ZA200603771B - Method of producing hemicellulase-containing enzyme compositions and use thereof - Google Patents

Description

4 GS

BACKGROUND OF THE INVENTION

The present invention relates to a method for the production of hemicellulolytic-, cellulolytic- and pectolytic-containing enzyme compositions and to their use in coffee production.

More particularly, the present invention relates to a method for providing yeast and 25s fungal strains adapted to produce enzyme compositions containing hemicellulolytic, cellulolytic and pectolytic activities, and to the use of these compositions in the production of coffee extracts.

Polysaccharides such as arabinogalactan, mannan, and cellulose constitute nearly 50 % of the green coffee bean weight (Nunes et al., 2001; Sachslehner et al., 2000).

The major polysaccharide of this fraction is a water-insoluble, crystalline mannan that forms approximately 20-30% of the dry weight of Arabica and Robusta beans (Sachslehner et al., 2000). These carbohydrates play an important role in the

Cf }

\ . : retention of volatile substances due to their capacity to bind aromatic compounds at the adsorptive sites (Nunes et al., 2001; Trugo, 1985).

Modern instant coffee production entails cleaning, roasting and grinding of coffee beans, followed by a split extraction and concentration to achieve a high solids concentrate for a low-energy spray-drying operation (Stoltze and Masters, 1979).

During the roasting procedure of the coffee beans, the physical and chemical properties of carbohydrates change drastically (Sachslehner et al, 2000). For instance, green coffees contain 62 % arabinogalactan, 24 % galactomannan, and glucans in the high-molecular weight material extracted with water, whereas roasted coffees contain 28 % arabinogalactan and 69 % galactomannan (Nunes et al., 2001).

After roasting and grinding, extraction is the key operation in the large-scale manufacture of instant coffee in which both soluble solids and volatile aroma/flavour compounds are extracted (Clarke, 1987). Technically produced extracts from roasted Arabica and Robusta coffee contain 20-36 % carbohydrates depending on the degree of extraction. They are predominantly composed of mannan and galactan in about the same proportions, with glucan and araban making up only 1-3 % of the extracts (Thaler, 1979).

Coffee extraction techniques lead to extracts of around 25 % w/w soluble solids concentration (Clarke, 1987). Spray-drying of these concentrations can provide coffee of the required physical form, although there will be substantial loss of volatile compounds. Consequently, pre-concentration methods for coffee extracts prior to drying have been introduced to circumvent extensive losses (Clarke, 1987). The extracts can be concentrated to 45 % w/w soluble solids concentration in vacuum evaporators, resulting in lower costs of removing water during drying (Stoltze and

Masters, 1979). However, concentrations above 42 % w/w solids are difficult to reach, due to the high viscosity of coffee extracts. Also highly viscous extracts will require longer pre-concentration times (Clarke, 1987). The viscosity of coffee extracts can be reduced by hydrolysing the mannan, xylan, cellulose and pectin to short oligosaccharides (Sachslehner et al, 2000; Wong and Saddler, 1993). This would make it possible to concentrate the extracts to concentrations higher than 42 % w/w soluble solids.

SUMMARY OF THE INVENTION

According to a first embodiment of the invention, there is provided a DNA expression : cassette for use in transforming a yeast or fungus so as to provide it with a capability of producing B-mannanase, the expression cassette including: a gene encoding a f-mannanase enzyme; and a suitable promoter for promoting transcription of the gene in the transformed yeast or fungus.

The expression cassette may include a suitable terminator sequence for promoting : efficient expression of the 3-mannanase gene.

The gene encoding B-mannanase may be the man? gene from a fungus such as

Aspergillus aculeatus MRC11624.

The yeast or fungus strain may be a Saccharomyces cerevisiae yeast strain (especially Saccharomyces cerevisiae Y294(pMESH1)), Yarrowia lipolytica, Pichia pastoris, Hansenula polymorpha, Aspergillus awamori or Aspergillus niger (especially

Aspergillus niger D15(pGT-man1)).

The yeast promoter and terminator sequences may be the PGK promoter and terminator DNA sequence, respectively.

The fungus promoter and terminator sequences may be the gpd promoter and glaA

Co 25 terminator DNA sequence, respectively.

According to a second embodiment of the invention, there is provided a DNA vector including the gene encoding the B-mannanase gene. The DNA vector containing the gene for B-mannanase, as well as promoter and terminator sequences, may be the yeast/Escherichia coli shuttle vector YEp352 or the Aspergillus/Escherichia coli shuttle vector pGT.

According to a third embodiment of the invention, there is provided a DNA expression cassette for use in transforming a yeast or fungus so as to provide it with a capability of producing B-endoglucanase 1, p-endoglucanase 2, -endoglucanase 3, B-xylanase 2, or B-cellobiohydrolase 1-4, the expression cassette including: one or more genes encoding one or more of a B-endoglucanase 1, B- endoglucanase 2, B-endoglucanase 3, B-xylanase 2, or [-cellobiohydrolase 1-4 enzyme, a suitable promoter for promoting transcription of the gene(s) in the transformed fungus or yeast strain.

The expression cassette may include a suitable terminator DNA sequence for promoting efficient expression of the p-endoglucanase 1, B-endoglucanase 2, B- endoglucanase 3, B-xylanase 2, or B-cellobiohydrolase 1-4 genes.

The recombinant B-endoglucanases 1, 2, and 3 may be produced from Aspergillus niger D15(pGT-eg1), Aspergillus niger D15(pGT-eg2), and Aspergillus niger

D15(pGT-eg3).

The recombinant B-xylanase may be produced from Aspergillus niger

D15(pGT-xyn2).

The recombinant p-cellobiohydrolase may be produced from Aspergillus niger

D15(pGT-cbh1-4).

The genes encoding B-endoglucanase 1, B-endoglucanase 2, f-endoglucanase 3, and B-xylanase 2 may be the eg?, eg2, eg3 or xyn2 genes, respectively, from a fungus such as Trichoderma reesei QM6a (see Table 1 for list of genes, donor organisms and Genbank accession numbers for the DNA sequences and deduced protein sequences). ] The gene encoding B-cellobiohydrolase 1-4 may be the cbh1-4 gene from a fungus such as Phanerochaete chrysosporium ATCC 24725.

