Process for the production of xylose by hydrolysis of hemicellulose by immobilized enzymes

The invention relates to a process for the hydrolysis of a hemicellulose substrate containing xylo-oligomers with an immobilized enzyme by adjusting the pH of an aqueous solution containing hemicellulose oligosaccharides and optionally ionic components to a value within a weakly acidic range, and contacting the solution with a hemicellulolytic enzyme or mixture of enzymes immobilized on a regenerable weak cation exchange carrier without fixing additives, and recovering xylose from the solution. The invention also concerns an enzyme preparation to be used in the process, comprising a weak cation exchange carrier and a hemicellulolytic enzyme or mixture of enzymes immobilized on said carrier.

The invention relates to a process for the hydrolysis of a hemicellulose 
substrate containing xylo-oligomers by hemicellulolytic enzymes 
immobilized by adsorbing on a solid carrier. The invention further 
concerns a combination of a solid carrier and a hemicellulolytic enzyme or 
mixture of enzymes immobilized on the carrier for realizing this process 
of hydrolysis. The process is well suited for the production of xylose 
from xylo-oligomers containing spent liquors of cellulose industries and 
from the water extracts of steam-exploded plant material. 
Hemicellulolytic enzymes, i.e. hemicellulases, include xylanase, 
.beta.-xylosidase and esterases, which actively cleave hemicellulosic 
material through hydrolysis. Among these xylanase and esterase enzymes 
cleave the xylan and acetyl side chains of birch xylan and the remaining 
xylo-oligomers are unsubstituted and can thus be hydrolysed with 
.beta.-xylosidase only. In addition, several less known side activities 
have been found in enzyme preparations which hydrolyse hemicellulose. 
Hemicellulolytic enzymes are produced by the fermentation of certain 
microorganisms, such as fungi of the Aspergillus or Trichoderma genera. 
The culture medium as such or enzyme fractions separated from it can be 
used as an enzyme preparation in the hydrolysis of hemicellulose. 
Under hydrolytic reaction conditions hemicellulolytic enzymes free in a 
solution retain their activity only for a relatively short period of time. 
They are expensive to use and difficult to recover. Enzymes immobilized on 
a solid carrier are more stable than free enzymes, and they are also more 
easily reusable, wherefore several processes have been developed for 
immobilizing hemicellulolytic enzymes. 
One major problem in the hydrolysis of a technical hemicellulose solution 
by immobilized enzyme is the high salt content of the solution. An ionic 
material shows a tendency to displace the enzyme from the surface of the 
carrier so that the column loses rapidly its enzyme activity. 
Hemicellulolytic enzymes have been immobilized, for example, on silica gel, 
alumina, or steel (Oguntimeim, G. B., Proc. Annu. Biochem, Eng. Symp. 
1978, vol. 8, p. 27-37), on porous glass (Rogalski, J. et al., Enzyme 
Microb. Technol. 7 (1985) No. 8, p. 27-37), on fibres (Tavobilov, I. et 
al.; Chemical Abstracts 104 (1986) 30924n) or on benzoquinone silochrome 
carrier (Balcere D. et al.; Chemical Abstracts 100 (1984) 99098f). The use 
of silica gel carrier has also been suggested by Shimizu (Biotechnology 
and Bioengineering, 29 (1987) p. 36-241) and Allenza et al. (Biotechnology 
and Bioengineering Symp. No. 17 (1986) John Wiley & Sons 1987). Puls J. et 
al. (Trans. Tech. Sect., Can. Pulp Pap. Assoc. 3 p. 64-72 and U.S. Pat. 
No. 4,275,159) have used porous glass beads, silica gel beads and sea sand 
as carrier in the immobilization of hemicellulase and xylanase fractions. 
In all these prior art immobilization techniques, the enzyme is fixed onto 
the carrier using an additive which activates the surface of the carrier 
and/or forms a covalent bond between the carrier and the enzyme. In the 
process described in the above-mentioned U.S. Pat. No. 4,275,159 (Puls et 
al.) an enzyme fraction containing mainly xylanase and an enzyme fraction 
containing mainly .beta.-xylosidase are immobilized separately on 
different carriers using in both cases glutaraldehyde, carbodiimide or 
TiCl.sub.4 as a fixing additive, and the resulting two enzyme preparations 
are used in the hydrolysis of xylan. 
Oguntimein et al. (Biotechnol. Bioeng. 22 (1980) p. 1127-1142) have studied 
the immobilization of .beta.-xylosidase purified from a commercial enzyme 
preparation produced by Aspergillus niger on ten different carriers. It 
was found that the immobilization was very difficult to perform and 
satisfactory results with regard to activity and stability were obtained 
only with an enzyme fixed with TiCl.sub.4 on alumina and with an enzyme 
fixed with glutaraldehyde on porous alkyl amine silica gel. 
