crops raise concerns about the diversion of farmlands or crops to biofuels production in detriment of the food supply. Biorefinery offers the potential to use a wide variety of non-food biomass resources such as agricultural residues, forestry and municipal wastes, to produce valuable biochemicals, biomaterials and biofuels. Production from this lignocellulosic biomass is thus an attractive alternative that does not interfere with food security. Biorefinery is essentially based on the conversion of lignocellulosic biomass into monomeric sugars that can be chemically transformed or fermented into various compounds such as biofuels or biochemicals.

In addition to lignocellulosic biomass, marine plant biomass is considered as a potential feedstock for producing biofuels since this biomass lacks the recalcitrant cell wall structures that are found in lignocellulosic biomass. In particular, macroalgae are attractive because of their wide geographical distribution and high growth rate. As example, a red seaweed (Gelidium amansii) abundant on the coastlines of Southeast Asia contains about 20% cellulose and 60% agar (galactan), while cellulosic biomass (switchgrass) consists of 31% cellulose, 20% hemicellulose, and 18% lignin (Ha et al., Appl. Environ. Microbiol. August 2011 , vol. 77, no. 16, 5822-5825). a-galactosidases are thus particularly useful for bioconversion processes from such galactose-rich biomass.

It should also be noted that, for some industrial and biotechno logical applications, using α-galactosidases with an acidic optimum pH may cause problems. In particular, removing the galactose-containing oligosaccharides from soy milk has to be conducted at neutral or basic pH since lowering the pH of the soy milk causes proteins to precipitate and leaves a sour taste to the milk. Similarly, removing raffmose from beet sugar or seroconversion of blood group type B are also preferably conducted at neutral or alkaline pH as the pH of beet molasses and blood is around 7.

In addition, because conversion of biomass at elevated temperature may provide many benefits, such as increased cellulase activity, less energy cost for cooling, and decreased risk of contamination, thermostable α-galactosidases or α-galactosidases exhibiting high optimal temperature would be particularly advantageous. In view of all these promising applications, there is thus an increasing need for a- galactosidases, in particular for a-galactosidases with neutral or alkaline optimum pH and/or exhibiting high optimal temperature.

SUMMARY OF THE INVENTION

The inventors herein identified a novel enzyme exhibiting alpha-galactosidase activity and showing interesting properties such as maximal activity at pH 7-8 and an optimal temperature at 70°C.

In a first aspect, the present invention thus relates to the use of a polypeptide comprising an amino acid sequence having at least 77 % identity to SEQ ID NO: 1 and exhibiting alpha- galactosidase activity, or a functional fragment thereof, or a recombinant host cell expressing said polypeptide, for hydrolyzing an a-D-galactoside.

The polypeptide may comprise an amino acid sequence having at least 80, 90, 95, 98, 99% identity to SEQ ID NO: 1 or may comprise the amino acid sequence of SEQ ID NO: 1.

Preferably, the hydrolysis is conducted at temperature from about 30°C to about 70°C and/or at pH from about 7 to about 8.

The a-D-galactoside to be hydro lyzed may be selected from the group consisting of galactose containing oligosaccharides, galactose containing polysaccharides and galactolipids.

In particular, the α-D-galactoside may be a galactose containing oligosaccharide selected from the group consisting of galactan, melibiose, raffmose, stachyose and verbascose.

The α-D-galactoside may also be a galactose containing polysaccharides selected from the group consisting of galactans, galactomannans, galactoglucomannans and arabinogalactans.

The α-D-galactoside may be contained in a substrate selected from the group consisting of cellulosic biomasses, legume and seed derived food and feed products, molasses (a byproduct made during sugar extraction of sugar cane and sugar beet), and group B or AB erythrocytes. Preferably, the substrate is a galactose-rich biomass, preferably a galactose rich cellulosic biomass, in particular a marine plant biomass such as biomass obtained from macroalgae or micro algae.

In a second aspect, the present invention also relates to a recombinant nucleic acid construct, expression cassette or vector comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence having at least 77 % identity to SEQ ID NO: 1 and exhibiting alpha-galactosidase activity, or a functional fragment thereof. It further relates to a recombinant host cell comprising a recombinant nucleic acid construct, expression cassette or vector according to the invention.

In a further aspect, the present invention relates to a method of producing a polypeptide comprising an amino acid sequence having at least 77 % identity to SEQ ID NO: 1 and exhibiting alpha-galactosidase activity, or a functional fragment thereof, comprising

(a) culturing the recombinant host cell according to the invention conditions conducive for production of said polypeptide; and

(b) recovering said polypeptide from the cell culture; and

(c) optionally, purifying said polypeptide.

In another aspect, the present invention relates to a composition or a kit comprising (i) a polypeptide comprising an amino acid sequence having at least 77 % identity to SEQ ID NO: 1, or a functional derivative thereof, and exhibiting alpha-galactosidase activity, and (ii) a divalent metal ion, preferably Mn²⁺, and/or the co-factor NAD⁺ or NADP+, preferably NAD⁺, and/or a reducing agent, preferably dithiothreitol.

In a further aspect, the present invention also relates to a method of producing a fermentation product or a compound of industrial interest from an a-galactoside containing substrate comprising contacting the substrate with a polypeptide comprising an amino acid sequence having at least 77 % identity to SEQ ID NO: 1 and exhibiting alpha-galactosidase activity, or a functional fragment thereof, or a host cell expressing said polypeptide. In particular, the fermentation product may be a biofuel, such as ethanol, butanol, propanol, glycerol methanol, isopropanol, propanediol, glycerol or 2-3 butanediol, an organic acid such as formate, acetate, lactate, butyrate, gluconate, xylonate, citrate, succinate, propionate, fumarate, malate, pyruvate, itaconic acid and kojic acid, and their salts or esters, an isopreno^'id compound, or a pharmaceutical compound such as antibiotics, bacteriostatic compounds, anti- metabolite, chemotherapeutic compounds, anti-parasitic agents, anti-fungal agents, anti-viral compounds, cytokine-activity compounds or cell-growth factors.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Determination of pH optimum of Gal4.

Figure 2: Determination of temperature optimum of Gal4.

Figure 3 : Hydrolysis of galacto-manno-oligo saccharides (substrate concentration 0.4 mg/ml, galactose content 20.9%) by Gal4. Galactose (Gal), mannobiose (Man2) and mannotriose (Man3) in unhydrolysed substrate (in white) and in substrate hydrolysed with Gal4 (in black) (0.2 U/mg substrate) for 24 h at pH 7, were quantified using HPLC.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel enzyme exhibiting alpha-galactosidase activity obtained from the bacterium Deinococcus cellulosilyticus 5516J-15^T firstly isolated from air sample in Jeju Island, Republic of Korea. Identifying such activity in said bacterium was particularly surprising since this bacterium was previously found negative for -galactosidase activity (Weon et al., IJSEM, 2007, vol. 57, no. 8, 1685-1688).

As shown in the examples, this enzyme is able to hydro lyze the / nitrophenyl-alpha-D- galactopyranoside and model substrate but also far more complex substrates such as galacto- manno-oligo saccharides from locust bean gum.

The inventors further found that the enzyme exhibits maximal activity in a pH range from 7 to 8 and has an optimal temperature at 70°C. This last property was particularly unexpected since most of known bacterial alpha-galactosidases exhibit a temperature optimum below 60°C and Deinococcus cellulosilyticus is a mesophilic bacterium with an optimum temperature range for growth at 25-35°C.

This enzyme thus combines a neutral/alkaline optimum pH and a high optimal temperature and is thus particularly advantageous for use in food, feed and medical fields, paper industry and in biomass conversion technologies to produce biofuels or other compounds of interest.

Accordingly, in a first aspect, the present invention relates to a polypeptide exhibiting alpha-galactosidase activity and comprising, or consisting of, an amino acid sequence having at least 77% identity to SEQ ID NO: 1, or a functional fragment thereof.

As used herein, the terms "peptide", "oligopeptide", "polypeptide" and "protein" are employed interchangeably and refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming said chain.