The fungus promoter and terminator sequences may be the gpd promoter and glaA terminator DNA sequence (Rose and van Zyl, 2002).

According to a further embodiment of the invention, there is provided a DNA vector including the gene encoding one or more of the B-endoglucanase 1, B- endoglucanase 2, B-endoglucanase 3, and B-xylanase 2 genes. The DNA vector may be the Aspergillus/Escherichia coli shuttle vector pGT.

According to a further embodiment of the invention, there is provided a method of producing a yeast strain which is capable of expressing B-mannanase, the method including the step of: "transforming a yeast strain with a nucleotide sequence including a gene encoding a B-mannanase enzyme and a suitable promoter for promoting transcription of the gene in the transformed yeast.

The transformation of the fungus strain may be effected by: (a) constructing the yeast expression vector containing the ADHZ - : 15 promoter and terminator DNA regions, called plasmid pDLG1 (La Grange et al., 1996); (b) amplifying the mani gene from a Aspergillus aculeatus MRC11624 culture by the PCR technique with the aid of oligodeoxyribonucieotide DNA primers designed by conventional techniques and cloning as a 1180-bp EcoRI/Xho1 DNA fragment into plasmid pDLG1 to generate plasmid pMES1 (Figure 12A) (Setati et al., 2001); (c) isolating total RNA from an Aspergillus aculeatus MRC11624 culture prepared on locust bean gum as carbon source; (d) purifying poly-A mRNA from the total RNA and preparing first strand complementary DNA (cDNA) for the poly-A mRNA; and (e) designing oligodeoxyribonucleotide DNA primers by conventional techniques.

The yeast strains may be selected from Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, and Hansenula polymorpha.

According to a further embodiment of the invention, there is provided a method of producing a fungus strain which is capable of expressing B-mannanase, the method including the step of:

transforming a fungus strain with a nucleotide sequence including a gene encoding a B-mannanase enzyme and a suitable promoter for promoting transcription of the gene in the transformed fungus.

The transformation may be effected by: (a) constructing the fungus expression vector containing the gpd promoter and glaA terminator DNA sequences, called plasmid pGT (Rose and Van Zyl, 2002); (b) amplifying the man1 gene from a Aspergillus aculeatus MRC11624 culture with the PCR technique with the aid of oligodeoxyribonucleotide DNA primers designed by conventional techniques and cloned as a 1180-bp EcoRI/Xho1 DNA fragment into plasmid pGT to generate plasmid pGT-man1 (Figure 12B).

According to a further embodiment of the invention, there is provided a method of ( producing a fungus strain which is capable of expressing one or more of B- " 15 endoglucanase 1, B-endoglucanase 2, p-endoglucanase 3, B-xylanase 2 and B- cellobiohydrolase 1-4, the method including the step of: : transforming a fungus strain with a nucleotide sequence including one or more genes encoding B-endoglucanase 1, B-endoglucanase 2, -endoglucanase 3,

B-xylanase 2 and B-cellobiohydrolase 1-4 and a suitable promoter for promoting transcription of the gene in the transformed fungus.

The transformation may be effected by: (a) constructing the fungus expression vector containing the gpd promoter and glaA terminator DNA sequences, called plasmid pGT (Rose and Van Zyl, 2002); and : (b) cloning the selected genes into plasmid pGT to generate plasmids pGT- egl, pGT-eg2, pGT-eg3, pGT-xyn2, pGT-cbh1-4 using similar methods described above for construction of pGT-manf.

The fungus strain may be an Aspergillus niger fungus strain. The recombinant B- endoglucanases 1, 2, and 3 may be produced from Aspergillus niger D15(pGT-eg1),

Aspergillus niger D15(pGT-eg2), and Aspergillus niger D15(pGT-eg3). The recombinant p-xylanase may be produced from Aspergillus niger D15(pGT-xyn2).

The recombinant B-cellobiohydrolase may be produced from Aspergillus niger

D15(pGT-cbh1-4).

According to a further embodiment of the invention, there is provided a host cell 5s which has been transformed as described above. The host cell may be a yeast or fungus, such as Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris,

Hansenula polymorpha, Aspergillus awamori or Aspergillus niger.

According to a further embodiment of the invention, there is provided p-mannanase enzyme produced by: causing a transformed host cell to express the B-mannanase gene, and recovering the B-mannanase enzyme.

According to a further embodiment of the invention, there is provided one or more of P-endoglucanase 1, B-endoglucanase 2, p-endoglucanase 3, B-xylanase 2 and - cellobiohydrolase 1-4 enzymes produced by: causing a host cell to express the gene(s) encoding the B-endoglucanase 1,

B-endoglucanase 2, B-endoglucanase 3, 3-xylanase 2 and/or B-cellobiohydrolase 1-4, and recovering the p-endoglucanase 1, -endoglucanase 2, -endoglucanase 3,

B-xylanase 2 and/or B-cellobiohydrolase 1-4 enzyme.

According to a further embodiment of the invention, there is provided a method of producing a coffee extract, the method including the step of subjecting roasted, ground coffee beans or leftovers thereof to an enzymatic hydrolysis extraction.

The extraction yield and/or quality of coffee extracts, soluble solids and/or volatile compounds obtained from the roasted, ground coffee beans, or leftovers thereof, ‘may be improved by the enzymatic hydrolysis.

The coffee extract may be used to produce coffee by percolation.

The hydrolytic enzyme extraction procedure may occur at a temperature of from : about 30 to about 100°C. More particularly, the extraction may occur at a temperature of from about 60 to about 80°C.

The enzymatic hydrolysis step may be performed for a period of up to about 24 hours.

Up to about 800 kg of one or more enzymes per ton dry spent ground or dry coffee beans may be used to perform the extraction step. However, an amount of as littie as about from 1 to about 7 kg of the enzymes per ton dry spent ground or dry coffee beans may be used to perform the extraction.

The method may include the step of drying the coffee extract.

The coffee extract may comprise between 8 % and 40 % w/v concentration. i

The coffee beans may be Arabica and/or Robusta beans.