Weckstrom, L. and Leisola, M. (Advances in Biotechnology. Edit. M. 
Moo-Young and C. W. Robinson, Proc. 6th Inter. Ferment. Symp. London 
Canada Jul. 20-25 1980, Pergamon Press 1981, p. 21-26) have studied the 
hydrolysis of xylan contained in sulphite spent liquor by an enzyme 
preparation produced by Aspergillus niger and fixed onto phenol 
formaldehyde resin (DUOLITE S 761) with glutaraldehyde. In a control 
experiment, the enzyme was adsorbed on the carrier without fixing 
additive. The experiments showed that the use of glutaraldehyde is 
necessary to maintain the adsorbed enzyme activity in the resin. With 
glutaraldehyde the half-life of the immobilized enzyme preparation was 
about 30 days. 
It is also well-known that ampholytic proteins such as enzymes can be 
adsorbed on ion exchangers. This adsorption has been utilized to isolate 
pure enzymes from various solutions. U.S. Pat. No. 4,168,250 discloses one 
way of immobilizing enzyme on an ion exchange carrier. From European 
Patent 222838 (Davies et al. 1987) it is known to immobilize gammaglobulin 
on a strong anion exchanger. 
A drawback of the prior art processes of immobilizing hemicellulolytic 
enzymes is that the enzyme activity level does not always remain 
satisfactory for a sufficiently long period of time; on the other hand, 
due to the fixing additive used in the immobilization, the regeneration of 
the carrier is too complicated to be applied in industrial processes. 
The object of the invention is to provide a process of hydrolysing a 
hemicellulose solution by immobilized enzyme in such a manner that the 
enzyme retains its activity substantially undeteriorated for long periods 
of time. The carrier is easy to regenerate and the hemicellulose substrate 
can be a hemicellulose solution containing high levels of ionic 
components, such as spent liquor from cellulose industries and an aqueous 
extract of a steam exploded plant material. 
These objects are achieved according to the invention by using a 
hemicellulolytic enzyme or a mixture of enzymes which is immobilized on a 
weak cation exchange carrier without fixing additive. 
Thus the invention concerns a process of hydrolysing a hemicellulose 
solution containing xylo-oligomers, which process is characterized by 
adjusting the pH of a solution of hemicellulose to a value within a weakly 
acidic range, and contacting the solution with a hemicellulolytic enzyme 
immobilized on a weak cation exchange carrier without the addition of 
fixing additives. The invention concerns further an enzyme preparation 
useful in this process and comprising a weak cation exchange carrier and a 
hemicellulolytic enzyme or mixture of enzymes immobilized on the carrier 
without fixing additives. 
The invention is based on the unexpected finding that a hemicellulolytic 
enzyme, preferably .beta.-xylosidase, can be successfully immobilized on a 
weak cation exchange carrier. Use of enzyme fixing additives, such as 
glutaraldehyde, is not necessary. 
Weak cation exchange carriers suited for use in the invention include 
macroporous acrylate resin DUOLITE C 464 and agglomerated carboxymethyl 
cellulose strengthened with polystyrene. 
Hemicellulolytic enzymes to be used in the invention belong to a 
hemicellulase group usually produced by fermenting a microorganism of the 
Trichoderma genus. The most important xylanolytic enzymes of this group 
are xylanase, .beta.-xylosidase, acetyl esterase and 
.alpha.-glucuronidase. These enzymes can be isolated from enzyme solutions 
by such known fractionating techniques as ammonium sulphate precipitation 
or ultrafiltration. The culture medium used in the fermentation of the 
microorganism as such or enzyme fractions separated from the solution can 
be used in the process of the invention. Preferably, the process of the 
invention uses a mixture of enzymes produced by the fungus Trichoderma 
longibrachiatum (former name T. reesei), or its fractions, such as 
.beta.-xylosidase, if the xylan contained in the hemi-cellulose substrate 
has first been prehydrolysed into xylo-oligomers. 
Immobilization is carried out simply by mixing the carrier with a buffered 
enzyme solution. The enzyme solution is buffered approximately within the 
pH range from 3.5 to 5. The enzyme solution is contacted with the carrier 
for 1 to 4 hours to ensure adequate fixation. If the hydrolysis is to be 
carried out in a column, the immobilization can be carried out in the 
column to be used. 
The obtained enzyme preparation is very stable even at high ion 
concentrations. This is highly unexpected since the prior art technique is 
to elute enzymes from the carrier by salt solutions; from the enzyme 
preparation of the invention, however, they could not be separated in this 
way. 