The terms "alpha-galactosidase", "alpha-D-galactosidase", "alpha-D-galactoside galactohydrolase", "melibiase" and " -galactoside galactohydrolase" are used herein interchangeably and refer to an enzyme that hydro lyzes terminal, non-reducing alpha-D- galactose residues in alpha-D-galactosides, including galactose oligosaccharides, galactomannans and galactolipids. These terms refer to an enzyme having an activity described as EC 3.2.1.22, according to the International Union of Biochemistry and Molecular Biology enzyme nomenclature. The alpha-galactosidase activity may be assessed by any method known by the skilled person. A number of well-known methods are available to assess this activity. In particular, the a-galactosidase activity may be routinely assayed by measuring the release of p- nitrophenol from the chromogenic substrate p-nitrophenyl-a-D-galactopyranoside. Alternatively, the a-galactosidase activity may be determined using a-D-galactopyranoside or locust bean gum prehydrolysed with mannase, as substrate. The assay may be carried out at different temperature and pH and for different times (e.g. from 10 min to 24 hours). Typically, the assay is carried out at about 40°C and at pH 7. Preferably, the reaction takes place in the presence of AD⁺, a source of Mn²⁺ such as MnCl₂, and in a reducing environment, for example in the presence of DTT.

As used herein, the term "sequence identity" or "identity" refers to the number (%) of matches (identical amino acid residues) in positions from an alignment of two polypeptide sequences. The sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithms (e.g. Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software available on internet web sites such as http:^last.ncbi.nlm.nih.gov/ or http://www.ebi.ac.uk/Tools/emboss/). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Preferably, for purposes herein, % amino acid sequence identity values refers to values generated using the pair wise sequence alignment program EMBOSS Needle that creates an optimal global alignment of two sequences using the Needleman-Wunsch algorithm, wherein all search parameters are set to default values, i.e. Scoring matrix = BLOSUM62, Gap open = 10, Gap extend = 0.5, End gap penalty = false, End gap open = 10 and End gap extend Preferably, the polypeptide comprises, or consists of, an amino acid sequence having at least 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 1. Preferably, the percentage of identity is determined over more than 10% of length of SEQ ID NO: 1, more preferably over more than 20, 30, 40, 50, 60, 70, 80, 90, 95, 99 or 100% of length of SEQ ID NO: 1. In a particular embodiment, the polypeptide comprises, or consists of, the amino acid sequence of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises, or consists of, a sequence that differs from the sequence set forth in SEQ ID NO: 1 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 substitutions, insertions and/or deletions of amino acid residues, preferably by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 substitutions, insertions and/or deletions of amino acid residues.

As used herein, the term "functional fragment" refers to a fragment of the polypeptide as defined above, comprising at least 100, 150, 200, 250 or 300, 350, 400 contiguous amino acids of said polypeptide, and exhibiting alpha-galactosidase activity. In a particular embodiment, the term "functional fragment" refers to a fragment of the polypeptide having the amino acid sequence of SEQ ID NO: 1, said fragment comprising at least 100, 150, 200, 250 or 300, 350, 400 contiguous amino acids of the amino acid sequence of SEQ ID NO: 1 and exhibiting alpha-galactosidase activity. Preferably, the functional fragment retains substrate specificity and/or substrate affinity and/or optimal pH and/or optimal temperature of the entire polypeptide. These properties can be easily assessed by the skilled person using well known methods.

The polypeptide of the invention may also be a hybrid polypeptide or fusion polypeptide in which the polypeptide exhibiting alpha-galactosidase activity and comprising, or consisting of, an amino acid sequence having at least 77% identity to SEQ ID NO: 1, is fused at its N- terminus and/or C-terminus to another polypeptide. Techniques for producing fusion polypeptides are well known in the art, and include ligating the coding sequences encoding the polypeptide and the addition region of another polypeptide so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. The addition region of the fusion polypeptide can be selected in order to enhance the stability of the enzyme, to promote the secretion (such as a N-terminal hydrophobic signal peptide) of the fusion protein from a cell (such as a bacterial cell or a yeast cell), or to assist in the purification of the fusion protein. More particularly, the additional region can be a tag useful for purification or immobilization of the enzyme. Such a tag is well-known by the person skilled in the art, for instance a His tag (Ηϊββ), a FLAG tag, a HA tag (epitope derived from the Influenza protein haemagglutinin), a maltose-binding protein (MPB), a MYC tag (epitope derived from the human proto-oncoprotein MYC) or a GST tag (small glutathione-S- transferase). A fusion polypeptide can further comprise a cleavage site for proteases or chemical agents, between the enzyme and the addition region. Upon secretion of the fusion protein, the site is cleaved releasing the two separate polypeptides.

In a particular embodiment, the polypeptide of the invention comprises, or consists of, an amino acid sequence having at least 77% identity to SEQ ID NO: 1 fused to a N-terminal hydrophobic signal peptide promoting secretion of the enzyme. The signal peptide may be a heterologous or endogenous signal peptide.

The polypeptide may also be fused at its N-terminus and/or C-terminus to one or several polypeptides exhibiting distinct enzymatic activity, preferably an enzymatic activity involved in hemicellulose hydrolysis such as amylase, laccase, glucosidase, cellulase, xylanase, mannanase, or mannosidase activity. The polypeptide may thus comprise one or several catalytic domains. Catalytic domains may be adjacent or may be separated by one or several CBM domains, dockerin or cohesin domains and/or SLH domains as described below.

The polypeptide may also be fused at its N-terminus and/or C-terminus to a Carbohydrate Binding Module (CBM) domain and/or to a dockerin or cohesin domain and/or to a SLH domain (surface layer homology domain). The fusion protein may comprise one or several CBM domains and/or one or several dockerin or cohesin domains and/or one or several SLH domains.

The CBM domain fused to the polypeptide may be from any enzyme comprising a CBM domain, preferably an enzyme from a Deinococcus bacterium. For example, the CBM domain may belong to the Carbohydrate-Binding Module Family 2, 4, 6, 12, 13, 17, 20, 28, 32, 41, 48, 50, 51 or 57 as defined on the Carbohydrate- Active enZYmes Database (http://www.cazy.org). In a particular embodiment, the CBM domain is selected from the group consisting of family- 2 CBM and family-3 CBM. The CBM domain may be linked to the C-terminus or N-terminus of the polypeptide, preferably though a peptide linker. The polypeptide may be fused to one or several CBM domains at its N-terminus and/or at its C-terminus.

Dockerin or cohesin domains allow construction of artificial cellulosomes or xylanosomes. A cellulosome or xylanosome system is a multi-enzyme complex characterized by a strong bi-modular protein-protein interaction between cohesin and dockerin modules that integrates the various enzymes into the complex. Typically, scaffoldin subunits (non-enzymatic protein components) contain the cohesin modules that incorporate the enzymes into the complex via their resident dockerins. Chimeric cellulosomes or xylanosomes can thus be constructed based on the very high affinity and specific interaction between cohesin and dockerin modules from the same microorganism species. Examples of dockerin and cohesin domains include, but are not limited to, dockerin or cohesin domains derived from Clostridium thermocellum, Clostridium acetobutylicum, Clostridium cellulovorans, Clostridium josui, Clostridium papyrosolvens, Acetivibrio cellulolyticus, Ruminococcus flavefaciens, Bacteroides cellulosolvens, Archaeoglobus fulgidus or Clostridium cellulolyticum. The polypeptide may be fused to one or several dockerin or cohesin domains at its N-terminus and/or at its C-terminus.

SLH domain are used in anchoring the polypeptide to the wall of a host cell expressing said polypeptide. Typically, a SLH domain contains one to three modules of about 55 amino acids and containing 10 to 15 conserved residues. These domains have been identified in various Gram positive and negative bacteria. The polypeptide may be fused to one or several SLH domains at its N-terminus and/or at its C-terminus.

Preferably, the polypeptide of the invention exhibits alpha-galactosidase in a wide pH range, i.e. from 6 to 9, and/or a wide temperature range, i.e. from 30°C to 90°C.