The coffee beans may contain 20-36% w/v carbohydrates, predominantly in the form of p-1,4-mannan and B-1,3-galactan.

B-1,4-mannan and B-1,3-galactan may be hydrolysed by the enzyme.

The coffee extract may have a concentration higher than 42% w/v.

One or more of galactomannan, cellulose, xylan and pectin present in the coffee beans or leftovers may be hydrolysed by the enzyme(s) during the enzymatic hydrolysis extraction step.

The enzyme(s) may be recombinant or native, and may be B-mannanase,

B-endoglucanase 1, B-endoglucanase 2, B-endoglucanase 3, B-xylanase 2, and/or p-cellobiohydrolase 1-4.

Recombinant B-mannanase may be produced from Saccharomyces cerevisiae (especially Saccharomyces cerevisiae Y294(pMES1)), Yarrowia lipolytica, Pichia pastoris, Hansenula polymorpha, Aspergillus awamori or Aspergillus niger (especially

Aspergillus niger D15(pGT-man1)).

The recombinant p-endoglucanases 1, 2, and 3 may be produced from Aspergillus niger D15(pGT-egt), Aspergillus niger D15(pGT-eg2), and Aspergillus niger

D15(pGT-eg3).

The recombinant B-xylanase may be produced from Aspergillus niger

D15(pGT-xyn2).

The recombinant B-cellobiohydrolase may be produced from Aspergillus niger

D15(pGT-cbh1-4). :

Natural fungal strains may be isolated from green or roasted coffee beans, or leftovers thereof. Natural fungal strains may also be isolated from nature or from decaying plant material.

B-mannanase, B-endoglucanase, B-xylanase, B-cellobiohydrolase, polygalacturonase, pectin lyase, and pectin esterase enzyme activities may also be complimented by additional (-mannanase, B-endoglucanase, B-xylanase, B- cellobiohydrolase, polygalacturonase, pectin lyase, and pectin esterase enzyme activities.

According to a further embodiment of the invention, there is provided a method for determining the fraction of monosaccharide represented in the total measurable neutral sugars for roasted ground coffee beans and spent ground.

- 2uu0/0377.L

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Pie charts indicating the fraction of minor sugars (1), arabinogalactan (2), xylan (3), mannan (4), and cellulose (5) in (A) roasted ground coffee, and (B) spent ground.

Figure 2: Total soluble solid yield increases measured in spent ground (Freeze- dry method) treated with recombinant enzymes followed by a thermal extraction process. The spent ground was treated with 3-mannanase (Man1) (e), B-endoglucanase 1 (Eg1) (MW), p-endoglucanase 2 (Eg2) (®@), and B-xylanase (Xyn2) (A) at concentrations of 0.2 nkat/mg, 0.8 nkat/mg, 1.6 nkat/mg, 2 nkat/mg and 10 nkat/mg spent ground.

Figure 3: Total soluble solid yield increases measured in spent ground (Freeze- 2 dry method) treated with recombinant enzymes followed by a thermal o extraction process. The spent ground was treated with B-mannanase (Man1) (), p-endoglucanase 1 (Eg1) (M), p-endoglucanase 2 (Eg2) (®), and B-xylanase (Xyn2) (A) at different protein concentrations.

Figure 4: Total soluble solid yield increases measured in spent ground (Freeze- dry method) treated with enzyme cocktails from natural fungal strains followed by a thermal extraction process. The spent ground was treated with cocktails from ABO 503 (®) and ABO 500 (Ml) at 0.2 nkat/mg, 0.6 nkat/mg, 1 nkat/mg, 2 nkat/mg or 8.8 nkat/mg spent ground.

Figure 5: Total soluble solid yield increases measured in spent ground (Freeze- dry method) treated with enzyme cocktails from natural fungal strains followed by a thermal extraction process. The spent ground was treated with cocktails from ABO 503 (@) and ABO 500 (HM) at different protein concentrations.

Figure 6: Soluble solid yield increases measured in spent ground treated with commercial enzyme cocktails. The amount of enzyme added was based on the amount of p-mannanase activity of the enzymes. An equivalent of 2 nkat/mg spent ground was added. The enzymes used are as follows; [1] Man1, [2] Cellulosin GMS (Anchor Biotechnologies and Hankyu Bioindustry Co., LTD., Japan), [3] Pectinex Ultra SP-L (Novozymes SA, Sandton, SA), [4] Gamanase (Novozymes SA,

Sandton, SA), [5] TP668L (Biocatalysts, Whales, UK), [6] D040L (Biocatalysts, Whales, UK), [7] D112L (Biocatalysts, Whales, UK), [8]

M263L (Biocatalysts, Whales, UK), [9] D670L (Biocatalysts, Whales,

UK), [10] GO15L (Biocatalysts, Whales, UK), [11] C013L (Biocatalysts,

Whales, UK), [12] M282L (Biocatalysts, Whales, UK), [13] TP692L (Biocatalysts, Whales, UK), and [13] D690L (Biocatalysts, Whales, Co

UK). - Figure 7: Soluble solid yield increases measured in spent ground (Freeze-dry. method) treated with commercial enzyme cocktails. The spent ground was treated with a total of 0.004 g total protein per 30 g spent ground.

The enzymes used are as follows; [1] Man1, [2] Cellulosin GM5 (Anchor Biotechnologies and Hankyu Bioindustry Co., LTD., Japan),

[3] Pectinex Ultra SP-L (Novozymes SA, Sandton, SA), [4] Gamanase (Novozymes SA, Sandton, SA), [5] TP668L (Biocatalysts, Whales,

UK), [6] DO40L (Biocatalysts, Whales, UK), [7] D112L (Biocatalysts,

Whales, UK), [8] M263L (Biocatalysts, Whales, UK), [9] D670L (Biocatalysts, Whales, UK), [10] GO15L (Biocatalysts, Whales, UK),

[11] CO13L (Biocatalysts, Whales, UK), [12] M282L (Biocatalysts,

Whales, UK), [13] TP692L (Biocatalysts, Whales, UK), and [13] D690L (Biocatalysts, Whales, UK).

