# Patent

## Title
Fructanase

## Abstract

The present invention relates to a novel protein and a method for the manufacture thereof. The novel protein according to the invention is a recombinant protein with fructanase activity. The recombinant protein according to the invention is an engineered protein derived from recombinant DNA encoding for the protein. The recombinant protein may be or may comprise a fragment of a naturally occurring protein, i.e. of a naturally occurring fructanase protein.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims benefit of the filing of U.S. provisional application 61/155,591 filed Feb. 26, 2009 and of UK application GB 0903227.


The sequence listing electronically filed herewith is hereby incorporates by reference in its entirety (File Name: 4091-55004Seq_ListCorrected3.txt; File Size: 18 KB; Date Created May 15, 2012).
FIELD OF THE INVENTION
The present invention relates to recombinant proteins. In particular, the present invention relates to proteins having fructanase activity and a method for their production.
INTRODUCTION
Fructans or fructooligosaccharides (FOS) are sugar polymers containing fructose molecules as well as fructose-glucose disaccharides. Fructans contain a core sucrose group (fructose and glucose) and an extension using fructose. Chemical bonds linking fructose and glucose differ from chemical bonds linking fructose to fructose. Fructans are often found in plants and play an important role in food industry and in probiotics or prebiotics (Prebiotics are carbon source for good bacteria. Humans can not metabolize these prebiotics). Examples of fructans are inulin or levan, both of which are fructose containing linear polysaccharides. Recent development indicates that fructans may also be used for the production of bio fuels, such as but not limited to bio-ethanol.
The production of bio-ethanol as well as many other processes utilising fructan as a carbon source for growth requires the hydrolysis of fructans in order to obtain monosaccharides such as fructose or glucose. The hydrolysis of fructans may be performed by naturally occurring enzymes such as a β-fructosidase precursor (fosE) as suggested in Goh et al., (“Functional Analysis of the Fructooligosaccharide Utilization Operon in Lactobacillus paracasei 1195”; Applied and Environmental Microbiology, September 2007, p. 5716-5724). Extracellular enzymes such as inulinase that hydrolyse fructans are extracted from Aspergillus niger and are commercially available. These extracellular enzymes are naturally occurring enzymes that are isolated or extracted from their natural environments. However, these fructanase extracellular enzymes are expensive and difficult to obtain in sufficient amounts and good purity for large scale applications.
SUMMARY OF THE INVENTION
The present invention relates to a novel protein and a method for the manufacture thereof. The novel protein according to the invention is a recombinant protein with fructanase activity. The recombinant protein according to the invention is an engineered protein derived from recombinant DNA encoding for the protein. The recombinant protein may be or may comprise a fragment of a naturally occurring protein, i.e. of a naturally occurring fructanase protein. The recombinant protein may be an enzyme. The fragment may have an amino acid sequence corresponding essentially to Seq. ID 3 or a homologue or variant thereof or may be a similar related sequence.
The fructanase activity of the recombinant protein may have advantageous properties for production and hydrolysis of fructans. The recombinant protein may hydrolyse at least one type of fructan to obtain sugar molecules. The fructose may be D-fructose and the glucose in fructan may be D-glucose. The protein may thus be termed fructanase. The protein may also be a levanase, and in this case the protein will hydrolyse levans, or an inulinase where the protein will hydrolyse inulin. Other polysaccharides may also serve as substrates.
Examples of fructans are inulin, levan, 1-kestose, nystose, raffinose, stachyose and melezitose or a combination thereof.
The recombinant protein may be a peptide. The protein may have a molecular weight of less than about 140 kDa. The protein may have a molecular weight of less than about 100 kDa. As a non-limiting an example, the protein may have a molecular weight of about 81.1 kDa.
The recombinant protein may comprise a portion of the amino acid sequence of a β-fructosidase precursor (fosE). The fosE may be fosE of lactic acid bacteri such as Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Streptococcus or the like. For example Lactobacillus paracasei, Lactobacillus casei, Lactobacillus rhamnosus or others may be used. The recombinant protein may also be from another organism e.g. prokaryote or eukaryote.