In a preferred embodiment, the polypeptide of the invention exhibits maximal alpha- galactosidase activity at pH from about 7 to about 8 and/or at temperature from about 60°C to about 70°C.

As used in this specification, the term "about" refers to a range of values ± 10% of the specified value. Preferably, the term "about" refers to a range of values ± 5 % of the specified value.

The present invention also relates to a nucleic acid encoding a polypeptide of the invention. The nucleic acid can be DNA (cDNA or gDNA), RNA, or a mixture of the two. It can be in single stranded form or in duplex form or a mixture of the two. It can comprise modified nucleotides, comprising for example a modified bond, a modified purine or pyrimidine base, or a modified sugar. It can be prepared by any method known to one skilled in the art, including chemical synthesis, recombination, and mutagenesis. The nucleic acid according to the invention may be deduced from the sequence of the polypeptide according to the invention and codon usage may be adapted according to the host cell in which the nucleic acid shall be transcribed. These steps may be carried out according to methods well known to one of skill in the art and some of which are described in the reference manual Sambrook et al. (Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, Third Edition Cold Spring Harbor).

In an embodiment, the nucleic acid of the invention comprises, or consists of, a nucleotide sequence having at least 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 2. Preferably, the percentage of identity is determined over more than 10% of length of SEQ ID NO: 2, more preferably over more than 20, 30, 40, 50, 60, 70, 80, 90, 95, 99 or 100% of length of SEQ ID NO: 2. In a particular embodiment, the nucleic acid comprises, or consists of, the amino acid sequence of SEQ ID NO: 2.

The present invention further relates to an expression cassette comprising a nucleic acid encoding a polypeptide according to the invention, operably linked to one or more control sequences that direct the expression of said nucleic acid in a suitable host cell under conditions compatible with the control sequences.

The term "expression cassette" denotes a nucleic acid construct comprising a coding region, i.e. a gene, and a regulatory region, i.e. comprising one or more control sequences, operably linked.

The term "control sequences" means nucleic acid sequences necessary for expression of a coding sequence. Control sequences may be native, homologous or heterologous. Well- known control sequences and currently used by the person skilled in the art will be preferred. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. Preferably, the control sequences include a promoter and a transcription terminator.

The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to a coding sequence, in such a way that the control sequence directs expression of the coding sequence.

The control sequence may include a promoter that is recognized by a host cell or an in vitro expression system for expression of a nucleic acid encoding a polypeptide of the invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The promoter may be a native, homologous or heterologous promoter. The promoter may be a strong, weak, constitutive or inducible promoter.

Preferably, the promoter is a polynucleotide that shows transcriptional activity in 5 Deinococcus bacteria. In this regard, various promoters have been studied and used for gene expression in Deinococcus bacteria. Examples of suitable promoters include VtufA and PtufB promoters from the translation elongation factors Tu genes tufA (DR0309) and tufB (DR2050), the promoter of the resU gene located in pI3, the promoter region PgroESL of the groESL operon (Lecointe, et al. 2004. Mol Microbiol 53: 1721-1730 ; Meima et al. 2001. J Bacteriol 10 183: 3169-3175), or derivatives of such promoters.

The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3 '-terminus of the nucleic acid encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention. Preferably, the terminator is functional in Deinococcus bacteria. 15 Examples of such terminator are disclosed in Lecointe et al, 2004, supra. Usually, the terminator is chosen in correlation with the promoter.

The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of the polypeptide and directs the polypeptide into the cell's secretory pathway, i.e. for secretion into the extracellular (or periplasmic) space. The 5'-end of 0 the coding sequence of the nucleic acid may contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may replace the natural signal peptide coding sequence in order to enhance secretion 5 of the polypeptide. Any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.

Optionally, the expression cassette may also comprise a selectable marker that permits easy selection of recombinant bacteria. Typically, the selectable marker is a gene encoding antibiotic resistance or conferring autotrophy.

30 Typically, the expression cassette comprises, or consists of, a nucleic acid according to the invention operably linked to a transcriptional promoter and a transcription terminator. The present invention also relates to an expression vector comprising a nucleic acid or an expression cassette according to the invention.

As used herein, the term "expression vector" means a DNA or RNA molecule that comprises an expression cassette. Preferably, the expression vector is a linear or circular double stranded DNA molecule.

The expression vector of the invention may be used to transform a host cell, preferably a Deinococcus host cell, and enable the expression of the nucleic acid of the invention in said cell. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.

The vector preferably comprises one or more selectable markers that permit easy selection of host cells comprising the vector. A selectable marker is a gene the product of which provides for antibiotic resistance, resistance to heavy metals, prototrophy to auxotrophy, and the like.

The vector preferably comprises an element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome. When integration into the host cell genome occurs, integration of the sequences into the genome may rely on homologous or non-homologous recombination. In one hand, the vector may contain additional polynucleotides for directing integration by homologous recombination at a precise location into the genome of the host cell. These additional polynucleotides may be any sequence that is homologous with the target sequence in the genome of the host cell. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The methods for selecting these elements according to the host cell in which expression is desired, are well known to one of skill in the art. The vectors may be constructed by the classical techniques of molecular biology, well known to one of skill in the art.

The present invention further relates to the use of a nucleic acid, an expression cassette or an expression vector according to the invention to transform, transfect or transduce a cell. The present invention also relates to a recombinant host cell comprising a nucleic acid, an expression cassette or an expression vector according to the invention.

As used herein, the term "recombinant host cell" designates a cell that is not found in nature and which contains a modified genome as a result of either a deletion, insertion or modification of genetic elements. In a particular embodiment, this term refers to a cell comprising a "recombinant nucleic acid", i.e. a nucleic acid which has been engineered and is not found as such in the wild type cell.

The host cell may be transformed, transfected or transduced in a transient or stable manner. An expression cassette or vector of the invention is introduced into a host cell so that the cassette or vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier. The term "host cell" also encompasses any progeny of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication.

The nucleic acid, expression cassette or expression vector according to the invention may be introduced into the host cell by any method known by the skilled person, such as electroporation, conjugation, transduction, competent cell transformation, protoplast transformation, protoplast fusion, biolistic "gene gun" transformation, PEG-mediated transformation, lipid-assisted transformation or transfection, chemically mediated transfection, lithium acetate-mediated transformation or liposome-mediated transformation.

Optionally, more than one copy of a nucleic acid, cassette or vector of the present invention may be inserted into the host cell to increase production of the polypeptide.

The host cell may be any cell useful in the production of a polypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium.

Examples of suitable bacterial expression hosts include, but are not limited to, Deinococcus and related bacteria, Escherichia (e.g. Escherichia coli), Pseudomonas (e.g. P. fluorescens or P. stutzerei), Proteus (e.g. Proteus mirabilis), Ralstonia (e.g. Ralstonia eutropha), Streptomyces, Staphylococcus (e.g. S. carnosus), Lactococcus (e.g. L. lactis), or Bacillus (subtilis, megaterium, licheniformis, etc.).

The host cell may also be an eukaryotic cell, such as a yeast, fungal, mammalian, insect or plant cell. Examples of suitable yeast expression hosts include, but are not limited to,

Saccharomyces (e.g. Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis), Schizosaccharomyces (e.g. Schizosaccharomyces pombe), Yarrowia (e.g. Yarrowia lipolytica), Hansenula (e.g. Hansenula polymorpha), Kluyveromyces (e.g. Kluyveromyces lactis), Pichia (e.g. Pichia pastoris) or Candida cell. Examples of suitable fungal expression hosts include, but are not limited to, Trichoderma, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium or Trametes cell.

Preferably, the host cell is a Deinococcus bacterium or related bacterium.