Figure 8: Saturation curve showing the soluble solid yield increases measured in spent ground (Total sugar method) after treatment with Mannanase-

L (River Biotech (Pty) Ltd, Milnerton, SA). The spent ground was treated with the enzyme cocktail at concentrations of 10 nkat/mg, 50 nkat/mg, 100 nkat/mg, and 200 nkat/mg spent ground.

Figure 9: Saturation curve showing the soluble solid yield increases measured : in spent ground (Total sugar method) after treatment with

B-mannanase (Man1) (¢), and total soluble solid yield increases after * and additional thermal extraction process (Hl). The spent ground was treated at concentrations of 10 nkat/mg, and 50 nkat/mg spent ground.

Figure 10: GC profiles of coffee samples. C-3 is the control sample, A-2 is the autoclaved sample, and E-1 is the enzyme treated sample.

Figure 11 Amino acid sequence of B-mannanase of Aspergillus aculeatus

MRC11624. The neutral Ser—Thr substitution in the p-mannanase of

A. aculeatus MRC11624 versus the published sequence (GenBank accession number L35487) of the B-mannanase of A. aculeatus

KSM510 is encircled. : s

Figure 12 diagrams of a man1 B-mannanase yeast expression cassette in (A) plasmid pMES1 and (B) plasmid pGT-man1.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a new method for producing yeast and fungal strains adapted to produce one or more enzymes containing hemicellulolytic, cellulolytic and/or pectolytic activities, and also provides for the use of hemicellulolytic-, cellulolytic- and/or pectolytic-containing enzymes in coffee production.

The invention also relates to a method for producing soluble coffee extracts, consisting of dissolved solids, from roasted coffee beans, or leftovers thereof. The purpose may be the production of an instant coffee product in an industrial process, or improving the extraction of soluble solids during percolation from roasted, ground coffee beans, sold as a final retail product. The method is based on the use of enzymes having hemicellulolytic, cellulolytic and/or pectolytic activity that may be used to improve either the yield and/or the quality of soluble solids and/or volatile compounds obtained from the roasted ground beans, or leftovers thereof. 12

N

The coffee extracts are typically obtained from Arabica and/or Robusta coffee beans.

The yeast strain or fungus strain (typically an Aspergillus fungus strain) is s transformed to have the capability of producing endo-1,4-B-mannanase (B- mannanases) (Man1), endo-1,4-B-endoglucanase (B-endoglucanases) (Eg1), endo- 1,4-p-endoglucanase (p-endoglucanases) (Eg2), endo-1,4-B-endoglucanase (B- endoglucanases) (Eg3), endo-1,4--xylanase (B-xylanases) (Xyn2), and/or exo-1,4-B- glucan cellobiohydrolase (B-cellobichydrolases) (Cbh1-4).

More particularly, the yeast strain has the capability of producing a B-mannanase (Man).

The fungus strain should have the capability of producing a B-mannanase (Man1),

P-endoglucanase (Eg1, Eg2 or Eg@3), B-xylanase (Xyn2) or pB-cellobiohydrolase (Cbh1-4).

DNA expression cassettes for use in transforming the yeast or fungus strains are also part of the invention.

Apart from transforming yeast or fungus strains to produce the enzymes, the enzymes may also be obtained by cultivating natural fungal strains. Any natural fungal strains, capable of producing B-mannanase, -endoglucanase, B-xylanase, p-cellobiohydrolase, polygalacturonase, pectin lyase, and/or pectin esterase, can be utilised for the production of the hydrolytic enzyme compositions. Such natural fungal strains may be isolated from nature, decaying plant material, green coffee beans, roasted coffee beans or the leftovers of roasted, ground coffee beans after water extraction.

The enzymes may also be obtained commercially.

Other enzymes containing hemicellulolytic, cellulolytic and/or pectolytic activities may be added to the recombinant, natural fungal strain compositions or commercial compositions containing B-mannanase to improve the efficiency thereof. For example, p-endoglucanase, p-xylanase, B-cellobiohydrolase, polygalacturonase, pectin lyase, and/or pectin esterase may be added to the composition containing B- mannanase to improve the efficiency thereof.

Other enzymes, such as those having hemicellulolytic, cellulolytic and pectolytic activities, may be added to the compositions containing f-mannanase, where the addition of the other enzymes provides a synergistic effect to improve the efficiency thereof.

The viscosity of soluble solid coffee extracts may be reduced by the addition of enzymes containing hemicellulolytic, ceilulolytic and pectolytic activities, such as those described above.

The present invention also relates to a method for determining the fraction of monosaccharide represented in the total measurable neutral sugars for roasted. ground coffee beans and spent ground.

The term “a mannanase yeast expression cassette”, as used herein, denotes a recombinant DNA molecule according to the invention which includes the mant gene, preferably the man? gene from Aspergillus aculeatus MRC11624 (Setati et al., 2001), and the yeast ADH2 promoter and terminator DNA sequences, preferably the

ADH?2 promoter and terminator DNA sequences resident on the yeast/Escherichia : coli shuttle vector pMES1 (Setati ef al., 2001).

The term “a mannanase fungus expression cassette”, as used herein, denotes a recombinant DNA molecule according to the invention which includes the man gene, preferably the man? gene from Aspergillus aculeatus MRC11624 (Setati et al., 2001), and the fungus gpd promoter and glaA terminator DNA sequences, preferably the gpd promoter and terminator DNA sequences resident on the fungus/Escherichia coli shuttle vector pGT-man1 (Figure 12B).

B-Mannanases hydrolyse linear mannan polysaccharides and complex substituted mannan polysaccharides such as glucomannan, galactomannan and galactoglucomannan into oligosaccharides of various chain lengths (Sabini et al.,

2000; Setati et al., 2001). Hydrolysis of substituted mannans is greatly affected by the degree and pattern of substitution so that as the galactose content increases, the rate of hydrolysis (Vmax) by B-mannanase decreases and the K,, increases (McCleary, 1983). B-mannanases can therefore be used to perform hydrolysis experiments in s which the intention is only to modify the properties of polysaccharides without complete degradation. The viscosity of polysaccharides is proportional to chain length, branching, and entanglement, and reduction of viscosity can be effected through partial hydrolysis.