The portion or fraction may be a domain or a core domain of fosE. The portion may be an amino acid sequence encoding for a certain region of fosE. However, modification may be made to this portion in a usual manner. Further amino acids or an amino acid sequence may be added to the portion or certain amino acids may be removed or replaced in a usual manner well known in the art. For example, the portion may have a modified N-terminal and a modified C-terminal amino acid sequence.
The protein may comprises one or more tags. For example, the tags may be used for purification. One or more hisitidine residues may be added to form one or more polyhistidine tags (his-tags). For example, a his-tag may be added at the C-terminal side of the portion. Other tags known in the art may also be used for the purification of the protein.
An expression vector, such as pET17b or other vectors known in the art may be attached to a DNA sequence encoding the protein to enable production in an appropriate host.
The protein of the invention may be used in the fermentation of fructans and to produce monosaccharides from fructans as, for example, a step in the production of ethanol from fructans. Other uses may be in processing of fructans to release fructose.
The invention also relates to DNA encoding for the recombinant protein. The invention further related to an expression system or expression construct for expressing the recombinant protein. The recombinant protein may be expressed in prokaryotic or eukaryotic cells, for example in Escherichia. coli (E. coli).
A preferred method for delivering the expression construct into the cell is transformation or transfection, wherein known substances for alleviating transfer of the expression construct through the cell membrane are within the scope of the present invention.
Genetic material comprising nucleic acids, polynucleotides, RNA and DNA, of either natural or synthetic origin, including recombinant RNA and DNA and antisense RNA and DNA; hammerhead RNA, ribozymes, antigene nucleic acids, both single and double stranded RNA and DNA and analogs thereof, either in combination or not with other elements such as, for example, without limitation, cell specific enhancers, and nuclear localization signals, can be introduced into prokaryotic or eukaryotic cells or organisms via transformation or transfection techniques. The present invention uses an “expression construct”, “nucleic acid construct” or alternatively a “nucleotide construct” or alternatively a “DNA construct”. The term “construct” is used herein to describe a molecule, such as a polynucleotide may optionally be chemically bonded to one or more additional molecular moieties, such as a vector, or parts of a vector. In a specific—but non-limiting—aspect, a nucleotide construct is exemplified by a DNA expression constructs suitable for the transformation of a cardiac host cell. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell/or tissue, including, for example, without limitation, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.
The invention further relates to a method for the manufacture of a recombinant protein with fuctanase activity, the method comprising: expressing a protein with fructanase activity in an expression system; isolating the protein with fructanase activity, wherein the protein is a recombinant protein that comprises a fragment or a partial sequence of a naturally occurring protein. The protein may be a protein as set out above, i.e the naturally occurring protein may be a naturally occurring fructanase protein. The recombinant protein may be an enzyme. The fragment may have an amino acid sequence corresponding essentially to Seq. ID 3 or a homologue or variant thereof or may be a similar related sequence.
Expressing the protein may be performed using prokaryotic cells such as bacteria, (i.e. Escherichia. coli (E. coli)). Other expression systems known to a person skilled in the art may also be used.
The isolated protein may be purified for example using a histidine-tag (his-tag) or other tags known in the art. The his-tag may be attached at the C-terminal of the protein.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows a SDS-PAGE of purified recombinant truncated β-fructosidase precursor (ΔfosE).
FIG. 1B shows an indication of the partial polypeptide sequence of ΔfosE expressed in E. coli compared to the native β-fructosidase precursor (fosE) protein's amino acid sequence (not underlined portion).
FIG. 2 shows temperature (A) and pH (B) profiles for activity of the recombinant ΔfosE activity using chicory inulin as a substrate.
FIG. 3 shows activity profiles using different carbon sources and activity of a recombinant ΔfosE protein.
FIG. 4 illustrates the growth of yeast on grass juice and in the presence of recombinant ΔfosE and when heat denatured.
FIG. 5. shows measurements of a) ethanol yield, b) optical density and c) cell numbers estimated by haemocytometer counts for Saccharomyces cerevisiae grown on untreated grass juice (GJ), GJ+recombinant ΔfosE, GJ+hd recombinant ΔfosE, GJ+Exo-Inulinase, GJ+Endo-Inulinase and GJ+Exo/Endo-Inulinase (t75 hr). *=significantly higher (Student's t-test, P<0.05, n=3) values than those seen on untreated GJ.