After the immobilized enzyme used in the hydrolysis has lost is activity, 
the column in use is regenerated. The exhausted enzyme is washed off, 
preferably with a diluted sodium hydroxide solution. Adsorbed impurities 
as well as the exhausted enzyme are removed in the wash. Then the carrier 
is restored into its original charging state by weak acid, and finally 
washed with water. Thereafter the column is filled with fresh enzyme 
solution. 
If required, the carrier can be washed with more efficient techniques. 
Polymeric carriers are chemically stable and can be washed at high 
temperatures with strong chemicals. In most cases a single wash with 
sodium hydroxide is sufficient to restore the carrier. 
When using the immobilized enzyme preparation of the invention, the carrier 
can be regenerated several times. For example, no notable changes were 
observed in the properties of the column after 6 regenerations. For 
process economy, the regeneration is a major advantage as the carrier 
materials are expensive. In the process of the invention the regeneration 
is easy to carry out as no fixing additives are used. 
The hydrolysing process of the invention is well suited for the production 
of xylose from steam-exploded plant material and spent liquor from 
cellulose industries. If individual purified enzyme fractions are used in 
place of mixtures of enzymes, individual immobilized enzymes can be used 
in successive columns, or the substrate can be pre-hydrolysed partially 
with a free mixture of enzymes to decrease the molecular weight of 
oligosaccharides. Excellent results have been obtained, e.g., when using 
immobilized .beta.-xylosidase in the hydrolysis of steam-exploded birch 
wood extract prehydrolysed enzymatically with xylanase and esterase. 
In the following the invention will be illustrated in greater detail by 
means of examples. 
The given enzyme activities are assayed as follows (Poutanen, K. and Puls, 
J., Characteristics of Trichoderma reesei .beta.-xylosidase and its use in 
the hydrolysis of solubilized xylans, Applied Microbiology and 
Biotechnology 1988; reprinted in Publications 47, Technical Research 
Centre of Finland, Espoo 1988, Appendix 4): 
.beta.-xylosidase activity was assayed using 5 mM 
p-nitrophenyl-.beta.-D-xylopyranoside (PNPX, Sigma N-2132) in 0.05 M 
citrate phosphate buffer, pH 4.5, as substrate. 200 .mu.l of enzyme sample 
was incubated with 1.8 ml of substrate at 50.degree. C. for 10 min. The 
reaction was stopped by adding 1 ml of 1 M sodium bicarbonate and the 
liberated p-nitrophenyl was measured at 400 nm. Activity was expressed in 
katals. 
Xylanase activity was assayed using 1% beechwood xylan (prepared according 
to the process of Ebringerova et al., Holzforschung 21 (1967) p. 74-77) as 
substrate. The pulverized xylan was suspended by homogenizing and warming 
in 0.05 M citrate-phosphate buffer, pH 5.3. The enzyme sample (0.2 ml) was 
incubated with 1.8 ml of the substrate solution at 50.degree. C. for 5 
min. The reducing sugars formed were assayed by adding 3 ml of DNS-reagent 
(Sumner, J. B. and Somers, G. F., Dinitrosalisylic method for glucose. In: 
Laboratory experiments in biological chemistry, Academic Press, New York 
1949, p. 38-39), boiling for 5 min, cooling and measuring the absorbance 
at 540 nm. The reference sugar was xylose and the activity was expressed 
in katals. 
Acetyl esterase was assayed using 1 mM .alpha.-naphtylacetate in 0.05 M 
citrate buffer, pH 5.3, as substrate. The substrate was first dissolved in 
a small volume of ethanol. 0.2 ml of the enzyme sample was incubated with 
1.8 ml of the substrate solution at 50.degree. C. for 10 min, after which 
1 ml of 0.01% Fast Corinth V Salt in 1 M acetate buffer, pH 4.3, 
containing 20% Tween 20 was added. The absorbance at 535 nm was read after 
10 min. The activity was expressed in katals. 
Suitable enzymes to be immobilized in the hydrolysing process of the 
invention include the enzyme preparation Multifect K, manufacturer Cultor 
Ltd., xylanase activity 84000 nkat/ml. 
The following carrier materials were used in the examples: 
Spezyme GDC, manufacturer Cultor Ltd.; an agglomerated diethylaminoethyl 
cellulose anion exchanger, abbreviated DEAE in the examples; 
an agglomerated carboxymethyl cellulose strengthened by polystyrene, weak 
cation exchanger, abbreviated CMC in the examples; 
DUOLITE S 761, manufacturer Duolite International, Rohm & Haas; adsorption 
resin, abbreviated DOULads in the examples; 
DUOLITE ES 562, manufacturer Duolite International, Rohm & Haas; anion 
exchanger, abbreviated DUOLani in the examples; 
DUOLITE C 464, manufacturer Duolite International, Rohm & Haas; weak cation 
exchanger, abbreviated DUOLcat in the examples; 
Diatomaceous Earth Standard Super Cell, manufacturer Johns-Manville; 
granular activated coal, manufacturer Chemviron CPG. 