Considering the high galactomannan content (69%) in roasted coffee beans, g-mannanases can also be considered for enzymatic pre-treatment of ground coffee beans before extraction (percolation) to (i) improve coffee solid yields and (ii) improve extraction of volatile substances. The leftovers of coffee beans after the first "extraction of soluble solids (percolation), often referred to as “spent ground” or “spent grain,” may similarly be treated with enzyme cocktails to improve the extraction of soluble coffee solids and/or volatile substances.

Table 1: List of recombinant DNA sequences used for the production of recombinant enzymes by yeast and Aspergillus strains

Gene description Gene Donor organism Accession

I 0 a

Cellobiohydrolase 1-4 cbh1-4 Phanerochaete chrysosporium ATCC L22656 mE fm

Examples: 2s The invention will now be described by way of example with reference to the accompanying schematic Figures.

(a) Method for isolation of natural fungal strains

Fungi able to produce B-mannanases were isolated from green coffee beans (Indonesia and Vietnam Robusta beans), as well as from coffee and chicory spent grain. A substrate consisting of coffee beans or spent grain, or chicory spent-grain, was incubated in a moisture chamber at 22°C for a period of two weeks. During this time fungal growth was periodically transferred to malt extract agar (MEA). The fungal isolates growing on MEA were purified by preparing single-spore cultures, followed by successive cultivation on MEA at 22°C. The pure cultures were deposited in the fungal culture collection of the Department of Microbiology at the University of

Stellenbosch. The isolates were maintained in this culture collection until they were tested for production of B-mannanase. Other fungal strains in the culture collection mentioned above, which were originally selectively isolated from the natural environment using complex plant materials (e.g. lignocellulose, mannan or xylan) as carbon source in selective media, were also screened for efficiency of growth and 3- mannanase on roasted, ground coffee beans, of leftovers thereof.

ABO500, ABO503, PPRI 5471 and PPRI 5469 represent four natural fungal strains that produce high levels of B-mannanase, and were isolated using the methods described above. (b) Enzyme production by recombinant Saccharomyces cerevisiae

The recombinant B-mannanase producing yeast strain Saccharomyces cerevisiae

Y294(pMES1) was cultivated in minimal synthetic complete medium supplemented with 4 % glucose, 0,17 % yeast nitrogen base without ammonium sulphate, and an amino acid drop-out mix without methionine. The cultures were incubated at 30°C on a rotary shaker for 4 days, after which the cells were removed by centrifugation. The supernatant (enzyme source) was concentrated through the Minitan cross-flow ultra filtration device (Millipore Corporation, Bedford, Massachusetts, USA). The filtrate was freeze-dried and used for hydrolysis experiments.

(c) Enzyme production by recombinant Aspergillus niger

The recombinant producing fungal strains of Aspergillus niger (Rose and van Zyl, 2002) were cultivated in double strength liquid minimal medium containing 10 % (w/v) glucose supplemented with 0.4 % (w/v) casamino acids, 0.08 % (w/v) MgS0,.7H:0, 1.2 % (wiv) NaNOs, 0.3 % (w/v) KH:PO4, 0.1 % (w/v) KC additives and trace elements. The cultures were incubated at 30°C on a rotary shaker for 6-9 days, after which the cells were removed by filtering cultures through Miracloth (Calbiochem biosciences inc., La Jolla, CA, USA). The supernatant (enzyme source) was : concentrated through either the Minitan cross-flow ultra filtration device (Millipore

Corporation, Bedford, Massachusetts, USA), or the Pellicon device (Millipore

Corporation, Bedford, Massachusetts, USA). (d) Enzyme production by natural fungal strains

The natural fungal strains were cultivated in liquid minimal medium containing 5 % (w/v) spent ground supplemented with 0.5 % (w/v) glucose, 0.2 % (w/v) casamino acids, 0.04 % (w/v) MgS0,.7H0, 0.6 % (wiv) NaNOs, 0.15 % (w/v) KH,POq, 0.05 % (wiv) KCI additives and trace elements. The cultures were incubated at 30°C on a rotary shaker after which the cells were removed by filtering cultures through

Miracloth (Calbiochem biosciences inc., La Jolla, CA, USA). The supernatant (enzyme source) was concentrated through either the Minitan cross-flow ultra filtration device (Millipore Corporation, Bedford, Massachusetts, USA) or the Pellicon device (Millipore Corporation, Bedford, Massachusetts, USA). (e) Determination of monosaccharide fraction

Polysaccharides were hydrolysed by subjecting 0.25 g of sample to two-stage sulphuric acid hydrolysis (Moore and Johnson 1967). After neutralization with CaCO; to pH ~5.5, samples were amended with 20 mg of myo-inositol (internal standard), then centrifuged (1,500 x g, 15 min.), and 10 ml of supernatant was lyophilised. The lyophilised samples were resuspended in 1000 ul deionised water, then centrifuged at 12,000 x g for 5 min. Supernatants were dried under an air stream and then subjected to reduction with Na borodeuteride, and acetylation with acetic anhydride, as described by Blakeney et al. (1983).

Gas-liquid chromatography of alditol acetates was performed using a Hewilett- 5s Packard 6890 Plus GC fitted with a flame ionisation detector and a Supelco SPB-225 : capillary column (30 m x 0.25 mm, with 0.25 pm film thickness). Helium was used as carrier gas. The temperature program was as follows: 215°C for 2 min., then increased at 4°C per min. for 3.75 min., and then held at 230°C for 11.25 min. (f) ~~ Hydrolysis of locust bean gum (galactomannan) :

A 1 % (w/v) locust bean gum solution was prepared in 50 mM citrate buffer pH 5 and used as a substrate for viscosity analysis. The viscosity analyses were performed : using a Brookfield viscometer model (Brookfield Engineering Laboratories, Inc.,.

Stoughton, Mass. USA). The initial viscosity was determined at 40°C, after which the..

B-mannanase enzyme was added to the final concentration of 2 nkat/mg substrate.