DETAILED DESCRIPTION
The following description of an embodiment of the invention is purely exemplary and the invention is by no means limited to this embodiment. A person skilled in the art will easily adapt the teachings to other biological systems.
The invention is demonstrated by the example of a truncated polypeptide cloned from the β-fructosidase precursor (fosE) from Lactobacillus paracasei (L. paracasei). It is evident to a person skilled in the art that the invention may be applied to fosE of other Lactobacillus species or other lactic acid bacteria as well as to other natural occurring proteins having fructanase activity.
The truncated polypeptide comprises the N-terminal deletion of signal sequences and the C-terminal deletion of cell-binding motif found in the native protein of L. paracasei. The truncated polypeptide was expressed successfully in Escherichia. coli (E. coli) using pET17b as expression vector and forming a ΔfosE-pET17b construct. Polymerase chain reaction using pfx polymerase was utilised to isolate the open-reading from strain 4134. The N-terminal amino acid sequence MAS comprised part of the NheI restriction site and ATG start triplet. A polyhistidine tag (his-tag) to facilitate purification, a stop codon and a BamHI restriction site were encoded in the primer for PCR corresponding to the C-terminal amino acid sequence.
Primers utilized in this work, referred to as Seq. ID 1 and Seq. ID 2:







(Seq.ID 1)


5′ ACGTAGCTAGCGCTACAAGTGCTTCGTCTAC


 


(Seq.ID 2)


5′ CGTAGGATCCTCAGTGGTGGTGGTGGTGGTGTTTTTCAGTTAGTTGA


 


CCAG






The truncated polypeptide comprises a portion of the amino acid sequence of fosE of L. paracasei. The amino acid sequence portion may be termed core domain of the β-fructosidase precursor (ΔfosE) having the amino acid sequence in, which is referred to as Seq. ID 3):
MASASSASSTQISQTNTGSQPNNETTGETAQSSVNSTATASSSSVADLPSSSDSKSSIGSTISQPTVDK KETSKSDTADNDLTKSVTTSDSDKALPTSKTTLPTSNEQVQSSVGQSQTDQPASSATIATNAVTSDVS QNDQYNEPYRNQYHYSSSQNWINDPNGLFYDSKTGLYNLYYQYNPEGNQWGNMSWGHAVSKDLI NWTQEDVAIPMLQNQGWEDFTYTNTTGSLKDKGEVRYVGVPTTNWGDADGKKAIFSGSIVVDTN NVSGLGKDAILAFYTADYQIATRKNDGAEDGWGTWIGLTEIQEQHLAYSLDGGKTFIQYSKDGNAA NPQAIIPTSMNQGGDAANFRDPSVVYDAVNKQYYLTVVSGQQALIYKSSNLLDWTYASKIERENDV GNGVWECPSLVPMKVAGTNETKWVFCISVQQGAHATGSGMQYYVGNMTADGTWVPESSKTLQN PMTMDSGEDFYAGIPFSNMPDGRTVMLAWQSNWSYVDEAKTSPWSGNMTLPRELSLKKNADTTD GYLLTNTVVKEIANNEEANVINKAESNFTVSRSDEQVQYEGKQYKISATFSWDEADKPKSVGFKLR VSDDQKYDMIVGYDLTTGLLYVQRLNTGEPNMGAPRDKMNATVNADGSITITVYVDETSIEAFAN DGEKSITQNFFMRPENIGDQATTGVYVYSNDGTTKISDLTINPITSIWNSTGQLTEK

An N-terminal amino acid sequence MAS has been added.

This is compared to the amino acid sequence of fosE of L. paracasei, referred to as Seq. ID 4:








>Q27J21|Q27J21_LACPA Beta-fructosidase -



Lactobacillus paracasei.