Production of CMC carrier 
Agglomerated carboxymethyl cellulose carrier strengthened with polystyrene 
can be produced as described in U.S. Pat. Nos. 3,823,133 and 4,168,250. 
The polymer, the purified fibrous cellulose, the filler (such as a metal 
oxide or silicate) and the lubricant (such as magnesium stearate, 
aluminium stearate, or suitable oil) are mixed, and the mixture is heated 
to a plastic state in an extruder machine. The mixture is then extruded 
and the cooled product is ground and screened to a mean particle size 
range of 100 to 1000 .mu.m, preferably 350 to 850 .mu.m. A carboxymethyl 
derivative is derived from the resulting agglomerated product strengthened 
with polystyrene by chloro acetic acid, using sodium sulphate to reduce 
the water activity in the reaction mixture. The ion exchange capacity of 
the prepared product is 0.1 to 0.2 mequiv/g. 
Carboxymethylation is illustrated by the following example: 
150 g of sodium suphate was dissolved in 335 ml of water at 40.degree. C. 
85 g 50% NaOH solution was added. 150 g of agglomerated cellulose resin 
strengthened with polystyrene and having a particle size of 350 to 850 
.mu.m was suspended into the resulting alkaline solution, and the 
suspension was heated to 70.degree. C. 75 g of a 50% chloro acetic acid 
solution in water was added slowly during 1 hour followed by 65 g of 50% 
NaOH and finally another 75 g of 50% chloro acetic acid also during 1 
hour. The suspension was then kept at 70.degree. C. for another 30 minutes 
and then cooled to 40.degree. C. The slurry was neutralized with 150 ml of 
1 M sulphuric acid to pH 6.5 and decanted with water until the supernatant 
was clear and free from fine particles. The product was washed in water at 
85.degree. C. overnight to dissolve soluble residues, decanted again and 
drained. The ion exchange capacity of the obtained product was 0.16 
mequiv/g.

EXAMPLE 1 
An enzyme solution from Trichoderma longibrachiatum culture (former name T. 
reesei) was immobilized on different carriers of which two (CMC and 
DUOLcat) were weak cation exchangers to be used according to the 
invention. The activity of the enzyme solution used was as follows: 
______________________________________ 
xylanase 13865 nkat/ml 
.beta.-xylosidase 4200 nkat/ml 
esterase 720 nkat/ml 
______________________________________ 
The immobilization was carried out using the following two compositions: 
______________________________________ 
1. 20 g carrier material 
96 ml 0.05 M sodium citrate buffer (pH 5.0) 
4 ml enzyme solution 
2. 20 g carrier material 
92 ml 0.05 M sodium citrate buffer (pH 5.0) 
8 ml enzyme solution. 
______________________________________ 
The carrier material was mixed with the enzyme solution at +4.degree. C. 
for 4 hours. Then the mixture was filtrated and the activity of bound 
enzyme was measured. 
For comparison, the enzymes were fixed in some experiments on 
glutaraldehyde (80 ml of 2.5% solution) by stirring for one hour. Residual 
aldehyde was washed off with about 2 1 of 0.05 M sodium citrate buffer. 
Enzyme activities were measured again after fixation with glutaraldehyde. 
Results from these immobilization tests are shown in the following TABLES 1 
to 3. In all the experiments, the enzyme activities were measured before 
("bound" in the tables) and after ("fixed" in the tables) fixation with 
glutaraldehyde. 
TABLE 1 
__________________________________________________________________________ 
Xylanase activity 
Carrier 
Activity Diatom. 
nkat/g 
DEAE CMC 
earth 
Act. coal 
DUOLads 
DUOLani 
DUOLcat 
__________________________________________________________________________ 
Offered 
1128 1128 
1128 
1128 1128 1128 1128 
Bound 
113 491 
397 609 1113 600 742 
Fixed 
98 452 
108 44 171 48 241 
Offered 
2256 2256 
2256 
2256 2256 2256 2256 
Bound 
226 1241 
835 1717 2195 957 1439 
Fixed 
179 1075 
182 64 246 138 570 
__________________________________________________________________________ 
TABLE 2 
__________________________________________________________________________ 
.beta.-xylosidase activity 
Carrier 
Activity Diatom. 