The viscosity of the reaction mixture was measured at different time points and samples were collected concurrently and analysed for reducing sugars. The reaction was terminated by boiling for 5 min. The reducing sugars were determined using the modified DNS method as previously described (Stalbrand et al., 1993). (9) The flow dynamics of coffee extracts

Ground Arabica coffee (Boveldt, pure South African) was supplied by SAPEKOE (Pty) Ltd. (Tzaneen, SA). Coffee extracts were prepared by pre-wetting 400 g of coffee in 5 mM citrate buffer at pH 5 for 5 hrs. Extraction was carried out overnight at 80°C with constant stirring. The soluble extract was collected by filtration through a

Miracloth (CALBIOCHEM), and freeze-dried. The dried extract was used to prepare different extract concentrations (20 %, 40 % and 60 % w/v) for viscosity analysis.

The flow dynamics of the extracts were studied at 30°C on a HAAKE RV12

Viscometer (HAAKE Mess-Technik GmbH & Co., Germany). The effect of B- mannanase on the viscosity, and flow dynamics of the coffee extract was evaluated.

The enzyme was added to a final concentration of 2 nkat/mg extract and 100 pg/ml

2000/0377 1)

BSA was added to stabilize the enzyme. The reaction was carried out at pH 5, 50°C for 3 h followed enzyme inactivation at 100°C for 5 min. (h) Extraction process on roasted coffee beans

Thirty grams of ground Arabica coffee was pre-wet to 50°C in 100 ml of sodium citrate buffer (50 mM, pH 4.5 — 5.0) for 5 hours, followed by hydrolysis with enzyme for 18 hours with constant stirring. An extraction was performed afterwards consisting of two autoclave cycles at 121°C for 20 min per cycle at 100 kPa. The soluble extracts were freeze-dried and volatile compounds were analysed by gas chromatography. (i) Extraction process on spent ground: Freeze-dry method

E 15 Thirty grams of spent ground was pre-wet at 50°C with agitation using 100 ml sodium citrate buffer (50 mM, pH 4.5 — 5.0). Enzyme was added for hydrolysis at different enzyme activities and protein concentrations. BSA (0.1 mg/ml) was added to certain extractions to stabilise the recombinant enzymes. Spent ground was hydrolysed by incubation with agitation at temperatures of 40°C-70°C for 18 hours. Supernatant was filtered through 150 mm diameter filter papers (Whatman, International Ltd.,

Maidstone, England) to remove spent ground and 50 ml supernatant was freeze- dried for soluble solid determination. The spent ground was re-suspended to a final : volume of 100 mi with sodium citrate buffer (50 mM, pH 4.5 — 5.0) and subjected to 2 liquid autoclave cycles for 15 minutes per cycle at 100 kPa (thermal extraction).

Supernatant was filtered and soluble solids determined after freeze-dry process. 1) Extraction process on spent ground: Total sugar method

Thirty grams of spent ground was pre-wet at 50°C with agitation using 100 ml sodium citrate buffer (50 mM, pH 4.5 — 5.0). Enzyme was added for hydrolysis at different enzyme activities and protein concentrations. BSA (0.1 mg/ml) was added to certain extractions to stabilise the recombinant enzymes. Spent ground was hydrolysed by incubation with agitation at temperatures of 40°C-70°C for 18 hours. Supernatant was filtered through 150 mm diameter filter papers (Whatman, International Ltd.,

Maidstone, England) to remove spent ground. Total sugars in the supernatant were determined using the phenol-sulphuric acid method. Shortly, samples were mixed with 5% phenol and mixed. Concentrated sulphuric acid was added and the optical density measured at 490 nm. Total sugars were then determined from a standard s curve. Soluble solids were calculated from a standard curve taking total sugars and soluble solids produced from control experiments (no enzymes added) into account.

The spent ground was re-suspended to a final volume of 100 ml with deionised water and subjected to 2 liquid autoclave cycles for 15 minutes per cycle at 100 kPa (thermal extraction). Supernatant was filtered and soluble solids calculated after total sugar determination. (k) Analytical methods

Gas chromatography analysis was performed on 3.57 g of coffee samples. SPME is fibre (100 um Polydimethylsiloxane) was used for sampling. Extraction was carried ., out for 120 min at 65°C (head space), and desorption at 230°C for 5 min.

Compounds were separated on PS089 (0.25 pM film), 40 m x 0.25 mm GC column using 38 cm/s He as a carrier gas. The GC program was 40°C (0 min) to 230°C at 4°C/min. (1) Monosaccharide fraction of total measurable neutral sugars

A large portion of the arabinogalactan and almost all of the xylan present in the roasted beans are hydrolysed during the coffee extraction. The residual monosaccharides present in the spent ground are largely mannan and cellulose (Figure 1A and 1B). (m) The effect of recombinant enzymes, commercial enzymes and natural fungal enzyme cocktails on soluble solid yield

An increase in soluble solids was measured after spent ground was treated with the different enzymes. Figure 2 indicates that f-mannanase showed the highest increase in soluble solids for the different recombinant enzyme activities used to treat spent ground. The p-xylanase enzyme however performs better when the experiment was based on the amount of protein added (Figure 3 ). The enzyme cocktail obtained by cultivation of ABO500 showed a higher increase in soluble solids than ABOS503 at the lower levels of enzyme treatment based on B-mannanase activity (Figures 4), whereas the ABO503 performed better when yields are observed for protein s concentration added (Figures 5). The Pectinex Ultra SP-L, M263L and M282L commercial enzymes showed the highest increase in soluble solid yields after treatment based on p-mannanase activity (Figure 6), but the recombinant

B-mannanase enzyme performed the best at protein level (Figure 7). The TP668L and M263L also compared very well with the recombinant 3-mannanase enzyme (Figure 7). Overall, the recombinant B-mannanase induced the highest soluble solids yield when performing experiments based on enzyme activity added, and was most effective for the intended purpose. Theoretical yields per gram total protein of enzyme added to treatments however indicate that the recombinant B-xylanase enzyme will induce the highest increase in soluble solid yield. Enzyme saturation is prevalent when using concentrations larger than 50 nkat/mg (100 nkat/mg and 200 nkat/mg) for Mannanase-L (Figure 8). An additional thermal extraction process further increased soluble solid yields after enzyme treatment (Figure 9). (n) The effect of enzyme-aided extraction on volatile compounds

