Reference to a Sequence Listing

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a sugar beet extraction method, said method comprising the steps of treating sugar beet cossettes with an enzyme composition comprising an effective amount of one or more pectin lyase enzyme (EC 4.2.2.10); and, optionally, recovering the sugar.

BACKGROUND OF THE INVENTION

Sugar beets are harvested and sent to a processing plant, where they are washed and mechanically sliced into thin strips called cossettes. The cossettes are passed to a machine called a diffuser to extract the sugar content into a water solution. Diffusers are long vessels of many metres in which the beet slices go in one direction while hot water goes in the opposite direction. The movement may either be caused by a rotating screw or the whole rotating unit, and the water and cossettes move through internal chambers. The flow rates of cossettes and water are usually in the ratio one to two. Typically, cossettes take about 90 minutes to pass through the diffuser, the water only 45 minutes. These countercurrent exchange methods extract more sugar from the cossettes using less water than if they merely sat in a hot water tank. The liquid exiting the diffuser is called raw juice. The colour of raw juice varies from black to a dark red depending on the amount of oxidation, which is itself dependent on diffuser design.

The used cossettes, or pulp, exit the diffuser at about 95% moisture, but low sugar content. Using screw presses, the wet pulp is then pressed down to 75% moisture. This recovers additional sugar in the liquid pressed out of the pulp, and reduces the energy needed to dry the pulp. The pressed pulp is dried and sold as animal feed, while the liquid pressed out of the pulp is combined with the raw juice, or more often introduced into the diffuser at the appropriate point in the countercurrent process. The final byproduct, vinasse, is used as fertilizer or growth substrate for yeast cultures.

During diffusion, a portion of the sugar breaks down into invert sugars. These can undergo further breakdown into acids. These breakdown products are not only losses of sugar, but also have knock-on effects reducing the final output of processed sugar from the factory. To limit (thermophilic) bacterial action, the feed water may be dosed with formaldehyde and control of the feed water pH is also practiced.

SUMMARY OF THE INVENTION

The instant invention provides a sugar beet extraction method comprising an enzymatic treatment of sugar beet cossettes with an effective amount of one or more pectin lyase enzyme (EC 4.2.2.10). Preferably the one or more pectin lyase enzyme has certain characteristics and is added alone or in combination with one or more additional enzyme, as outlined below.

Accordingly, in a first aspect the present invention relates to a sugar beet extraction method, said method comprising the steps of:

a) treating sugar beet cossettes with an enzyme composition comprising an effective amount of one or more pectin lyase enzyme (EC 4.2.2.10); and, optionally,

b) recovering the sugar.

In a second aspect, the invention provides a sugar beet extraction enzyme composition as defined herein.

A final aspect of the invention relates to the use of an enzyme composition in a method as defined in the first aspect; preferably to improve processing, improve the sugar yield, reduce the amount of sugar in the molasses or increase the amount of dry substance in the beet pulp, compared with a method without enzyme treatment.

BRIEF DESCRIPTION OF THE FIGURES

The present invention and in particular preferred embodiments according to the invention will be described in more detail with reference to the accompanying figures. The figures show ways of implementing the present invention and are not to be construed as being limiting to other possible embodiments falling within the scope of the attached claim set.

Figure 1 shows the filtration speeds at pH 5.5 after 65°C incubation in Example 1 of the different enzyme-treated samples plus the negative control.

Figure 2 shows the filtration speeds at pH 5.5 after 65°C incubation in Example 2 of the different enzyme-treated samples plus the negative control.

DEFINITIONS

Sugar Beet Cossettes: After harvesting, sugar beets are sent to a processing plant, where they are washed and mechanically sliced into thin strips called cossettes.

Pectin lyase: The term“pectin lyase enzyme”,“pectin lyase” and polypeptide with pectin lyase activity are used interchangeably herein and refer to one or more {e.g. several) EC 4.2.2.10 enzymes of the PL1 subfamily 4 domain (PL1_4) as well as clusters such as the clades. A phylogenetic tree was constructed herein of polypeptide sequences containing a PL1 subfamily 4 domain, as defined in CAZy (PL1 , Polysaccharide Lyase Family 1 , subfamily 4, The Carbohydrate-active enzymes database (CAZy) in 2013, Lombard V et al, Nucleic Acids Research, Database Issue 42).