MEMDEKKHYKMYKSKSVWVFACLSTCLIVSFFNDGQNVSAATSASSTQISQTNTGSQPNN


 


ETTGETAQSSVNSTATASSSSVADLPSSSDSKSSIGSTISQPTVDKKETSKSDTADNDLT


 


KSVTTSDSDKALPTSKTTLPTSNEQVQSSVGQSQTDQSASSATIATNAVTSDVSQNDQYN


 


EPYRNQYHYSSSQNWINDPNGLFYDSKTGLYNLYYQYNPEGNQWGNMSWGHAVSKDLINW


 


TQEDVAIPMLQNQGWEDFTYTNTTGSLKDKGEVRYVGVPTTNWGDADGKKAIFSGSIVVD


 


TNNVSGLGKDAILAFYTADYQIATRKNDGAEDGWGTWIGLTEIQEQHLAYSLDGGKTFIQ


 


YSKDGNAANPQAIIPTSMNQGGDAANFRDPSVVYDAVNKQYYLTVVSGQQALIYKSSNLL


 


DWTYASKIERENDVGNGVWECPSLVPMKVAGTNETKWVFCISVQQGAHATGSGMQYYVGN


 


MTADGTWVPESSKTLQNPMTMDSGEDFYAGIPFSNMPDGRTVMLAWQSNWSYVDEAKTSP


 


WSGNMTLPRELSLKKNADTTDGYLLTNTVVKEIANNEEANVINKAESNFTVSRSDEQVQY


 


EGKQYKISATFSWDEADKPKSVGFKLRVSDDQKYDMIVGYDLTTGLLYVQRLNTGEPNMG


 


APRDKMNATVNADGSITITVYVDETSIEAFANDGEKSITQNFFMRPENIGDQATTGVYVY


 


SNDGTTKISDLTINPITSIWNSTGQLTEKFVDENGNTIASDKIQTGRVGQSYTSESATIP


 


GYVFVKENTDHINSNQLYTTQNQTITYTYRASQASVVTKDTTLAAGPSAAWNAADNLVGA


 


TDADGNALAVSDLTVNGAVDPKTPGTYTVTYSYTDATGNKISKKATVTVIASKADIVTKD


 


TTMVAGASTIWNAADNFVEAKNADGNALTVSDLMINGTVDSKTPGTYTVTYSYTDAAGNK


 


INKEAIVTVIASKADIVTKDTTMVAGPSAAWNAVDNFVEATGADGNALALSDLTVNGAVD


 


PKTPGTYTVTYSYTDPAGNKISKEATVTVIASKADIVTKDTTMVAGPSATWNAVDNFVEA


 


TGADGNALALSDLTVNGAVDPKTPGTYTVTYSYTDVAGNKISKEAIVTVIASKADIVTKD


 


TTKVAGPSATWNAADNLVIATDAKGNALALSNLTVTGSVDSKTPGTYTVTYSYTDAAGNK


 


ISKEATVTVIASKADIVTKDTTMVAGPSAAWNAANNLVSATDADGNALAMSNLTVTGTVD


 


LKTQGTYTVTYTYTDVAGNKISKEATVTVLTEKETNIEDNTGSSISNDRENPPASITGKG


 