It has previously been shown that hydrolysis of coffee galactomannan with f- mannanase results in reduction of viscosity, and that the viscosity remained constant after a few hours of hydrolysis (Sachslehner et al., 2000). Therefore, it was expected in the experiment that hydrolysis of coffee extracts at 50°C for 5 h would generate high concentrations of oligosaccharides which together with high molecular weight arabinogalactan have the capacity to bind volatile compounds (Nunes et al., 2001;

Trugo, 1985). Due to the mildness of the treatment, retention of higher concentrations of volatile compounds would be achieved. Figure 10, shows the chromatograms obtained from coffee extract samples that were extracted by enzyme-aided treatment (E-1), and through autoclaving at 121°C (A-2). The profiles of volatile compounds were compared to those detected in ground coffee. The three samples displayed similar profiles, which, indicated that they contained the same type of volatile compounds. However, the differences in peak sizes indicate that the autoclaved sample had a significant loss of volatile compounds, relative to the control. Enzyme treatment does also seem to affect the amount of compounds retained, resulting in improved retention of some compounds and slight losses of others.

The preliminary results indicate that B-mannanase is capable of hydrolysing coffee galactomannan, leading to substantial reduction in viscosity. In addition the results seem to indicate that enzyme-aided extraction might be a valuable technique for instant coffee production since this could allow extractions at lower temperatures, ~ and therefore, better retention of volatile compounds. In addition, lower extract viscosity would allow recovery of concentrates higher than 42 % after pre- concentration, and also improve the drying process due to the lower water content of the extracts.

Construction of recombinant yeast and fungal strains : : (a) Isolation of B-mannanase (mani) gene

For the purpose of isolating the man1 gene, the fungus Aspergillus aculeatus

MRC11624 was cultivated in minimal medium containing: 0,3% locust been gum [Sigma], 0.1% bacto tryptone; 0.5% yeast extract; 0.1% NaNO3; 0.001%

FeS04.7H20; pH 5.5 in shaking flasks for 48 hours at 30°C. Total RNA was isolated essentially according to Crous et al (1995). The poly (AY'mRNA was purified from total RNA using the Oligotex™ mRNA isolation kit (Qiagen, Hilden, Germany). First strand cDNA was synthesised from 116 ng of mRNA using a first strand cDNA "25 synthesis kit (Roche Molecular Biochemicals, Ottweiler, Germany), and used as template for amplification of the B-mannanase encoding gene mani by PCR on a

Biometra Trio Thermoblock TB1 (Biometra Biomedizinische Analytik, Géttingen,

Germany). The primers used were designed based on the sequence of the man1 gene of A. aculeatus strain KSM510 (Accession. No. L35487) (Christgau et al., 1994). ) 1. MANR (28-mer, the EcoR restriction site is underlined) (5'-GATCGAATTCCACCACCACACAACCAAG-3)

2. MANL (28-mer; the Xhol restriction site is underlined) (5-CTAGCTCGAGCGCCAACAGTCTACTTCG-3').

The cDNA amplified man? gene was ligated to pBLUESCRIPT and sequenced, and the nucleotide sequence showed 99.7% identity with the sequence of the A. aculeatus KSM510 as reported by Christgau et al. in 1994 (GenBank accession number L35487). Three base-pair discrepancies were detected on the DNA level and one resulted in an amino acid sequence difference (Ser — Thr) at position 225 (Figure 16). This region is variable according to sequence alignment by Hilge et al. in 1998 and is not crucial for its structure and function. The neutral Ser—Thr substitution is thus not likely to affect the enzyme activity. - (b) Construction of recombinant yeast and fungal strains

For the purpose of constructing a yeast strain capable of producing p-mannanase, plasmid pMES1 was engineered. The PCR product was pre-digested with the restriction enzymes EcoRI and Xhol, the DNA purified through agarose gel electrophoresis and ligated as a 1180-bp EcoRIl/Xhol DNA fragment into the EcoRI and Xhol sites between the ADH2 promoter and terminator in plasmid pDLGH, thereby generating plasmid pMES1, which constitutes a B-mannanase yeast expression cassette according to the invention (Figure 12A). Plasmid pMES1 was transformed into Saccharomyces cerevisiae strain Y294 following the DMSO-Ilithium acetate method described by Hill et al. in 1991. Auto-selective Saccharomyces cerevisiae strains contain this plasmid was constructed by replacing the FUR? gene on the chromosome with a fur1::LEUZ2 disruptive allele, as described by La Grange et al. in 1996.

For the purpose of constructing a fungus strain capable of producing endo-1,4-(3- mannanase, plasmid pGT-man1 was engineered. The man/ gene was retrieved as an EcoRI-Xhol DNA fragment from plasmid pMES1 (Setati et al., 2001) and cloned into the corresponding sites of Escherichia coli plasmid pBLUESCRIPT. The man1 gene was retrieved from pBLUESCRIPT-man1 as a Notl-Xhol DNA fragment and cloned into the Notl and Sall sites of plasmid pGT (Rose and Van Zyl, 2002), generating plasmid pGT-man1 (Figure 12B). Plasmid pGT-man1 was transformed

[] < into Aspergillus niger strain D15 by the spheroplasting method of Ballance et al. of 1983. Plasmid pGT-man1 was transformed, together with plasmid p32R2 and selection for successful transformants was performed on medium containing acetamide as nitrogen source (Rose and Van Zyl, 2002). (c) Enzymatic assays

Mannanase, endoglucanase, xylanase and polygalacturonase activities were measured using the modified DNS method (Stalbrand et al., 1993). The substrates used for liquid assays were 0.25% Locust bean gum (Sigma-Aldrich Co., Missouri,