nkat/g 
DEAE CMC 
earth 
Act. coal 
DUOLads 
DUOLani 
DUOLcat 
__________________________________________________________________________ 
Offered 
393 393 
393 393 393 393 393 
Bound 
42 294 
392 265 261 140 383 
Fixed 
41 220 
181 1 129 56 69 
Offered 
786 786 
786 786 786 786 786 
Bound 
700 610 
776 606 478 277 753 
Fixed 
70 439 
370 0.5 
276 158 226 
__________________________________________________________________________ 
TABLE 3 
__________________________________________________________________________ 
Esterase activity 
Carrier 
Activity Diatom. 
nkat/g 
DEAE CMC 
earth 
Act. coal 
DUOLads 
DUOLani 
DUOLcat 
__________________________________________________________________________ 
Offered 
72.5 72.5 
72.5 
72.5 72.5 72.5 72.5 
Bound 
32.5 57.6 
63.1 
59.0 70.9 64.4 41.9 
Fixed 
30.0 72.3 
62.0 
0.2 55.3 34.1 32.0 
Offered 
145 145 
145 145 145 145 145 
Bound 
65.4 97.7 
114.0 
134.0 
136.9 
133.0 
87.4 
Fixed 
54.0 136.0 
109.0 
0.2 135.0 
96.5 56.4 
__________________________________________________________________________ 
As appears from TABLES 1 to 3, all enzymes are adsorbed on the weak cation 
exchangers (CMC and DUOLcat), and the immobilized products show high total 
activities. The fixing with glutaraldehyde did not improve the system. 
EXAMPLE 2 
The hydrolysing effect of the immobilized enzymes was tested in column 
experiments. The column experiments were carried out in 4 laboratory scale 
columns. The column size was 50 ml or 100 ml. The enzyme was immobilized 
on the columns by immersing the carriers in the enzyme solution for 4 
hours. The carriers were then washed with a citrate buffer solution. The 
50 ml columns were filled with the immobilized enzyme/carrier preparation 
without further treatment. The 100 ml columns were filled with an 
enzyme/carrier preparation which for comparison was treated with 
glutaraldehyde (GA) (1 hour) to fix the enzymes. 
Analysis of the enzyme solution used in the immobilization: 
______________________________________ 
.beta.-xylosidase 552 nkat/ml 
xylanase 25000 nkat/ml 
esterase 240 nkat/ml 
______________________________________ 
Immobilization: 
______________________________________ 
50 ml enzyme solution 
350 ml 0.05 M citrate buffer pH 5 
100 g carrier 
______________________________________ 
The characteristics of the resulting immobilized systems appear from the 
following TABLE 4. 
TABLE 4 
______________________________________ 
Immobilization of enzymes on CMC and 
DUOLITE C 464 carriers 
Enzyme activity, nkat/g carrier 
.beta.-xylosidase 
xylanse esterase 
______________________________________ 
CMC carrier 
Enzyme 520 14700 200 
added 
Enzyme 374 9540 150 
adsorbed 
Activity in 346 2970 126 
carrier 
Activity after 
300 3180 137 
GA fixation 
DUOLITE carrier 
Enzyme 364 10288 140 
added 
Enzyme 357 8375 84 
absorbed 
Activity in 236 3272 68 
carrier 
Activity after 
250 2413 75 
GA fixation 
______________________________________ 
For the continuous hydrolysis the following columns were prepared using the 
above-mentioned enzyme preparations: 
1. A 100 ml column was filled with 19.9 g of dry enzyme/CMC carrier 
composition. Activities after fixing with glutaraldehyde were: 
______________________________________ 
.beta.-xylosidase 
300 nkat/g 
xylanase 3180 nkat/g 
esterase 137 nkat/g 
______________________________________ 
2. A 100 ml column was filled with 26.3 g of dry enzyme/DUOLITE 
composition. Activities after fixing with glutaraldehyde were: 
______________________________________ 
.beta.-xylosidase 
250 nkat/g 
xylanase 2413 nkat/g 
esterase 75 nkat/g 
______________________________________ 
3. A 50 ml column was filled with 12.5 g of dry enzyme/CMC carrier 
composition. No fixing was carried out. Activities were 
______________________________________ 
.beta.-xylosidase 
346 nkat/g 
xylanase 2970 nkat/g 
esterase 126 nkat/g 
______________________________________ 
4. A 50 ml column was filled with 14.5 g of dry enzyme/DUOLITE composition. 
No fixing was carried out. Activities were: 
______________________________________ 
.beta.-xylosidase 
236 nkat/g 
xylanase 3272 nkat/g 
esterase 68 nkat/g 
______________________________________ 
The substrate to be hydrolysed was a steam-exploded birch wood extract 
(steam-explosion conditions 210.degree. C., 4 minutes) containing 11 g/l 
xylose and 23 g/l xylan oligomers. The total dry substance content of the 
solution was 70 g/l, and the pH was adjusted to 5. The filtrated solution 
was fed to the bottom of each column and its flow rate in the columns 1 to 
4 was 14.4 ml/h, 15.0 ml/h, 6.9 ml/h and 6.8 ml/h, respectively. The 
temperature was 45.degree. C. The xylose in the eluate was measured after 
2, 4 and 7 days. The results are shown in the following TABLE 5. 