GDDIHQNAKTTMTKKKTETLPQAGNHVNELAIVLGQMILAICVGGILWLKRRVKRV






A direct comparison of ΔfosE sequence and fosE sequence is shown in FIG. 1B. The complete sequence corresponds to the sequence of fosE while the sequences left away for ΔfosE are underlined. Consequently, the non-underlined portion corresponds to the ΔfosE protein sequence.
The ΔfosE sequence is expressed in bacterial system and the recombinant ΔfosE protein is isolated and eventually purified as described below. The obtained ΔfosE protein is en enzyme having fructanase activity, i.e. the ΔfosE protein is a levanase or an inulinase or both, hydrolysing levan and inulin and other fructans.
Heterologous Expression in E. Coli and Isolation of Recombinant ΔfosE protein.
The ΔfosE-pET17b construct was transformed into E. coli strain BL21 (DE3) and positive transformants selected using ampicillin. Overnight cultures (10 ml) of transformants were used to inoculate one litre volumes of Terrific Broth supplemented with 20 g·l−1 peptone and 0.1 mg·ml−1 sodium ampicillin. Cultures were grown at 37° C., 230 rpm for 7 hours prior to induction with 1 mM IPTG and expression at 30° C., 190 rpm for 18 hours. Recombinant ΔfosE protein was isolated according to the method of Arase et al (Arase M, Waterman M R, Kagawa N; Biochem Biophys Res Commun 2006 May 26; 344(1):400-5. Epub 2006 Mar. 20) except that 2% (w/v) sodium cholate and no Tween20 were used in the sonication buffer. The solubilized ΔfosE protein was purified by affinity chromatography using Ni2+-NTA agarose with the modification that 0.1% (w/v) L-histidine in 50 mM sodium phosphate, pH 7.5, 25% (w/v) glycerol was used to elute non-specifically bound E. coli proteins after the salt washes and elution of P450 protein was achieved with 1% (w/v) L-histidine in 50 mM sodium phosphate, pH 7.5, 25% (w/v) glycerol. Isolated ΔfosE protein fractions were stored at −80° C. Protein purity was assessed by SDS polyacrylamide gel electrophoresis and the identity of the purified protein confirmed by trypsin digestion followed by nano-LC/MS/MS of the tryptic peptides released. A SDS page of purified recombinant ΔfosE is illustrated in FIG. 1A.
Determination of Fructanase Activity.
Fructanase activity, in this example exo-fructanase activity, was determined using a discontinuous assay system with the levels of fructose produced being determined at fixed time intervals. The standard assay system consisted of a 1 ml reaction volume of saccharide solution in 0.1 M sodium acetate, pH 5, containing 1.62 ng/ml Ni-NTA agarose purified ΔfosE. Incubation was for 30 minutes at 37° C. prior to the withdrawal of 100 μl for colour development with 0.9 ml of 1 mg/ml 2,3,5-triphenyl tetrazolium chloride in 1 M NaOH (15 minutes at 37° C.). The pink-red colour produced was monitored by the absorbance at 520 nm. The colorimetric assay was standardised against 100 μl of fructose solutions (0 to 6 mM) in 0.1 M sodium acetate, pH 5. Each assay was performed in triplicate. The colorimetric reaction with 2,3,5-triphenyl tetrazolium chloride was found to be 20-fold more sensitive for D-fructose than D-glucose using the conditions described above. Exo-fructanase activity was expressed as nmoles of fructose produced per minute per μg ΔfosE protein.
Protein concentrations were determined by the Coomassie Blue 8250 dye-binding method (BioRad, Hemel Hempstead, UK) using bovine serum albumin standards. Spectral determinations were made using a Hitachi U-3310 UV/VIS spectrophotometer (San Jose, Calif.).
Thermostability Determinations.
Stock solutions (162 μg/ml) of Ni-NTA agarose purified ΔfosE protein were incubated for ten minutes at temperatures ranging from 22 to 90° C. These enzyme solutions were then incubated on ice prior to the commencement of the exo-fructanase assay described above in 10% (w/v) chicory inulin, 0.1 M sodium acetate, pH 5. The inulin was solubilised by warming to 70° C. for 5 minutes followed by cooling to room temperature prior to use.
pH-Profile Determinations.
The pH-profile of ΔfosE protein was determined between pH 3 and 11 using 10% (w/v) chicory inulin dissolved in 0.1 M buffer as described previously. The buffers used were 0.1 M sodium acetate (pHs 3, 3.5, 4, 4.5, 5, 5.5, 6), 0.1 M sodium phosphate (pHs 6.5, 7, 7.5), 0.1 M Tris-HCl (pHs 8, 8.5) and sodium bicarbonate/carbonate (pHs 9, 9.5, 10, 10.5, 11).
Substrate Saturation Determinations.
Substrate specificity for ΔfosE protein was determined using chicory inulin (0.125 to 20% w/v), sucrose (0.025 to 2 M), 1-kestose (0.005 to 0.4 M), nystose (0.0045 to 0.35 M), rafinose (0.02 to 0.48 M), levan (from Zymomonas mobilis 0.088 to 3.5% w/v), stachyose (0.04 to 0.4 M) and melezitose (0.04 to 0.4 M) in 0.1 M sodium acetate, pH 5 using the exo-fructanase assay system described previously.
Chemicals.
All chemicals, unless otherwise stated, were obtained from Sigma Chemical Company (Poole, UK). DIFCO growth media were obtained from Becton Dickinson Ltd (Cowley, UK).
Experimental Results
Heterologous Expression and Purification of Recombinant ΔfosE Protein.
Expression of truncated fosE levanase (recombinant ΔfosE protein) in E. coli followed by purification using affinity chromatography on Ni-NTA agarose yielded 22.5 nmoles fosE levanase from 1 litre of cell culture which was over 95% pure as resolved by SDS-PAGE (lane 3—FIG. 1A). However, only 35% of the total exo-fructanase activity (as determined using 10% w/v chicory inulin) detected in the cytosolic fraction was recovered, albeit with a 5-fold increased in specific activity. Less than 1% of the exo-fructanase activity was found not to bind to the Ni-NTA agarose matrix. SDS-PAGE (FIG. 1A) indicated that the recombinant ΔfosE protein had an apparent molecular weight of 100 kDa, some 20 kDa greater than that predicted from the amino acid sequence of the truncated fosE enzyme. The identity of the purified ΔfosE protein was confirmed by trypsin digestion of the 100 kDa SDS-PAGE band (FIG. 1A—lane 3) followed by nano-LC/MS/MS, identifying 21 peptides present in ΔfosE protein (Q27J21—full length sequence) which accounted for 50.7% coverage of the truncated protein (FIG. 1B) with a MASCOT score of 3609. Gel exclusion chromatography of ΔfosE protein on Sephacryl S-400HR indicated that the native molecular weight of the ΔfosE protein was 85 kDa (data not shown) when compared against several protein standards ranging in molecular weight from 12 to 700 kDa. While the polypeptide used in the invention had a predicted molecular weight of 81.1 kDa the predicted molecular weight of the native protein is 147 kDa.