In the context of the invention, the term "Deinococcus" includes wild type or natural variant strains of Deinococcus, e.g., strains obtained through accelerated evolution, by DNA- shuffling technologies, mutagenesis or recombinant strains obtained by insertion of eukaryotic, prokaryotic and/or synthetic nucleic acid(s), strains genetically and/or chemically modified by any process known per se in the art or any genetic engineering technology. Deinococcus bacteria can designate any bacterium of the genus Deinococcus, such as without limitation, D. geothermalis, D. cellulolysiticus, D. radiodurans, D. proteolyticus, D. radiopugnans, D. radiophilus, D. grandis, D. indicus, D. frigens, D. saxicola, D. maricopensis, D. marmoris, D. deserti, D. murrayi, D. aerius, D. aerolatus, D. aerophilus, D. aetherius, D. alpinitundrae, D. altitudinis, D. apachensis, D. aquaticus, D. aquatilis, D. aquiradiocola, D. aquivivus, D. caeni, D. claudionis, D. daejeonensis, D. depolymerans, D. ficus, D. gobiensis, D. hohokamensis, D. hopiensis, D. misasensis, D. navajonensis, D. papagonensis, D. peraridilitoris, D. pimensis, D. piscis, D. radiomollis, D. reticulitermitis, D. roseus, D. sonorensis, D. wulumuqiensis, D. xibeiensis, D. xinjiangensis, D. yavapaiensis, D. citri, D. guilhemensis, D. phoenicis, D. soli, D humi, D. sahariens, D. mumbaiensis, or D. yunweiensis bacterium, or any combinations thereof. Preferably, the term "Deinococcus" refers to D. geothermalis, D. cellulolysiticus, D. deserti, D. murrayi, D. maricopensis or D. radiodurans.

As used herein, the term "related bacterium" refers to a bacterium "related" to Deinococcus, i.e. a bacterium which (i) contains a 16S rDNA which, upon amplification using primers GTTACCCGGAATCACTGGGCGTA (SEQ ID NO: 3) and GGTATCTACGCATTCCACCGCTA (SEQ ID NO: 4), generates a fragment of about 158 base pairs and/or (ii) resists a UV treatment of 4 mJ/cm². In a particular embodiment, Deinococcus-related bacteria are bacteria having a 16S rDNA molecule which is at least 70%, preferably at least 80%> identical in sequence to a Deinococcus 16S rDNA sequence. In particular, the term "related bacterium" may refer to a Deinobacterium, Truepera, Thermus, Meiothermus, Marinithermus, Oceanithermus, Vulcanithermus, Bacillus, Microbacterium, Cellulosimicrobium, Methylobacterium, Sphingobacterium, Pseudomonas, Caldimonas, Paenibacillus, Gordonia, Rhodococcus, Stenotrophomonas, Novosphingobium, Sphingomonas, Flavobacterium, Sphingobium, Sphingopyxis, Tepidimonas, Exiguobacterium, Nocardia, Arthrobacter, Kineococcus, Williamsia, Porphyrobacter, Geodermatophylus, Hymenobacter, Kineococcus, Kocuria, Methylobacterium, Halobacterium salinarum, Chroococcidiopsis, Pyrococcus abissis or Lactobacillus plantarum bacterium Preferably, this term refers to a bacterium belonging to the phylum of Deinococcus-Thermus such as Deinobacterium, Truepera, Thermus, Meiothermus, Marinithermus, Oceanithermus or Vulcanithermus bacteria.

In an embodiment, the host cell expresses one or several additional amylolytic, cellulo lytic or hemicellulo lytic enzymes. These additional enzymes may be endogenous or heterologous enzymes. These enzymes may be, for example, amylases, laccases, glucosidases, cellulases, xylanases, pectinases, esterases, acetyl xylan esterases, ferulic acid esterase, p- coumaroyl esterases, alpha-arabinofuranosidase, beta-galactosidases, mannanase, mannosidase and/or glucuronidases.

The host cell may also express endogenous or heterologous enzymes involved in production of compounds of interest by fermentation of monomeric sugars. In a particular embodiment, the host cell expresses an endogenous or heterologous enzyme selected from acetaldehyde dehydrogenases, alcohol dehydrogenases (ADH) and/or pyruvate decarboxylase (PDC). The present invention further relates to a cell extract of the recombinant host cell according to the invention. Preferably, the cell extract comprises a polypeptide comprising an amino acid sequence having at least 77 % identity to SEQ ID NO: 1 and exhibiting alpha- galactosidase activity, or a functional fragment thereof. As used herein, the term "cell extract" refers to any fraction obtained from a bacterium, such as a cell supernatant, a cell debris, cell walls, DNA extract, enzymes or enzyme preparation or any preparation derived from bacteria by chemical, physical and/or enzymatic treatment, which is essentially free of living bacteria.

In another aspect, the present invention also relates to a method of producing a polypeptide of the invention, i.e. a polypeptide comprising an amino acid sequence having at least 77 % identity to SEQ ID NO: 1 and exhibiting alpha-gal actosidase activity, or a functional fragment thereof.

In an embodiment, the method comprises (a) culturing a recombinant host cell of the invention in conditions conducive for production of said polypeptide; and (b) recovering said polypeptide from the cell culture; and (c) optionally, purifying said polypeptide.

The recombinant host cells of the invention are cultivated in a nutrient medium suitable for production of polypeptides using methods known in the art. For example, they may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed- batch, or solid state fermentations) in laboratory or industrial fermenters, performed in a suitable medium and under conditions allowing the enzyme to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide of the invention is secreted into the nutrient medium, it can be recovered directly from the culture supernatant. If the polypeptide is not secreted, it can be recovered from cell lysates or after permeabilisation. The polypeptide may be detected using any method known in the art. In particular, the polypeptide may be detected by alpha-galactosidase activity assay or, if the protein is a tagged recombinant protein, using antibodies directed against this tag with techniques well-known in the art. The polypeptide may be recovered using any method known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. Optionally, the polypeptide may be partially or totally purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain substantially pure polypeptides.

Alternatively, the method may comprise (a) contacting a nucleic acid, expression cassette or expression vector of the invention with an in vitro expression system; and (b) recovering the polypeptide; and (c) optionally, purifying said polypeptide. In vitro expression systems are well-known by the person skilled in the art and are commercially available.

In another aspect, the present invention also relates to a method for preparing a polypeptide of the invention immobilized on a solid support comprising producing the polypeptide as detailed above and immobilizing the polypeptide on a solid support. The present invention also relates to a solid support, a polypeptide according to the present invention being immobilized on the solid support. Immobilization means are well-known to the person skilled in the art (see e.g. 'Enzyme Technology' by Martin Chaplin and Christopher Bucke, Cambridge University Press, 1990). The polypeptide according to the present disclosure can be immobilized on the solid support by any convenient mean, in particular adsorption, covalent binding, entrapment or membrane confinement. A wide variety of insoluble materials may be used to immobilize the polypeptide. These are usually inert polymeric or inorganic matrices. The solid support can be for instance membranous, particulate or fibrous. More particularly, the solid support is preferably a bead, e.g., micro- or nanobeads. The polypeptide can be immobilized on a polyurethane matrix, on activated sepharose, alginate, amberlite resin, Sephadex resin or Duolite resin. Other solid supports useful for the invention include resins with an acrylic type structure, polystyrene resins, macroreticular resins and resins with basic functional groups. The immobilized polypeptide may then be used in a reactor. Examples of reactor include, but are not limited to, an enzyme reactor, a membrane reactor, a continuous flow reactor such as a stirred tank reactor, a continuously operated packed bed reactor, a continuously operated fluidized bed reactor, and a packed bed reactor.

The inventors showed that the activity of the polypeptide having the amino acid sequence of SEQ ID NO: l is dependent on the presence of co-factors NAD(+) and a divalent metal ion such as Mn2+. They also demonstrated that optimal activity of this enzyme can be obtained when the reaction takes place in reducing conditions.

Thus, in a further aspect, the present invention relates to a composition comprising (i) a polypeptide of the invention as defined above, i.e. a polypeptide comprising an amino acid sequence having at least 77 % identity to SEQ ID NO: 1, or a functional derivative thereof, and exhibiting alpha-galactosidase activity, and (ii) a source of divalent metal ion and/or the co- factor NAD(+) (nicotinamide adenine dinucleotide) or NADP(+) (nicotinamide adenine dinucleotide phosphate) ,and/or a reducing agent.