TABLE 5 
______________________________________ 
Xylose content in the eluate 
(grams per liter of solution and % of dry substance) 
DUOLcat/ 
Time CMC/GA GA CMC DUOLcat 
days g/l % g/l % g/l % g/l % 
______________________________________ 
2 29.2 41.6 33.4 47.8 25.6 36.6 35.1 50.1 
4 26.9 38.4 31.0 44.2 24.3 34.7 32.9 47.0 
7 22.5 32.1 33.1 47.3 25.4 36.2 32.4 46.2 
______________________________________ 
The results show that the column activities were not significantly impaired 
during the 7-day experiment. The fixation with glutaraldehyde had no 
effect on the column stability. 
EXAMPLE 3 
Immobilized enzymes were tested in the hydrolysis of a hemicellulose 
solution from steam-exploded birch-wood. 
The immobilization was carried out as described in Example 1, except that 
the temperature was 22.degree. C. No fixation with glutaraldehyde was 
carried out. 
Carriers: 
1. Cellulose-based CMC carrier; particle size 350-850 .mu.m; ion-exchange 
capacity 0.15 mequiv/ml; density 0.25-0.38 g/ml. 
2. DUOLITE C 464; pH 3.8; water content 57-62%; density 0.75 g/ml. 
Enzyme solution: 
______________________________________ 
xylosidase 5400 nkat/ml 
esterase 400 nkat/ml 
xylanase 99100 nkat/ml. 
______________________________________ 
Immobilization: 
______________________________________ 
1. 50 ml enzyme solution 
25 ml 0.05 M citrate buffer pH 5 
37.5 g CMC carrier 
2. 50 g enzyme solution 
25 ml 0.05 M citrate buffer pH 5 
37.5 g DUOLITE C 464 carrier 
______________________________________ 
For each experiment the column was filled with the immobilized enzymes and 
the birch wood hydrolysate was fed through the column. The concentration 
of the hydrolysate was 9.0% by weight and pH 5. The composition contained 
xylose 10 g/l and oligomers 32 g/l. The temperature was 45.degree. C. The 
solution was fed slowly through the column so that the total hydrolysis 
time was 4 hours. The column experiment was continued 26 days in order to 
check possible in activation of the immobilized enzyme, and the xylose 
concentration in the solution was determined. The theoretical xylose yield 
is 55% on dry substance. 
The results are shown in the following TABLE 6. 
TABLE 6 
______________________________________ 
Hydrolysis of xylan to xylose in a continuous 
process utilizing immobilized enzymes 
Time CMC carrier DUOLITE carrier 
days % xylose of d. s. 
% xylose of d. s. 
______________________________________ 
1 56 53 
4 48 
5 57 
9 49 42 
14 42 
15 40 
20 40 
21 49 42 
26 44 
______________________________________ 
The conversion rate is very high, close to the theoretical value (55% with 
free enzymes). No significant inactivation was observed during the 
experiment period. 
The carriers can be regenerated by washing out the inactivated enzyme with 
alkaline solution, activating with acid, washing with water and feeding 
fresh enzyme into the column. The enzyme is immobilized on the carrier and 
the process can continue. 
EXAMPLE 4 
Hydrolysis with immobilized enzyme was carried out on two xylo-oligomer 
solutions: Solution 1 was obtained from a chromatographic separation of a 
birch wood hydrolysate, solution 2 was a steam-exploded birch wood extract 
which was subjected to pre-hydrolysis with free (not immobilized) enzymes. 
Before prehydrolysis the compositions of the solutions were as follows: 
______________________________________ 
Solution 1 
Solution 2 
______________________________________ 
Dry subst., % by weight 
7.0 12.5 
pH 5.3 3.3 
Xylose, g/1 7.4 16 
Xylo-oligomers g/l 
42 43 
Conductivity, mS/cm 
2.75 8.0 
______________________________________ 
Solution 2 was prehydrolysed with free (not immobilized) enzyme to reduce 
the size of the polymers. A similar enzyme solution as in the following 
immobilization was used; the prehydrolysis temperature was 40.degree. C. 
and pH 5.0. 0.015 ml of enzyme solution was added per g substrate. After 
pre-hydrolysis the solution was filtrated. The obtained prehydrolysed 
solution 2 contained xylose 30 g/l. 