Biochemical Characterisation of ΔfosE Protein.
Thermostability studies (FIG. 2A) indicate that the recombinant ΔfosE protein was stable up to 46° C. for 10 minutes. At temperatures higher than this, the fructanase activity is rapidly lost, with the ΔfosE protein effectively deactivated by temperatures higher than 55° C. The T0.5 value for the ΔfosE protein was calculated to be 49° C. under the stated conditions. The pH profile (FIG. 2B) of the ΔfosE protein, using 10% (w/v) chicory inulin, indicated an optimal pH of 5 to 5.5 for the exo-fructanase activity measured, with the activity of the ΔfosE protein falling sharply as the pH fell below 5.0. The decrease in the observed exo-fructanase activity as the pH is increased above 5.0 is gradual, with the ΔfosE protein effectively becoming inactive at pH values above 8.
Substrate saturation experiments with the polyfructans chicory inulin and especially bacterial levan (FIG. 3A) were hampered by the relative insolubility of these compounds in 0.1 M sodium acetate buffer, pH 5. This relative insolubility was partially solved by warming the solutions for 5 minutes at 70° C., which increased the solubility of these the compounds, even when cooled back to room temperature. However, incubation for periods longer than 5 minutes at 70° C. were avoided at this caused the release of free fructose from the polyfructans (probably by acidic hydrolysis of the glycosidic bonds). The bacterial levan became extremely viscous at concentrations 3.5% (w/v) preventing higher concentrations from being used. The chicory inulin solutions above 6% (w/v) became progressively more viscous with the chicory inulin solution progressively changing in appearance from a colourless solution to a viscous white slurry at 25% (w/v). A saturating concentration of bacterial levan could not be obtained due to solubility problems of the bacterial levan, with 3.5% (w/v) levan yielding an exo-fructanase specific velocity of 66 nmol/min/μg. A saturating concentration of 8% (w/v) chicory inulin (78 nmol/min/μg) was obtained above which, the observed enzyme velocity fell with further increase in the chicory inulin concentration. This is indicative of substrate inhibition and km and ki values for chicory inulin of 7.8% and 11.2%, respectively, were calculated by non-linear regression of the Michaelis-Menten single substrate inhibition equation [v=(Vmax·[S])/(km+([S]2/ki)+[S])]. The inhibition caused by chicory inulin concentrations above 8% (w/v) is likely to be due in part to viscosity effects rather than ‘classical’ substrate inhibition alone.
Substrate saturation experiments with the oligosaccharides (FIG. 3B) 1-kestose, nystose, rafinose, stachyose and melezitose were less problematic, with no solubility problems encountered up to 0.5 M. The stachyose and the melezitose could not be hydrolysed to produce free fructose by the ΔfosE protein under the stated exo-fructanase assay conditions at concentrations up to 0.5 M saccharide. The rafinose was a relatively poor substrate with a specific velocity of just 2 nmol/min/μg observed in 0.32 M rafinose. This was is in contrast to both the 1-kestose and the nystose. The nystose gave a ‘biphasic’ substrate saturation curve with the first Michaelis-Menten phase extending up to 0.2 M, yielding a specific velocity of 39 nmol/min/μg and a km value of 15.5 mM. At higher nystose concentrations, the velocity significantly increases well beyond what is predicted by either the Michaelis-Menten or Hill equations. The substrate saturation curve obtained with the 1-kestose achieved a maximum velocity of 47 nmol/min/μg at 0.1 M kestose, with further increases in kestose concentration causing a progressive reduction in the observed exo-fructanase velocity. This velocity curve is indicative of substrate inhibition and can be described using the Michaelis-Menten single substrate inhibition equation (see above) with km and ki values of 50 mM and 210 mM, respectively, being obtained for 1-kestose.
The substrate saturation velocity curve obtained with sucrose (FIG. 3C) obeyed Michaelis-Menten kinetics yielding a km value of 398 mM and an observed specific velocity of 62 nmol/min/μg with 2 M sucrose. The ability to hydrolyse sucrose into free fructose and glucose is indicative of an invertase/sucrase enzyme. Therefore, the ΔfosE protein exhibits wide-ranging substrate specificity for the exo-fructanase reaction, suggesting that this ΔfosE protein could be a levan(o)sucrase rather than a typical exo-fructanase/levanase/inulinase.
Table 1 shows further characterisation of the ΔfosE protein for the release of fructose from the fructan in grass juice with potential for the optimised production of bioethanol from grass. The table 1 shows efficient release of fructose in column 2 that is abolished on addition of heat denatured protein in column 3 The ΔfosE protein compares well with commercial endo- and exo-inulinase and a combination of these. The release of sugars fermentable by Saccharomyces cerevisiae as opposed to the fructan polymer could provide a route to optimal bioethanol production using the novel ΔfosE protein or derivatives thereof made by genetic improvements.