The divalent metal ion may be selected from the group consisting of Mn(2+), Ca(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Ni(2+), and Zn(2+), preferably is Mn(2+). The source of divalent metal ion may be a metallic salt. Preferably, the source of divalent metal ion is selected from MnCl₂ or MnS04. More preferably, the source of divalent metal ion is MnCh.

The reducing agent may be easily selected by the skilled person. This reducing agent may be, for example, dithiothreitol (DTT) or 2-mercaptoethanol, preferably DTT.

Preferably, the composition comprises (i) a polypeptide of the invention as defined above, and (ii) a source of divalent metal ion, preferably Mn(2+), and/or the co-factor NAD(+) or NADP(+), preferably NAD(+), and optionally a reducing agent. In particular, the composition may comprise (i) a polypeptide of the invention as defined above, (ii) a source of divalent metal ion, preferably Mn(2+), the co-factor NAD(+) or NADP(+), preferably NAD(+), and a reducing agent.

The composition may further comprise components suitable for enzyme preservation such as stabilisers like glycerol, sorbitol or monopropylene glycol, preservatives or buffering agents. Preferably, the composition comprises a buffering agent maintaining the pH between 7 and 8. The polypeptide of the invention may be free or immobilized on a solid support. The composition can be liquid or dry. In a particular embodiment, the composition is liquid and comprises at least 10, 20, 30, 40 or 50 % (w/v), preferably between 20 and 50 % (w/v), of glycerol, sorbitol or monopropylene glycol, preferably glycerol. The composition may also further comprise one or more additional proteins of interest, in particular one or several amylolytic, cellulolytic or hemicellulotic enzymes such as amylases, laccases, glucosidases, cellulases, xylanases, pectinases, esterases, acetyl xylan esterases, feruloyl esterase, p- coumaroyl esterases, alpha-arabinofuranosidase, beta-galactosidases, mannases, mannosidases and/or glucuronidases. In a particular embodiment, the composition comprises one or more additional enzymes requiring a source of divalent metal ion, preferably Mn(2+), the co-factor NAD(+) or NADP(+), preferably NAD(+), and/or a reducing agent. Examples of such enzymes include, but are not limited to, amylases and laccases.

The present invention also provides a composition comprising a recombinant host cell of the invention. The composition can be liquid (e.g. suspension) or dry (e.g. freeze-dried composition). Preferably, the composition comprising the host cell is kept frozen (e.g at about -20°C) until use. Preferably, the composition further comprises components suitable for cell preservation, in particular if cells are frozen. The composition of the invention may comprise one or several host cells of the invention, and optionally one or several additional cells.

The present invention also relates to a kit comprising (i) a polypeptide or a recombinant host cell of the invention and as defined above, and (ii) a source of divalent metal ion and/or the co-factor NAD(+) or NADP(+) and/or a reducing agent. In a particular embodiment, the kit comprises (i) a polypeptide or a recombinant host cell of the invention, and (ii) a source of divalent metal ion, preferably Mn(2+), and/or the co-factor NAD(+) or NADP(+), preferably NAD(+), and optionally a reducing agent.

The present invention also relates to a cellulosome or xylanosome comprising a polypeptide of the invention exhibiting alpha-galactosidase activity and comprising, or consisting of, an amino acid sequence having at least 77% identity to SEQ ID NO: 1, fused at its N-terminus and/or C-terminus to a dockerin or cohesin domain. Optionally, the fusion protein may further comprise one or several CBM domain. The fusion protein may be constructed as detailed above. Methods for constructing and producing chimeric cellulosomes are well known by the skilled person, and are described for example in the articles of Fierobe et al, 2002, J. Biol. Chem. 277, 49621-49630, and Fierobe et al, 2001, J. Biol. Chem. 276, 21257-21261.

The inventors showed that the polypeptide having the amino acid sequence of SEQ ID NO:l is able to hydro lyze the a-D- galactopyranoside /?-nitrophenyl model substrate but also far more complex substrates such as galacto-manno-oligo saccharides from locust bean gum. They also found that the enzyme exhibits maximal activity in a pH range from 7 to 8 and has an optimal temperature at 70°C.

Accordingly, in another aspect, the present invention relates to the use of a polypeptide or recombinant host cell of the invention expressing said polypeptide, or cell extract thereof, for hydrolyzing an alpha-D-galactoside. It also relates to a method of hydrolyzing an a-D- galactoside present in a substrate comprising contacting said substrate with a polypeptide or a recombinant host cell of the invention, or an extract thereof, and optionally recovering the hydro lyzed substrate.

As used herein, the term "alpha-D-galactoside" refers to a glycoside comprising galactose and in particular a glycoside comprising a terminal non-reducing a-D-galactose residue.

Examples of alpha-D-galactosides include, but are not limited to, galactose containing oligosaccharides such as melibiose, rafmose, stachyose and verbascose, galactose containing polysaccharides such as galactans, galactomannans, galactoglucomannans and arabinogalactans, galactolipids, or blood group B trisaccharide (Galal-3(Fucal-2)Gal) and tetrasaccharide (Galal-3(Fuccd-2)Gaipi-4GlcNAc).

As used herein, the term "galactolipid" refers to a glycolipid whose sugar group is galactose.

In an embodiment, the alpha-D-galactoside is selected from the group consisting of galactose containing oligosaccharides and galactose containing polysaccharides, and combinations thereof.

As used herein, the term "galactose containing oligosaccharide" refers to carbohydrates that are composed of two to nine monosaccharide residues joined through glycosidic linkage.

As used herein, the term "galactose containing polysaccharide" refers to a polymer of monosaccharides containing ten or more monosaccharide residues. The polysaccharide may be a homopolysaccharide, i.e. composed of a single type of monosaccharide, e.g. galactan, or a heteropolysaccharide, i.e. containing more than one kind of monosaccharide residue, e.g. galactomannans comprising galactose and mannose residues, galactoglucomannans comprising galactose, glucose and mannose residues, arabinogalactans comprising galactose and arabinose residues, or pectins.

In an embodiment, the alpha-D-galactoside is a galactose containing oligosaccharide selected from the group consisting of melibiose, rafmose, stachyose and verbascose, and combinations thereof.

In another embodiment, the alpha-D-galactoside is a galactose containing polysaccharide selected from the group consisting of galactan, galactomannans, galactoglucomannans and arabinogalactans, and combinations thereof. In a particular embodiment, the alpha-D-galactoside is a galactomannan selected from the group consisting of guar gum, tara gum, fenugreek gum, bean gum.

In a further embodiment, the alpha-D-galactoside is selected from the group consisting of melibiose, rafmose, stachyose, verbascose, galactan, galactomannans, galactoglucomannans and arabinogalactans, and combinations thereof.

The a-D-galactoside hydro lyzed by the polypeptide or recombinant host cell of the invention may be provided in the form of a raw, partially or totally purified substrate (e.g. partially or totally purified galactose containing oligo- or polysaccharides). In particular, the a- D-galactoside may be contained in a substrate selected from the group consisting of cellulosic biomasses, legume and seed derived food and feed products and molasses (i.e. by-products made during extraction of sugars from sugarcane and sugar beets).

As used herein, the term "cellulosic biomass" refers to any biomass material, preferably vegetal biomass, comprising cellulose, hemicellulose and/or lignocellulose, preferably comprising cellulose and hemicellulose. In some embodiments, the cellulosic biomass further comprises starch. Cellulosic biomass includes, but is not limited to, plant material such as forestry products, woody feedstock (softwoods and hardwoods), agricultural wastes and plant residues (such as corn stover, shorghum, sugarcane bagasse, grasses, rice straw, wheat straw, empty fruit bunch from oil palm and date palm, agave bagasse, from tequila industry), perennial grasses (switchgrass, miscanthus, canary grass, erianthus, napier grass, giant reed, and alfalfa); municipal solid waste (MSW), aquatic products such as algae and seaweed, wastepaper, leather, cotton, hemp, natural rubber products, and food processing by-products. Preferably, the cellulosic biomass is a galactose-containing biomass.

