Enzyme-composition for hydrolyzing biomass

Abstract

The present invention is directed to an enzyme-composition for hydrolyzing biomass containing comprising at least one cellulase, at least one hemicellulases and/or at least one pectinases. In a further aspect, the present invention is directed to a process for hydrolyzing biomass implementing this enzyme-composition and the use of the enzyme-composition for hydrolyzing biomass.

Claims

1. An enzyme composition comprising: at least one cellulase, at least one hemicellulase and at least one pectinase, wherein said at least one cellulase comprises at least one enzyme selected from the group consisting of cellobiohydrolases and endo-1,4--glucanase; wherein said at least one hemicellulase comprises an Arabinan endo-1,5-alpha-L-arabinosidase (EC 3.2.1.99) and the arabinan degrading activity of the Arabinan endo-1,5-alpha-L-arabinosidase (EC 3.2.1.99) is at least 8 times as much as the cellulose degrading activity of the at least one cellulase; wherein said at least one pectinase comprises at least one enzyme selected from the group consisting of pectinesterases, polygalacturonases, pectinlyases, mannosidases and rhamnogalacturonases; wherein the arabinan degrading activity of the Arabinan endo-1,5-alpha-L-arabinosidase (EC 3.2.1.99) is at least 10 times as much as the pectin degrading activity of the at least one pectinase; wherein said at least one cellulase, said one least one hemicellulase, and said at least one pectinase are in a non-naturally occurring ratio; and wherein said enzyme-composition provides improved arabinose yield and liquefaction.

2. The enzyme composition enzyme composition of claim 1, wherein said enzyme-composition further comprises at least one hemicellulase selected from the group consisting of xylanases, xylosidases, esterases, arabinofuranosidases, galactanases, peroxidases and oxidases.

3. The enzyme-composition of claim 1, wherein the arabinan degrading activity of the Arabinan endo-1,5-alpha-L-arabinosidase (EC 3.2.1.99) is at least 10 times as much as the pectin degrading activity of the at least one pectinase.

4. The enzyme composition of claim 2, wherein the arabinan degrading activity of the Arabinan endo-1,5-alpha-L-arabinosidase (EC 3.2.1.99) is at least 0.25 times as much as the hemicellulose degrading activity of the at least one xylanase, xylosidase, esterase, arabinofuranosidase, galactanase, peroxidase and/or oxidase.

5. The enzyme composition of claim 1, wherein the hemicellulases, cellulases and/or pectinases are produced by expression in Trichoderma reesei.

Description

EXAMPLES AND FIGURES

(1) The present invention is now described by the following examples and figures. All examples and figures are for illustrative purposes only and are not to be understood as limiting the invention.

(2) FIG. 1 shows the improved liquefaction of sugar beet roots within the first 5 hours for the enzyme-composition according to the present invention (EC) compared to the reference enzyme-composition at 50 C.

(3) FIG. 2 shows the improved arabinose release from sugar beet roots within the first 5 hours for the enzyme-composition according to the present invention (EC) compared to the reference enzyme-composition at 50 C.

(4) FIG. 3 shows the improved liquefaction of sugar beet roots after 24 hours at 50 C. depending on the arabinan endo-1,5-alpha-L-arabinase added to the reference enzyme composition.

(5) FIG. 4 shows the improved arabinose release from sugar beet roots after 24 hours at 50 C. depending on the arabinan endo-1,5-alpha-L-arabinase added to the reference enzyme composition.

EXAMPLE 1: ENZYMATIC LIQUEFACTION OF WHOLE SUGAR BEET AT 50 C.

(6) Whole sugar beet material was prepared from fresh sugar beet roots sampled in Sulzemoos, Germany. Beet roots were washed to remove remaining soil and cut into approx. 10 mm10 mm pieces using a Waring blender. The sugar beet material had an average d.m. content of 23%.

(7) The reaction mixture (20 mL) contained 0.1% E/S of the enzyme-composition according to the present invention (EC) or of the reference enzyme-composition and a d.m. content of 15% sugar beet in 50 mM sodium acetate buffer (pH5). The reaction mixture was incubated for 30 min to 5 hours at 50 C. After liquefaction and hydrolysis the reaction mixture was centrifuged for 30 min at 3200 g and the liquid supernatant was separated and weighted. 1 ml of the supernatant was heat inactivated at 95 C. for 10 min and the amount of sugar released was analyzed by HPLC (Agilent, Germany) with an Aminex HPX 87 (BioRad Labs, Hercules, USA) ion exchange column (Eluent: 100% water, T: 85 C., Flow: 0.6 ml/min, RI detection).

(8) The liquefaction was determined according the formula:

(9) $\frac{\mathrm{net}\mathrm{weight}\mathrm{of}\mathrm{supernatant}}{20}100.$

(10) The results are shown in FIGS. 1 and 2.

EXAMPLE 2: ENZYMATIC LIQUEFACTION OF WHOLE SUGAR BEET AT DIFFERENT ACTIVITIES OF ARABINAN ENDO-1,5-ALPHA-L-ARABINANASE

(11) The reaction mixture (20 mL) contained 0.05% E/S of Reference enzyme blend and variable amounts arabinan endo-1,5-alpha-L-arabinanase. The d.m. content was set to 15% by addition of 50 mM sodium acetate buffer (pH5). The reaction mixture was incubated for 24 h at 50 C. After liquefaction and hydrolysis the reaction mixture was centrifuged for 30 min at 3200 g and the liquid supernatant was separated and weighted. 1 ml of the supernatant was heat inactivated at 95 C. for 10 min and the amount of sugar released was analyzed by HPLC (Agilent, Germany) with an Aminex HPX 87 (BioRad Labs, Hercules, USA) ion exchange column (Eluent: 100% water, T: 85 C., Flow: 0.6 ml/min, RI detection).

(12) The liquefaction was determined according the formula:

(13) $\frac{\mathrm{net}\mathrm{weight}\mathrm{of}\mathrm{supernatant}}{20}100.$

(14) The results are shown in FIGS. 3 and 4.