Contact time: For one or more enzymes to react with a substrate, the one or more enzymes have to be in contact with the substrate.“Contact time” refers to the time period in which an effective amount of one or more enzymes is in contact with at least a fraction of a substrate mass. The enzymes may not be in contact with all of the substrate mass during the contact time, however mixing the one or more enzymes with a substrate mass allows the potential of enzymatically catalyzed hydrolysis of a fraction of the substrate mass during the contact time.

Mature enzyme: The term “mature enzyme” or“mature polypeptide” means an enzyme or polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.

Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter“sequence identity”.

For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. Version 6.1.0 was used.

The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labelled“longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment).

DETAILED DESCRIPTION

It is an object of the present invention to provide a sugar beet extraction method, said method comprising the steps of treating sugar beet cossettes with an enzyme composition comprising an effective amount of one or more pectin lyase enzyme (EC 4.2.2.10); and, optionally, recovering the sugar.

In a preferred embodiment of the first aspect of the invention, the enzyme composition comprises an effective amount of one or more pectin lyase enzyme of the Polysaccharide Lyase Family 1 subfamily 4 domain (PL1_4), as defined in the Carbohydrate-active enzymes database; preferably the amino acid sequence of the one or more pectin lyase comprises the conserved motif: [FLA]X[GS][VAT]T[GA]GGX[AT] (SEQ ID NO:4); more preferably the one or more pectin lyase comprises the conserved motif: [FLA]X[GS][VAT]T[GA]GGX[AT] (SEQ ID NO:4) and the conserved motif: T[GA][AT][GA]C (SEQ ID NO:5).

In another preferred embodiment of the first aspect of the invention, the enzyme composition comprises one or more mature pectin lyase having an amino acid sequence that is at least 70% identical to the mature amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3; SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1 1 or SEQ ID NO: 12; preferably at least 75% identical, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or most preferably at least 98% identical to the mature amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3; SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1 1 or SEQ ID NO:12.

It is highly preferred that the enzyme composition of the invention comprises at least one pectin lyase having a melting point temperature, Tm, at both pH 5 and 6 that is at least 50°C; preferably at least 52°C; more preferably at least 54°C, 56°C, 58°C, 60°C, 62°C, 64°C, 66°C, 68°C, 70°C, 72°C, 74°C, or even more preferably at least 76°C; preferably the melting point temperature, Tm, is determined by the thermal shift assay described herein.

In another preferred embodiment of the first aspect of the invention, the enzyme composition also comprises an effective amount of one or more rhamnogalacturonan lyase enzyme (EC 4.2.2.23), one or more pectate lyase enzyme (EC 4.2.2.2), one or more rhamnogalacturonan a cetyl esterase enzyme (EC 3.1.1.86); galactanase enzyme (EC 3.2.1.89); one or more polygalacturonase enzyme (EC 3.2.1.15); one or more rhamnogalacturonan hydrolase enzyme (EC 3.2.1.171 ); and/or one or more arabinase enzyme (EC 3.2.1.15).

It is preferred that the enzyme composition of the invention also comprises an effective amount of one or more rhamnogalacturonan lyase enzyme (EC 4.2.2.23); preferably the one or more one or more rhamnogalacturonan lyase enzyme comprises at least one mature rhamnogalacturonan lyase having an amino acid sequence that is at least 70% identical to the mature amino acid sequence of SEQ ID NO: 13; preferably at least 75% identical, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or most preferably at least 98% identical to the mature amino acid sequence of SEQ ID NO:13.

It is preferred that the enzyme composition of the invention also comprises an effective amount of one or more pectate lyase enzyme (EC 4.2.2.2); preferably the one or more pectate lyase enzyme comprises at least one mature pectate lyase having an amino acid sequence that is at least 70% identical to the mature amino acid sequence of SEQ ID NO: 14; preferably at least 75% identical, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or most preferably at least 98% identical to the mature amino acid sequence of SEQ ID NO: 14.

It is also preferred that the enzyme composition of the invention also comprises an effective amount of one or more rhamnogalacturonan acetylesterase enzyme (EC 3.1.1.86); preferably the one or more rhamnogalacturonan acetylesterase enzyme comprises at least one mature rhamnogalacturonan acetylesterase having an amino acid sequence that is at least 70% identical to the mature amino acid sequence of SEQ ID NO: 15; preferably at least 75% identical, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or most preferably at least 98% identical to the mature amino acid sequence of SEQ ID NO: 15.