The hemicellulase enzymes obtained by fermentation with Trichoderma were 
immobilized on a weak cation exchanger (DUOLITE C 464) by mixing for 4 
hours in a pH-5 citrate buffer at room temperature (.apprxeq.20 to 
24.degree. C.). Enzyme activities were measured: 
______________________________________ 
xylanase 47000 nkat/g 
esterase 174 nkat/g 
.beta.-xylosidase 1200 nkat/g 
______________________________________ 
The .beta.-xylosidase immobilization yield was 100%, the xylanase yield 76% 
and the esterase yield 70%. 
The hydrolysis was carried out in a column as a continuous process under 
the following conditions: 
______________________________________ 
Column volume 10 ml 
Flow rate 1 column volume per hour 
Temperature 45.degree. C. 
______________________________________ 
Results when the column had been in use 15 days: 
______________________________________ 
Solution 1 
Solution 2 
______________________________________ 
Xylose, g/l 
feed solution 7.4 30 
after hydrolysis 
36 51 
Glucose, g/l 2 3 
after hydrolysis 
Arabinose, g/l 3 4 
after hydrolysis 
Galactose, g/l 1 3 
after hydrolysis 
Oligomers, g/l 12 18 
after hydrolysis 
______________________________________ 
There was no serious deterioration or exhaustion of the column during the 
15-day experiment. In order to examine the regeneration the column was 
washed with sodium hydroxide, activated with acid, washed with water and 
loaded with fresh enzyme. 
EXAMPLE 5 
Duolite C 464 and CMC carriers were used in the regeneration experiments. 
The carrier was washed in the column with 0.5 M sodium hydroxide (4 column 
volumes) at 50.degree. C. The carrier was then restored with 0.5 M 
sulphuric acid, rinsed with water and buffered with citrate buffer to pH 
5. 
Fresh enzyme wad added to the column and immobilized (no fixing additives). 
100 column volumes of steam-exploded birch wood extract was fed into the 
column. 
The cycle was repeated 6 times. The results are shown in the following 
TABLE 7. 
TABLE 7 
______________________________________ 
Repeated immobilization (regeneration of columns) 
Enzyme activity, nkat/g carrier 
.beta.-xylosidase 
esterase 
xylanase 
______________________________________ 
CMC carrier 
Experiment 1 
offered 1936 264 319200 
bound 1312 150 163300 
Experiment 2 
offered 2420 330 415800 
bound 1092 129 139300 
Experiment 3 
offered 1632 192 163800 
bound * 44 * 
Experiment 4 
offered 2750 155 415800 
bound 1367 * 193900 
Experiment 5 
offered 2750 275 300200 
bound 1014 98 21300 
DUOLITE C 464 carrier 
Experiment 1 
offered 962 133 319200 
bound 956 93 144500 
Experiment 2 
offered 1285 178 2151000 
bound 1278 169 205000 
Experiment 3 
offered 844 101 84700 
bound 791 57 15700 
Experiment 4 
offered 1426 80 215600 
bound 1282 47 179900 
Experiment 5 
offered 1419 142 155000 
bound 1375 92 134000 
Experiment 6 
offered 1165 142 186400 
bound * 51 38500 
______________________________________ 
*not measured 
It appears from the results that the binding of .beta.-xylosidase to the 
DUOLITE carrier remained almost unchanged whereas the binding of the two 
other enzymes was slightly deteriorated. The regeneration of the CMC 
carrier was not quite as successful but the binding of all the enzymes 
deteriorated to some extent. The experiments showed that the 
immobilization could be repeated several times on the same carrier without 
significant reduction in the absorption capacity. 
EXAMPLE 6 
This experiment was carried out to study the binding of an individual 
hemicellulase enzyme, .beta.-xylosidase on a weak cation exchange carrier 
and its ability to hydrolyse xylo-oligomers. 
An enzyme solution produced by Trichoderma longibrachiatum and having the 
following activities: 
______________________________________ 
Xylanase 200000 nkat/ml 
Acetyl esterase 1000 nkat /ml 
.beta.-xylosidase 4200 nkat/ml 
______________________________________ 
was separated into two fractions (A and B) by adding ammonium sulphate 20% 
on the weight of the solution, stirring lightly and allowing to stand at 
+4.degree. C. for 24 hours. The fractions were separated by centrifugation 
for 5 minutes at 2000 rpm. The .beta.-xylosidase was enriched in the 
solution fraction A with a yield of 54%, the obtained purity being 
3.3-fold. The amounts of other xylanolytic enzymes in this fraction were 
very small (see TABLE 8). The .beta.-xylosidase was further purified by 
ultrafiltration (UF) of the fraction A with an A Amicon ultra filter PM 10 
having a separating range of MP 10,000, until the salt content of the 
solution was 4%. The removal of the salt involved a further increase in 
the degree of purity of the .beta.-xylosidase (see TABLE 8). 