Preferably, if the cellulosic biomass comprises lignocellulose, this biomass is pre- treated before hydrolysis using the polypeptide of the invention. This pretreatment is intended to open the bundles of lignocelluloses in order to access the polymer chains of cellulose and hemicellulose. Pretreatment methods are well known by the skilled person and may include physical pretreatments (e.g. high pressure steaming, extrusion, pyrolysis or irradiation), physicochemical and chemical pretreatments (e.g. ammonia fiber explosion, treatments with alkaline, acidic, solvent or oxidizing agents) and/or biological pretreatments.

Examples of legume and seed derived food and feed products include, but are not limited to, soybean and soymilk. Indeed, removing raffmose and stachyose sugars from soy derived food and feed products greatly improves their digestibility by humans and monogastric animals. In a preferred embodiment, the substrate containing the a-galactoside to be hydrolyzed is a galactose rich substrate, preferably a galactose rich biomass, more preferably a galactose riche cellulosic biomass.

As used herein, the term "galactose rich substrate" refers to a substrate comprising at least 10% -D-galactoside, preferably 20, 30, 40, 50, 60, 70, 80, 90, 95, 99 or 100% a-D- galactoside. The substrate may be a solid or liquid substrate, a partially or totally purified substrate or a raw substrate. Preferably, percentages are weight/weight (w/w) percentages.

As used herein, the term "galactose rich biomass" refers to a biomass, preferably a vegetal biomass, comprising at least 10% a-D-galactoside, preferably 20, 30, 40, 50 or 60% a- D-galactoside. Preferably, the galactose rich biomass is a galactose rich cellulosic biomass comprising cellulose and at least 10% α-D-galactoside, preferably 20, 30, 40, 50 or 60% a-D- galactoside. Preferably, percentages are weight/weight (w/w) percentages. In particular, the galactose rich cellulosic biomass may be a marine plant biomass. Marine plant biomass may be obtained from macroalgae or microalgae. The macroalgae include red algae, brown algae, and green algae, while examples of microalgae include, but are not limited to, chlorella and spirulina. Preferably, the galactose rich cellulosic biomass is a biomass obtained from red algae. This biomass may be raw or pre-treated before to be subjected to hydrolysis with the polypeptide of the invention.

Examples of red algae include, but are not limited to, Gelidium amansii, Gracilaria verrucosa, Bangia atropurpurea, Porphyra suborbiculata, Porphyra yezoensis, Galaxaura falcate, Scinaia japonica, Gelidium divaricatum, Gelidium pacificum, Lithophylum okamurae, Lithothammion cystocarpideum, Amphiroa anceps, Amphiroa beauvoisii, Corallina officinalis, Corallina pilulifera, Marginisporum aberrans, Carpopeltis prolifera, Grateloupia filicina, Grateloupia elliptica, Grateloupia lanceolanta, Grateloupia turtuturu, Phacelocarpus japonicus, Gloiopeltis furcata, Hypnea charoides, Hypnea japonitca, Hypnea saidana, Chondrus cripspus, Chondracanthus tenellus, Gracilaria textorii, Lomentaria catenata, Heterosiphonia japonica, Chondria crassicaulis and Symphyocladia latiuscula. Preferably, the red algae is Gelidium amansii which contains approximately 15-25% cellulose and 50-70% agar which is composed mostly of galactan, based on the total dry weight.

Alpha-galactosidase enzymes were shown to exhibit specificities for blood group B trisaccharide (Galal-3(Fucal-2)Gal) and tetrasaccharide (Galal-3(Fucal-2)Gaipi-4GlcNAc) (Balabalova et al., 2010, Mar Biotechnol (NY), 12, 111-20). These enzymes can thus be used to remove of the immunodominant monosaccharides specifying the blood group B antigens, namely al,3-D-galactose.

Thus, in another embodiment, the a-D-galactoside hydrolyzed by the polypeptide or recombinant host cell of the invention is a blood group B or AB substrate.

The present invention also relates to a method for seroconversion of blood group B erythrocytes to blood group O erythrocytes or blood group AB erythrocytes to blood group A erythrocytes comprising contacting the blood group B or AB erythrocytes with a polypeptide of the invention, so as to remove the immunodominant B antigens, and optionally recovering modified erythrocytes.

Methods of seroconversion using a-galactosidase have been already described and are well known by the skilled person (e.g. Balabalova et al., 2010, Mar Biotechnol (NY), 12, 111- 20; US 2011/0014170; EP 1436387). Typically, group B or AB erythrocytes are suspended in a buffer solution having an approximately neutral pH (about pH 7 to about pH 8), and are contacted with an alpha-galactosidase polypeptide of the invention. Removing of immunodominant B epitopes may be controlled by serological typing or hemagglutination assays.

Removing raffmose and stachyose sugars from food and feed products greatly improves their digestibility by humans and monogastric animals.

Thus, the present invention also relates to an animal feed additive or a food additive comprising a polypeptide of the invention in combination with one or several other hydrolases. For example, hydrolases may be selected from the group consisting of lactases, amylases, β- galactosidases, phytases, β-glucanases, mannanases, mannosidase, xylanases, proteases and cellulases.

The present invention also relates to an animal feed additive or a food additive comprising a recombinant host cell of the invention or an extract thereof.

The additive of the invention may be prepared in accordance with methods known in the art and may be in the form of a dry or a liquid preparation. The additive of the invention may be supplemented to the animal before or simultaneously with the diet, preferably simultaneously with the diet. The additive of the invention is particularly useful when the diet comprises substantial amounts of leguminous, in particular soybean. Preferably, the animal is a monogastric animal such as poultry, pigs and calves. The additive of the invention may also be prepared in a suitable carrier or excipient so as to be in the form of a tablet, a capsule, a powder, a liquid, or in a soft-gel capsule form, to be administered to a mammal, and in particular to a human. This additive can thus be used as digestive aid and/or dietary supplement.

The present invention also relates to a method of pretreating animal feed or food, comprising subjecting the animal feed or food to the action of a polypeptide of the invention. The polypeptide of the invention may be used in the form of isolated or purified enzyme, optionally in combination with one or several other hydrolases such as lactases, β- galactosidases, phytases, β-glucanases, mannanases, xylanases, proteases and cellulases. Alternatively, the animal feed or food may be contacted with a recombinant host cell of the invention expressing said polypeptide, or a cell extract thereof. Preferably, the animal feed or food comprises substantial amounts of leguminous, in particular soybean, and is to be administered to monogastric animals.

The present invention further relates to a feed or food composition comprising a cellulosic biomass hydro lyzed or partially hydrolyzed by a polypeptide of the invention, or a recombinant host cell of the invention or cell extract thereof. The present invention also relates to a method of producing such feed or food composition comprising subjecting the cellulosic biomass to the action of a polypeptide of the invention. The polypeptide of the invention may be used in the form of isolated or purified enzyme, optionally in combination with one or several other hydrolases such as lactases, β-galactosidases, phytases, β-glucanases, mannanases, xylanases, proteases and cellulases. Alternatively, the cellulosic biomass may be contacted with a recombinant host cell of the invention expressing said polypeptide, or cell extract thereof. Preferably, the cellulosic biomass is a galactose-containing cellulosic biomass.

The present invention relates to the use of a polypeptide, recombinant host cell, or cell extract thereof, of the invention to remove alpha-galactose from galactose-containing oligosaccharides or polysaccharides present in a substrate, or to modify a substrate containing a terminal non-reducing a-D-galactose residue. It also relates to a method of removing alpha- galactose from galactose-containing oligosaccharide present in a substrate or modifying a substrate containing a terminal non-reducing α-D-galactose residue, said method comprising contacting the polypeptide, recombinant host cell, or cell extract thereof, of the invention with said substrate, and optionally recovering the modified substrate. The present invention further relates to a method of converting a substrate comprising galactose-containing oligosaccharides or polysaccharides or a substrate containing a terminal non-reducing -D-galactose residue to monomeric sugars comprising contacting the substrate with the polypeptide, the recombinant host cell, or cell extract thereof, of the invention.