Also, it is preferred that the enzyme composition of the invention also comprises an effective amount of one or more galactanase enzyme (EC 3.2.1.89); preferably the one or more galactanase enzyme comprises at least one mature galactanase having an amino acid sequence that is at least 70% identical to the mature amino acid sequence of SEQ ID NO: 16; preferably at least 75% identical, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or most preferably at least 98% identical to the mature amino acid sequence of SEQ ID NO:16.

Preferably, the enzyme composition of the invention also comprises an effective amount of one or more polygalacturonase enzyme (EC 3.2.1.15); preferably the one or more polygalacturonase enzyme comprises at least one mature polygalacturonase having an amino acid sequence that is at least 70% identical to the mature amino acid sequence of SEQ ID NO: 17; preferably at least 75% identical, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or most preferably at least 98% identical to the mature amino acid sequence of SEQ ID NO: 17.

The enzyme composition of the invention also preferably comprises an effective amount of one or more rhamnogalacturonan hydrolase enzyme (EC 3.2.1.171 ); preferably the one or more rhamnogalacturonan hydrolase enzyme comprises at least one mature rhamnogalacturonan hydrolase having an amino acid sequence that is at least 70% identical to the mature amino acid sequence of SEQ ID NO: 18; preferably at least 75% identical, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or most preferably at least 98% identical to the mature amino acid sequence of SEQ ID NO:18.

In a preferred embodiment, the enzyme composition of the invention also comprises an effective amount of one or more arabinase enzyme (EC 3.2.1.15); preferably the one or more arabinase enzyme comprises at least one mature arabinase having an amino acid sequence that is at least 70% identical to the mature amino acid sequence of SEQ ID NO: 19; preferably at least 75% identical, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, or most preferably at least 98% identical to the mature amino acid sequence of SEQ ID NO: 19.

In a preferred embodiment of the first aspect of the invention, the processing is improved, the sugar yield is improved, the amount of of sugar in the molasses is reduced, and/or the amount of dry substance in the beet pulp is increased, as compared with the same method without enzyme composition treatment.

In another preferred embodiment of the first aspect of the invention, the cossettes are allowed to react with the enzyme composition for at least 15 minutes, preferably for at least 30 minutes, more preferably for at least 45 minutes. Preferably the cossettes are allowed to react with the enzyme composition at a temperature of at least 50°C; preferably at least 52°C; more preferably at least 54°C, 56°C, 58°C, 60°C, 62°C; 64°C, 66°C, 68°C, 70°C, 72°C, 74°C, or even more preferably at least 76°C. Also preferably, the cossettes are allowed to react with the enzyme composition at a pH in the range of 4 to 7, preferably in the range of 5 to 6.

Preferably, the dosage of the enzyme composition is adjusted so that the effective amount of one or more enzyme is in the range of 0.5g to 5kg enzyme protein per metric tonne sugar beet cossettes per day.

EXAMPLES

Example 1. Determination of filtration rate of enzyme treated cossettes

Filtration assay

Substrate for the assay:

Frozen cossettes (a term for sliced sugar beets well known to people skilled in the art) were thawed and cut into 1 cm pieces. Procedure:

50 g cossettes + 50 g water were added to a 250 ml flask, 1000 ppm Ca++ were added to the flask (as CaC ) and pH adjusted to 5,5 with 1 M HCI.

Enzymes were added to the slurry and the flasks (with lid) were incubated for 1 hour at 65°C in a shaking incubator

After incubation, the content of the flasks was poured into a funnel with a paper filter (320 nm) and the filtration rate was measured.

The sugar content of the filtrates were measured as Brix (measurement of the refractometer index - a procedure wellknown to people skilled in the art). Table 1. Enzymes used in this example

Table 2. The enzyme dosages listed below are as pg purified enzyme protein per gram of cossette weight (as is). Results (mean value of two repeats, standard deviation in parenthesis):

Table 3. Brix measurements of filtrate after 5 minutes

The results show a slower filtration rate with dosage of pectin lyase which indicates a higher sugar content in the filtrate.

Example 2. Determination of filtration rate of enzyme treated cossettes

The procedure described in Example 1 was also used in this example.

