1. Enzymatic hydrolysis of PHWE birch and spruce
permeates to monosaccharides and acetic acid
Suvi Teräslahti1
Anne Kallioinen2
Matti Siika-aho2
Tenkanen, Maija1
1Dep. of Food and Environmental Sci.,
University of Helsinki, Finland
2VTT, Finland
HELSINGIN YLIOPISTO
HELSINGFORS UNIVERSITET
UNIVERSITY OF HELSINKI
MAATALOUS-METSÄTIETEELLINEN TIEDEKUNTA
AGRIKULTUR-FORSTVETENSKAPLIGA FAKULTETEN
FACULTY OF AGRICULTURE AND FORESTRY
Figure 1. Optimization of enzyme mixture for hydrolysis of birch permeate. Effect of
the ratio of commercial enzymes Celluclast, Viscozyme and Depol740 on acetic acid
(left) and xylose yield (right). Hydrolysis at 40°C, 5 h, pH 5.0 with total protein dosage
of 20 mg / g of dry matter.
CONCLUSIONS
MATERIALS
RESULTS
This research is conducted as part of Fubio JR2, WP1, Task 6:
Controlled hydrolysis of hemicelluloses, as subtask enzymatic
hydrolysis. The main objective of the research is to obtain fundamental
information about the production of monosaccharides via
hydrolysis process from the low molar mass hemicelluloses
obtained from Task 5. The final aim of the study is to maximise the
production of sugars for example for fermentation to hydroxy acids or
ethanol.
INTRODUCTION
Hydrolysis of birch permeate was first optimised in using three commercial
enzymes produced by different fungi. Celluclast alone resulted in good xylose
yield. Best acetic acid release was obtained with a mixture of Celluclast and
Depol 740 (Fig. 1).
Figure 2. Formation of xylose during 48 h hydrolysis with Celluclast (C) alone or
mixed with Depol740 (D) (0.65:0.35). Total protein dosage was 10 or 20 mg / g dry
matter. Addition of α-glucuronidase (GLUR) was 1000 nkat/g of dry matter.
• The yield of xylose from birch permeate in 24 h hydrolysis was
65 - 70% with Celluclast1,5L alone.
• The xylose yield could be improved by 5% by mixing Depol740L to
Celluclast whereas the addition of α-glucuronidase resulted in 10%
increase.
• The hydrolysis of spruce permeate is more challenging due to lack of
some essential activities in the commercial enzyme preparations.
Figure 3. Formation of galactose, glucose and mannose with different enzyme
combinations (1:1) of Mannanase500 (M), Gamanase1,0L (G) and Novozym188 (N).
Hydrolysis at 40°C, 24 h, pH 5.5 with total protein dosage of 20 mg / g of dry matter.
Further hydrolysis experiments were conducted using Celluclast and the
mixture of Celluclast 0.65 : Depol740 0.35 with two protein dosages. The yield
of xylose was followed for 48 hours. Effect of pure α-glucuronidase (GLUR)
from S. commune on the action of Celluclast was also tested. After 24 h the
xylose yield was 65-70% with the Celluclast alone (Fig. 2). Effect of Depol740
was visible only with the higher 20 mg/g protein dosage, when the yield was
increased about 5%. Addition of GLUR increased the yield about 10%
demonstrating the shortage of this enzyme in the commerical preparations .
Commercial enzymes used in the hydrolysis experiments.
The hydrolysis experiments of spruce permeate (GGM) with commercial
enzymes have been started. The enzymatic hydrolysis was clearly more limited
than in the case of birch. Spruce GGM has a complex structure and thus several
different enzyme activities are needed for its hydrolysis to monosaccharides.
One limiting enzyme is estease, and chemical deacetylation prior to enzymatic
hydrolysis increased especially the mannose yield to 55% (Fig. 3). However,
chemical deacetylation results in reduced solubility of GGM which may limit
the enzyme action.
Sample Ara Gal Glc Xyl Man meGlcA Sum Ac
Birch permeate, FuBio2-3, 14.12.11 0.2 0.3 0.1 5.3 0.2 0.4 6.1 11.3
Spruce permeate, FuBio2-2, 24.11.11 0.3 0.6 0.5 1.2 1.9 0.1 4.6 7.2
Name Manufacturer Produced by
Protein
(g/l)
Enzyme activities (nkat/ml)
Xylanase βX αGLUR AXE
Birch perm.
Cellulast 1.5L Novozymes T. reesei 85 12005 623 856 218
Depol 740L Biocatalysts Humicola sp. 21 11230 95 454 59
Viscozyme Novozymes Aspergillus sp. 89 4094 nd nd nd
Spruce perm.
Novozym 188 Novozymes A. niger 104
Mannanase 500 Adv. Enzymes Aspergillus spp. 8
Gamanase 1.0L Novozymes A. niger 18
Composition of birch and spruce permeates (g/l).
βX = β-xylosdiase, αGLUR = α-glucuronidase, AXE = acetyl xylan esterase
Mannan hydrolysing
activities will be analysed
later.
Tekes and Finnish Bioeconomy Cluster are thanked for the financial support.
ACNOWLEDGEMENTS
0
10
20
30
40
50
60
70
80
90
0 5 10 15 20 25 30 35 40 45 50
Xyloseyield-%oftheoretical
Time (h)
C10
C20
CD10
CD20
C10+GLUR
C20+GLUR
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
MG_24h MN_24h GN_24h de_MG_24h de_MN_24h de_GN_24h
Monosaccharideyield-%oftheoretical
Galactose
Glucose
Mannose
Untreated permeate Deacetylated permeate