The present invention also relates to a method of producing a fermentation product from a substrate comprising galactose-containing oligosaccharides or polysaccharides or a substrate containing a terminal non-reducing a-D-galactose residue comprising (a) contacting the substrate with the polypeptide, the recombinant host cell, or cell extract thereof, of the invention, thereby degrading the substrate into monomeric sugars; and (b) fermenting monomeric sugars obtained in step (a) into said fermentation product.

In a preferred embodiment, the substrate is a cellulosic biomass. Preferably, the cellulosic biomass is a galactose containing cellulosic biomass and more preferably a galactose- rich cellulosic biomass.

The fermentation may be carried out by the recombinant host cell of the invention used in step (a) or by another microorganism.

In an embodiment, step (a), i.e. enzymatic hydrolysis of the substrate, is separated from step (b), i.e. fermentation step. In this case, the substrate is hydrolyzed to monomeric sugars, and subsequently fermented in separate units.

In another embodiment, step (a) and step (b) are conducted simultaneously in the same reactor. This process may be SSF (Simultaneous Saccharification and Fermentation) or CBP (Consolidated Bioprocessing) process. In such process, monomeric sugars produced by the hydro lyzing enzymes is consumed immediately by the fermenting microorganism present in the culture.

The fermentation is a metabolic process carried out by a microorganism wherein monomeric sugars are converted to a product. This metabolic pathway may be naturally encoded by the microorganism, or said microorganism may have been genetically engineered to carry out such pathway.

The fermentation product may be any compound of industrial interest. Examples of fermentation products include, but are not limited to, biofuel such as ethanol, butanol, propanol, glycerol methanol, isopropanol, propanediol, glycerol or 2-3 butanediol, organic acids such as formate, acetate, lactate, butyrate, gluconate, xylonate, citrate, succinate, propionate, fumarate, malate, pyruvate, itaconic acid, muconic acid and kojic acid, and their salts or esters, isoprenoid compounds, or pharmaceutical compounds such as antibiotics, bacteriostatic compounds, antimetabolite, chemotherapeutic compounds, anti-parasitic agents, anti-fungal agents, anti-viral compounds, cytokine-activity compounds or cell-growth factors. Preferably, the fermentation product is a bio fuel, more preferably ethanol.

The present invention also relates to a method of producing a fermentation product from a substrate comprising galactose-containing oligosaccharides or polysaccharides or a substrate containing a terminal non-reducing a-D-galactose residue comprising contacting the biomass with a polypeptide comprising an amino acid sequence having at least 77 % identity to SEQ ID NO: 1 and exhibiting alpha-galactosidase activity, or a functional fragment thereof, or a host cell expressing said polypeptide, or cell extract thereof.

The host cell may be a cell naturally expressing the polypeptide or a recombinant host cell of the invention.

The present invention further relates to the use of a polypeptide of the invention, a host cell expressing said polypeptide, or cell extract thereof, for paper pulp bleaching. It also relates to a method of bleaching paper pulp comprising contacting the paper pulp with a polypeptide of the invention or a host cell expressing said polypeptide, or cell extract thereof. The variant of the invention may be used in combination with one or several additional enzymes such as xylanases, β-mannanases and/or β-glucosidases enzymes.

Depending on the conditions, the biomass or substrate can be contacted with polypeptide, recombinant host cell, or cell extract thereof, of the invention alone or in combination with other enzymes or cells. It should be understood that the precise amounts of polypeptide or host cell used initially in order to efficiently transform biomass or substrate can be adjusted by the skilled artisan depending on the type of cells, the type of biomass or substrate, and the culture conditions. In a particular embodiment, the method of the invention is performed in a reactor of conversion of biomass. By "reactor" is meant a conventional fermentation tank or any apparatus or system for biomass conversion, typically selected from bioreactors, biofilters, rotary biological contactors, and other gaseous and/or liquid phase bioreactors. The apparatus which can be used according to the invention can be used continuously or in batch loads. Depending on the cells used, the method may be conducted under aerobiosis, anaerobiosis or microaerobiosis.

The present invention further relates to a polypeptide of the invention for the treatment of Fabry disease. It also relates to a method of treating Fabry disease comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide of the invention, thereby treating Fabry disease. It further relates to a pharmaceutical composition comprising a polypeptide of the invention and a pharmaceutically acceptable carrier.

Fabry disease (also known as Fabry's disease, Anderson-Fabry disease, Angiokeratoma Corporis Diffusum and alpha-galactosidase A deficiency) is a rare X- linked recessive lysosomal storage disease caused by a mutational deficient activity of a-galactosidase A. Enzyme replacement therapy has been shown to successfully treat the disease.

As used herein, the term "treatment" refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease. In certain embodiments, such term refers to the amelioration or eradication of the disease, or symptoms associated with said disease. In other embodiments, this term refers to minimizing the spread or worsening of the disease resulting from the administration of one or more therapeutic agents to a subject with said disease.

As used herein, the term "subject" or "patient" refers to any mammal, preferably a human being.

The polypeptide of the invention may be formulated in accordance with standard pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known by a person skilled in the art. It may also be associated with other drugs used for the treatment of Fabry disease. By a "therapeutically effective amount" is intended an amount of the polypeptide of the invention administered to a patient that is sufficient to provide a therapeutic effect. It is to be understood that this amount may vary depending on physiological characteristics of the patient. A therapeutically effective amount of the polypeptide of the invention may range from about 50 to about 10,000 units a-galactosidase activity per kg body weight per day. The polypeptide of the invention may be administered in a convenient manner such as by oral or parenteral route.

Further aspects and advantages of the present invention will be described in the following examples, which should be regarded as illustrative and not limiting.

EXAMPLES MA TERIALS AND METHODS

Deinococcus strains

Deinococcus cellulosilyticus was obtained from DSMZ collection under the following reference DSM-18568.

Gene cloning and heterologous expression

The gene encoding the α-galactosidase was synthetized according to the genomic sequence determined by the inventors, and cloned into eurogentec plasmid PUC57. The resulting plasmid containing the alpha-galactosidase has been used has DNA matrice to amplify the gene by PCR using the following primers:

PCR products were subcloned on pDONR201 vector and then in a pET-DEST42 gateway vector system using the Gateway Technology as described by the manufacturer (Life technology). The resulting construct was checked by sequencing. E. coli BL21(DE3)pLysS strain was transformed with the pET-DEST42 vector containing the gene encoding the a-galactosidase. This transformed strain was then used to inoculate 4L of sterile LB medium containing 10(^g/ml ampicillin. The cultures were performed at 37°C under shaking and 1 mM IPTG was added when the cultures reached OD 5 6oonm equal to 0.6. Then, the cultures were shaken overnight to induce recombinant protein expression at 37°C. After centrifugation of induced cultures, supernatants were discarded.