Table 4. Enzymes used in this example Table 5. The enzyme dosages listed below are as pg purified enzyme protein per gram of cossette weight (as is). Results (mean value of three repeats, standard deviation in parenthesis)

Example 3. Construction of clades and phylogenetic trees

This example provides polypeptides of the invention having pectin lyase activity and comprising the PL1 subfamily 4 domain (PL1_4) as well as clusters such as the clades. A phylogenetic tree was constructed of polypeptide sequences containing a PL1 subfamily 4 domain, as defined in CAZy (PL1 , Polysaccharide Lyase Family 1 , subfamily 4, The Carbohydrate-active enzymes database (CAZy) in 2013, Lombard V et al, Nucleic Acids Research, Database Issue 42). The phylogenetic tree was constructed from a multiple alignment of mature polypeptide sequences containing at least one PL1_4 domain. The sequences were aligned using the MUSCLE algorithversion 3.8.31 (Edgar, 2004. Nucleic Acids Research 32(5): 1792-1797), and the trees were constructed using FastTree version 2.1.8 (Price et al., 2010, PloS one 5(3)) and visualized using iTOL (Letunic & Bork, 2007. Bioinformatics 23(1 ): 127-128).

The polypeptides of the PL1_4 domain comprise several motifs; one motif is [FLA]X[GS][VAT]T[GA]GGX[AT] (SEQ ID NO:4) situated in positions corresponding to positions 1 1 to 20 in the mature pectin lyase of Aspergillus niger (SEQ ID NO:1 ), where the G in position 18 is fully conserved in the clade.

Generation of GGG clade

A phylogenetic tree was constructed, of polypeptide sequences containing a PL1_4 domain, as defined above. The phylogenetic tree was constructed from a multiple alignment of mature polypeptide sequences containing at least one PL1_4 domain. The sequences were aligned using the MUSCLE algorithm version 3.8.31 (Edgar, 2004. Nucleic Acids Research 32(5): 1792-1797), and the tree was constructed using FastTree version 2.1.8 (Price et al., 2010, PloS one 5(3)) and visualized using iTOL (Letunic & Bork, 2007. Bioinformatics 23(1 ): 127-128). Using the phylogenetic tree, the polypeptides in PL1_4 can be separated into distinct sub-clusters, one which we denoted GGG.

A characteristic motif for this sub-cluster is the motif [FLA]X[GS][VAT]T[GA]GGX[AT] (SEQ ID NO:4), corresponding to the amino acids in positions 1 1 to 20 in SEQ ID NO: 1. An additional motif of the GGG clade is T[GA][AT][GA]C (SEQ ID NO:5), corresponding to the amino acids in positions 59 to 63 in the mature pectin lyase polypeptide of A. niger (SEQ ID NO:1 ). Example 4. Melting point determination of PL1_4 pectin lyases by Thermal Shift Assay

To determine the thermostability of a pectin lyases at pH 5 and pH 6, a Thermal Shift Assay (TSA) (Lo, M.C. el al. (2004). Analytical Biochemistry. 332 (1 ): 153-9) was used to determine the melting point (Tm) or temperature at which the enzymes denature at the selected pH. Purified pectin lyase samples were prepared for TSA by diluting to a standard concentration, 0.24 mg/ml, in Milli- Q water, and SYPRO Orange dye (Life Technologies, S6650) was diluted 1 :200 in Assay buffer. Assay buffers used were 0.1 M succinic acid, 0.1 M HEPES, 0.1 M CHES, 0.1 M CAPS, 0.15 M KCI, 1 mM CaCh, 0.01 % Triton X100, pH adjusted to either 5 or 6.

For measurement, 10 pi of diluted enzyme sample was combined with 20 pi of diluted dye in the well of a TSA assay plate (Roche LightCycler 480 Multiwell plate 96, white) and the plate was covered with optic sealing foil (Roche LightCycler 480 Sealing foil). Thermal ramping and fluorescence measurements were run in a Roche Lightcycler 480 II machine running with a ramp from 25 to 99 °C at a rate of 200 °C/h. The data collected was analyzed by Roche LightCycler 480 software (release 1.5.0 SP4). All samples were analyzed in duplicate and averaged. The reported readout is Tm, defined as the midpoint value of the protein melting curves.

Table 6. Pectin lyase melting temperatures at pH 5 and 6.