TABLE 8 
______________________________________ 
Fractionation of hemicellulase with ammonium sulphate 
Specific activity nkat/mg protein 
Fractions Ultrafiltration 
Hemicellulase 
A B fraction 
______________________________________ 
Xylanase 1430 130 1680 42 
.beta.-xylosidase 
30 100 12 195 
Acetylesterase 
7.5 0.4 9 0.6 
______________________________________ 
.beta.-xylosidase was immobilized on DUOLITE C 464 using 60 ml of 
ultrafiltration fraction A (.beta.-xylosidase 4600 nkat/ml) and 20 ml 
resin which were mixed for 1 hour at 25.degree. C. and ph 3.7. After 
washing the resin contained bound .beta.-xylosidase 11000 nkat/ml. 
The .beta.-xylosidase so immobilized was used in the hydrolysis of 
xylo-oligomers from a steam-exploded birch wood extract which was 
prehydrolysed enzymatically to cleave xylan and acetyl side chains. In the 
prehydrolysis the enzyme amounts per g dry substance were: xylanase 1280 
nkat, .beta.-xylosidase 25 nkat and acetyl esterase 7 nkat; the hydrolysis 
period was 24 hours, pH 5 and temperature 45.degree. C. Composition of the 
extract used in the prehydrolysis was: 
______________________________________ 
Dry substance, % 10 
pH 4.3 
Conductivity, mS/cm 7 
Carbohydrates 
xylose, % on weight of sol. 
1.3 
glucose, % on weight of sol. 
0.1 
oligosaccharides % on weight of sol. 
4.5 
______________________________________ 
Prehydrolysed extract was fed through a carrier column containing 
immobilized .beta.-xylosidase under the following conditions: 
______________________________________ 
Column volume 10 ml 
Flow rate 2 column volumes/h 
Temperature 45.degree. C. 
pH 4.3 
______________________________________ 
Hydrolysis with immobilized enzyme was continued as a continuous process 
for 30 days, whereafter the solution was assayed for its xylose and 
oligosaccharide content. The results were as follows: 
______________________________________ 
Concentration, g/100 g 
xylose 
oligosaccharides 
______________________________________ 
Steam-exploded 
birch wood extract 
as such 1.3 4.5 
enzymatically prehydrolysed 
2.5 3.3 
prehydrolysate hydrolysed 
5.0 0.8 
further with immobilized 
.beta.-xylosidase 
______________________________________ 
Reduction in the xylose yield during the observing period (30 days) was 
only 5% as compared with the initial values. 
EXAMPLE 7 
Immobilized hemicellulase was tested in the hydrolysis from xylo-oligomers 
contained in a beech prehydrolysate from a side product obtained from 
Celuloza Swiecie, Poland. The xylo-oligomers of the beech prehydrolysate 
were purified by chromatographic separation before hydrolysis with 
immobilized hemicellulase, and its composition was as follows: 
______________________________________ 
Dry substance, % 7.3 
pH 5.4 
Color (Icumsa, pH 5) 
184000 
Conductivity, mS/cm 
4 
Carbohydrates 
xylose 0.7%* 9.5%** 
glucose 0.5%* 7.0%** 
oligosaccharides 
3.9%* 53.5** 
______________________________________ 
*on the weight of the solution 
**on dry substance 
The immobilization was carried out with the hemicellulase preparation 
mentioned in Example 6. In the immobilization, hemicellulase was used 0.5 
ml/g DUOLITE C 464 carrier, and the binding was effected by stirring for 4 
hours at 25.degree. C. at pH 4.5. Amounts bound to the DUOLITE C 464 were 
______________________________________ 
Xylanase 78960 nkat/g resin on native weight 
.beta.-xylosidase 
2050 nkat/g resin on native weight 
Acetyl esterase 
250 nkat/g resin on native weight 
______________________________________ 
The beech prehydrolysate was hydrolysed in a continuously operating column 
under the following conditions: 
______________________________________ 
Column volume 10 ml 
Flow rate 2 column volumes/h 
pH 5.5 
Temperature 40.degree. C. 
______________________________________ 
The following xylose yields were obtained during the running of the column: 
______________________________________ 
Running Xylose, w - 
Xylose, w - 
time, days % on solution 
% on d. s. 
______________________________________ 
1 2.9 40.1 
3 3.0 41.0 
6 2.65 36.3 
7 2.65 36.3 
14 2.65 36.3 
18 2.65 36.3 
______________________________________