Recombinant 6(His)-C-terminal tagged alpha-galactosidase Gal4 was purified onto Nickel-affinity chromatography column using AKTA purifier™ 10 as described below. The bacterial cell pellet from the induced cultures were resuspended in lysis buffer containing

10 50mM pH7.5 phosphate buffer or 50 mM Tris-HCl pH8 and 300mM NaCl, 5mM Imidazole, lmg/ml lysozyme, 0.5mM PMSF) and lysed by ultra sonication on ice. The broken cell suspensions were then centrifugated at 13000rpm 20 minutes at 4°C. The supernatant fraction containing the recombinant enzyme was loaded on pre equilibrated Colum Ni Hi trap HP column (histrap HP coloumn 1 ml 17-5247-01 from GE healthare). The column was washed

15 with 50 mM TRIS-Cl pH 7.5, 300 mM NaCl and 10 mM Imidazole to remove unspecific binding. Recombinant proteins were eluted with 200-300 mM Imidazole. Proteins were then applied onto Desalting Hi trap column (Hitrap desalting column 17- 1408-01 from GE healthare) to remove imidazole and NaCl excess using the following desalting buffer, 50 mM pH 7.5 phosphate 50mM NaCl. Proteins were purified to 90% homogeneity as visualized onto SDS-

20 PAGE and western blotting using antibodies directed against 6 His tag.

Alpha-galactosidase activity

α-galactosidase activity was determined using 1.0 mM p-nitrophenyl-alpha-D- galactopyranoside as substrate. The reaction mixture consisted of 180 of substrate in 0.1 M sodium citrate buffers (pH 5 and 7) and 20 μΕ of enzyme dilution. After 10 min incubation at 25 40°C, 100 of 1.0 M Na2C03 were added. Released /?-nitrophenol was quantified by measuring absorbance at 400 nm. Awonm values were converted in U/ml using p-nitrophenol as standard curve, α-galactosidase activity assays were performed in the presence or absence of 0.9 mM NAD, 1 mM MnCl₂ and 50 mM DTT, which were added to the buffer solutions. α-galactosidase activity was also determined using locust bean gum 30 (galactoglucomannan) prehydrolysed with mannanase as substrate. The hydrolysis was assayed in following conditions: substrate concentration 0.4 mg/ml, enzyme dosage 2000 nkat/g substrate, pH 7, temperature 40°C, hydrolysis time 24 h in the presence of 0.9 mM NAD, ImM MnC12, and 50 mM DTT. Hydrolysis was terminated by heating (98°C 10 min) and hydrolysis products were analyzed using HPLC and "L-Arabinose/D-Galactose" test kit (Megazyme). pH and temperature optima

For determination of pH optimum, a-galactosidase activity assays were carried out in Mcllvaine buffers pH 3-8 and in Glycine-NaOH buffers at pH 9-11, in the following conditions: p-nitrophenyl alfa-D-galactopyranoside (2 mM), enzyme: 2000 nkat/g of substrate, temperature : 40°C, hydrolysis time : 10 min in the presence of 0.9 mM NAD, 1 mM MnC12 and 50 mM DTT.

Similarly, temperature optimum was determined in the following conditions: 0.4 mg/ml prehydrolyzed galactoglucomannan from locust beam gum, enzyme : 2000 nkat/g of substrate, pH 8, temperature : 30-80°C, hydrolysis time : 10 min in the presence of 0.9 mM NAD, 1 mM MnC12 and 50 mM DTT.

RESULTS

Biochemical characterization

The inventors identified and amplified the gene encoding the alpha-galactosidase enzyme of Deinococcus cellulosilyticus (herein named Gal4). This gene was cloned in E. coli and the recombinant enzyme was purified.

The activity of recombinant Gal4 was shown to be dependent on the presence of co- factors NAD(+) and a divalent metal ion (e.g. Mn). Furthermore, optimal activity was obtained in a reducing environment, e.g. in the presence of DTT. In the presence of NAD(+), MnCl, and DTT, at pH 7, Gal4 exhibits a specific activity of 10.2 U/mg on ?-nitrophenyl-alpha-D- galactopyranoside.

Temperature and pH optima determination

The inventors found that Gal4 enzyme has maximal activity at pH 7-8 (Figure 1) and that the enzyme has an optimal temperature at 70°C (Figure 2). pH stability

The pH stability of Gal4 was determined at different pH values after 24h at 30°C by measuring the residual activity on pNP-alpha-D-galactopyranoside (pH 8, 40°C). It was shown that the enzyme is more stable at pH 6 but retains more than 60% of its activity between pH 7 and 8 (data not shown).

Substrates specificity

The inventors showed that the enzyme is active on/7-nitrophenyl-a-galactopyranoside, an artificial model substrate of -galactosidase.

To confirm the alpha-galactosidase activity on real substrate, hydrolysis of galactomannan oligosaccharides (obtained after hydrolysis of Locust bean galactoglucomannan with T. reesei mannanase) was investigated in the presence of NAD, Mn and DTT. Released galactose was analyzed by L-arabinose/D-galactose assay kit (Megazyme; based on galactose dehydrogenase) and HPLC. Hydrolysis results showed that, in addition to the artificial model substrate /?-nitrophenyl-a-galactopyranoside, Gal4 is able to hydro lyse galactose in galacto- manno-oligossaccharides (Figure 3). Furthermore, compared to unhydrolyzed galacto-manno- oligo mixture, the Gal4 treatment produced free galactose but also increased the amount of mannobiose (Man2) and mannotriose (Man3), indicating that galactomannans with mannose- to-galactose ratios of 2: 1 and 3: 1 are also substrates of Gal4 (Figure 3).

Claims

1. Use of a polypeptide comprising an amino acid sequence having at least 77 % identity to SEQ ID NO: 1 and exhibiting alpha-galactosidase activity, or a functional fragment thereof, or a recombinant host cell expressing said polypeptide, for hydrolyzing an -D-galactoside.

2. The use of claim 1 wherein the polypeptide comprises an amino acid sequence having at least 80, 90, 95, 98, 99% identity to SEQ ID NO: 1.

3. The use of claim 1 or 2, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

4. The use of claim 1 to 3, wherein the hydrolysis is conducted at temperature from about 30°C to about 70°C and/or at pH from about 7 to about 8.

5. The use of any one of claims 1 to 4, wherein the a-D-galactoside is selected from the group consisting of galactose containing oligosaccharides, galactose containing polysaccharides and galactolipids.

6. The use of claim 5, wherein the α-D-galactoside is a galactose containing oligosaccharide selected from the group consisting of galactan, melibiose, raffinose, stachyose and verbascose.

7. The use of claim 5, wherein the α-D-galactoside is a galactose containing polysaccharides selected from the group consisting of galactans, galactomannans, galactoglucomannans and arabinogalactans.

8. The use of any one of claims 1 to 7, wherein the -D-galactoside is contained in a substrate is selected from the group consisting of cellulosic biomasses, legume and seed derived food and feed products, molasses, and group B or AB erythrocytes.

9. The use of claim 8 wherein the substrate is a galactose-rich biomass, in particular a marine plant biomass such as biomass obtained from macroalgae or microalgae.

10. A recombinant nucleic acid construct, expression cassette or vector comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence having at least 77 % identity to SEQ ID NO: 1 and exhibiting alpha-gal actosidase activity, or a functional fragment thereof.

11. A recombinant host cell comprising a recombinant nucleic acid construct, expression cassette or vector of claim 10.

12. A method of producing a polypeptide comprising an amino acid sequence having at least 77 % identity to SEQ ID NO: 1 and exhibiting alpha-gal actosidase activity, or a functional fragment thereof, comprising

(a) culturing the host cell of claim 11 conditions conducive for production of said polypeptide; and

(b) recovering said polypeptide from the cell culture; and

(c) optionally, purifying said polypeptide.

13. A composition or a kit comprising (i) a polypeptide comprising an amino acid sequence having at least 77 % identity to SEQ ID NO: 1, or a functional derivative thereof, and exhibiting alpha-galactosidase activity, and (ii) a divalent metal ion, preferably Mn²⁺, and/or the co-factor NAD⁺ or NADP⁺, preferably NAD⁺, and/or a reducing agent, preferably dithiothreitol.

14. A method of producing a fermentation product from an a-galactoside containing substrate comprising contacting the substrate with a polypeptide comprising an amino acid sequence having at least 77 % identity to SEQ ID NO: 1 and exhibiting alpha-galactosidase activity, or a functional fragment thereof, or a host cell expressing said polypeptide or a cell extract thereof.

15. The method of claim 14, wherein the fermentation product is a bio fuel, such as ethanol, butanol, propanol, glycerol methanol, isopropanol, propanediol, glycerol or 2-3 butanediol, an organic acid such as formate, acetate, lactate, butyrate, gluconate, xylonate, citrate, succinate, propionate, fumarate, malate, pyruvate, itaconic acid, muconic acid and kojic acid, and their salts or esters, an isopreno^'id compound, or a pharmaceutical compound such as antibiotics, bacteriostatic compounds, anti-metabolite, chemotherapeutic compounds, antiparasitic agents, anti-fungal agents, anti-viral compounds, cytokine-activity compounds or cell- growth factors.