Carbohydrate content of growth and fermentation media (t0hr)
























n.d = not determined


Δ = change (±) in glc or fru content following enzyme addition


Glucose concentrations quantified using a Glucose Assay Kit (GAGO-20, SIGMA).


Fructose concentrations quantified using a colorimetric assay for six-carbon ketohexoses (not glucose).






This is supported by the examination of growth characteristics in FIG. 4 which illustrates the growth of yeast on grass juice and in the presence of recombinant ΔfosE protein and when heat denatured. The presence of the active protein allows enhanced growth compared to a heat denatured form.
FIG. 5 shows measurements of a) ethanol yield, b) optical density and c) cell numbers estimated by haemocytometer counts for Saccharomyces cerevisiae grown on untreated grass juice (GJ), GJ+recombinant ΔfosE, GJ+hd recombinant ΔfosE, GJ+Exo-Inulinase, GJ+Endo-Inulinase and GJ+Exo/Endo-Inulinase (t75 hr). *=significantly higher (Student's t-test, P<0.05, n=3) values than those seen on untreated GJ.

## Claims

1. An isolated protein with fructanase activity, wherein the protein comprises the amino acid sequence of SEQ ID NO:3.

2. The isolated protein according to claim 1, wherein the protein is isolated from Lactobacillus paracasei.

3. The isolated protein according to claim 1, wherein the protein further comprises at least one histidine residue.

4. The isolated protein according to claim 1, wherein the protein comprises a tag.

5. The isolated protein according to claim 1, wherein the fructanase activity comprises the ability to hydrolyse a fructan.

6. The isolated protein according to claim 5, wherein the fructan is at least one of inulin, levan, 1-kestose, nystose, rafinose, stachyose and melezitose or a combination thereof.

7. The isolated protein according to claim 1, wherein the protein is soluble in an aqueous solution.

8. The isolated protein according to claim 1, recombinantly expressed in a prokaryotic or an eukaryotic cell.