USA), 1% CMC (Sigma-Aldrich Co., Missouri, USA), 1% Birchwood xylan (Carl Roth

GmbH, Karlsruhe, Germany), and 0.05% Polygalacturonic acid (Sigma-Aldrich Co.,

Missouri, USA). Cellobiohydrolase activity was quantified using the chromophoric substrate PNPC, essentially as described for p-glucosidase activity determinations . 15 with the chromophoric substrate PNPX (La Grange et al., 1997). -

Total protein concentration of enzyme cocktails was determined using the Bio-Rad protein assay (Bio-Rad Laboratories GmbH, Miinchen, Germany). (d) Production levels of recombinant B-mannanase with S. cerevisiae

The maximum B-mannanase activity obtained from the recombinant fur?::LEU2

Saccharomyces cerevisiae Y294(pMES1) strain (521 nkat/ml at 50°C) and recombinant Aspergillus niger D15(pGT-man1) strain (6 000 nkat/ml at 50°C) compares very well with that of Aspergillus aculeatus MRC11624 (about 500 nkat/ml) and other recombinant strains reported in literature (Table 2), if one takes into account that the Aspergillus aculeatus MRC11624 culture supernatant contains the auxiliary enzymes involved in mannan degradation.

Table 2: p-Mannanase activity levels measured from expression in different hosts.

S. lividans 1AF10-164 1450 Arcand et al., 1993

S. lividans 1326 1917 Marga et al., 1996 v oC 1

E. coli IM109 81.7 Mendoza et al. 1995

E. coli RR28 1.33 Gibbs et al., 1992

S. cerevisiae DBY746 0.22 Stalbrand et al., 1995

S. cerevisiae Y294(pMES1) 521 This patent application

S. cerevisiae Y294 6000 This patent application (e) Production levels of recombinant 3-mannanase with A. niger

An average activity of 1700 nkat/ml was reached after 6 days of cultivation. (f) Production levels of B-mannanase in cocktails from natural fungal strains

Natural strains ABO 500 and ABO 503 showed a maximum B-mannanase activity of 120 nkat/ml and 2000 nkat/ml after 9 days of cultivation, respectively.

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Claims (22)

CLAIMS: | ll lI I oa }

1. A method of producing a coffee extract, the method including the step of subjecting leftovers of roasted, ground coffee beans, from which a substantial fraction of the water-soluble matter has been extracted, to an enzymatic hydrolysis extraction using a mannanase enzyme.

2. A method according to claim 1, wherein the extraction yield and/or quality of coffee extracts, soluble solids and/or volatile compounds obtained from leftovers of the roasted ground coffee beans is improved by the enzymatic hydrolysis.

3. A method according to either of claims 1 or 2, wherein the hydrolytic enzyme extraction step is performed at a temperature of from about 30 to about 100°C.

4, A method according to claim 3, wherein the extraction is performed at a temperature of from about 60 to about 80°C.

5. A method according to any one of claims 1 to 4, wherein the enzymatic hydrolysis step is performed for a period of up to about 24 hours.

6. A method according to any one of claims 1 to 5, wherein up to about 800 kg of one or more enzymes per ton dry spent ground or dry coffee beans is used to perform the enzymatic hydrolysis extraction step.

7. A method according to any one of claims 1 to 6, which includes the step of drying the coffee extract.

8. A method according to any one of claims 1 to 7, wherein the coffee beans are Arabica and/or Robusta beans.

9. A method according to any one of claims 1 to 8, wherein the leftovers of the roasted, ground coffee beans contain 20-36% w/v carbohydrates, predominantly in the form of B-1,4-mannan and B-1,3-galactan. 29 Amended sheet 02/03/2007

10. A method according to any one of claims 1 to 9, wherein 3-1,4-mannan and 3- 1,3-galactan are hydrolysed by the enzyme during the enzymatic hydrolysis extraction step.

1. A method according to any one of claims 1 to 10, wherein one or more of galactomannan, cellulose, xylan and pectin present in the leftovers of the roasted, ground coffee beans is hydrolysed by the enzyme(s) during the enzymatic hydrolysis extraction step.

12. A method according to any one of claims 1 to 11, wherein the enzyme or enzymes are recombinant or native.

13. A method according to any one of claims 1 to 12, wherein the enzyme or enzymes are selected from one or more of mannanase, endoglucanase, xylanase, cellulase, pectinase and/or cellobiohydrolase.

14. A method according to claim 13, wherein the recombinant mannanase is produced from Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Hansenula polymorpha, Aspergillus awamori or Aspergillus niger.

15. A method according to either of claims 13 or 14, wherein the recombinant endoglucanase is produced from Aspergillus niger D15(pGT-eg1), Aspergillus niger D15(pGT-eg2), and Aspergillus niger D15(pGT-eg3).

16. A method according to any one of claims 13 to 15, wherein the recombinant xylanase is produced from Aspergillus niger D15(pGT-xyn2).

17. A method according to any one of claims 13 to 16, wherein the recombinant cellobiohydrolase is produced from Aspergillus niger D15(pGT-cbh1-4).

18. A method according to any one of claims 1 to 17, wherein additional enzymes are used for the hydrolysis extraction, the additional enzymes being selected by a process which comprises the steps of: (i) determining the monosaccharide composition of leftovers of the roasted, ground coffee beans; Amended sheet 02/03/2007

(ii) deriving the polysaccharide composition of the leftovers from the monosaccharide composition obtained in step (i); and (iii) selecting the additional enzymes based on the polysaccharide composition obtained in step (ii).

19. A method according to any one of claims 1 to 18, wherein prior to the enzymatic hydrolysis step, the leftovers of the roasted, ground coffee beans are obtained by: (i) subjecting coffee beans to dry or wet roasting; and (ii) grinding and water extraction of the roasted coffee beans.

20. A coffee extract or coffee product produced by the method of any one of claims 1 to 19.

21. A method according to claim 1, substantially as herein described with reference to any one of the illustrative examples.

22. A coffee extract or coffee product according to claim 20, substantially as herein described with reference to any one of the illustrative examples. Dated this 11" day of May 2006 SPOOR & FISHER APPLICANT'S PATENT ATTORNEYS Amendment dated this 2" day of March 2007 SPOOR & FISHER APPLICANT'S PATENT ATTORNEYS 31 Amended sheet 02/03/2007