Pilot-Scale Production and Properties of Lignin Peroxidases - ACS

Chapter 18

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 29, 2016 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0460.ch018

Pilot-Scale Production and Properties of Lignin Peroxidases K. Polvinen, P. Lehtonen, M. Leisola, and Κ. Visuri Cultor Ltd., Technology Center, SF-02460 Kantvik, Finland

3

Lignin peroxidase was produced in a1m bioreactor. The mycelia of Phanerochaete chrysosporium was immobilized on a 12 m carrier lamellae bound on steel wire mesh. The enzyme was harvested with ultrafiltration and the permeate with added nutrients was fed back to the bioreactor through a sterile filter. A total of 1.5 million units of lignin peroxidase was harvested during one month. Lignin peroxidase was purified by ion exchange chromatography. The purified lignin peroxidase was most stable at pH 4.5-5.0. As expected, hydrogen peroxide rapidly inactivated the enzyme. Veratryl alcohol had no observable stabilizing effect on lignin peroxidase. 2

Lignin together with cellulose and hemicellulose is a structural component of woody plants. It is an aromatic irregular polymer having no repetitive linkages and no definite structure. Lignin effectively protects the woody plants against microbial attack and only a few organisms including rot-fungi and some bacteria are able to degrade it. The discovery of a lignin degrading enzyme in 1983 (7,2) created excitement and enthusiasm among both the scientific and industrial community. Great expectations were laid on the enzyme's capability for lignin degradation and modification in e.g. pulp and paper industry (3). However, as usual, the story was not at all as simple as expected. The ligninase proved to be a peroxidase (4 ) and the in vitro reaction with lignin led to polymerization reactions (5). Furthermore, the enzyme was produced only in tiny amounts during secondary (growth limited) metabolism of slowly growing non-agitated cultures of some white-rot fungi, the best producer being Phanerochaete chrysosporium. Initially lignin peroxidases were produced in shake flasks in very small quantities, which made it impossible to study their industrial applications. Early lignin peroxidase research has been reviewed by Kirk and Farrell (6 ), and the mechanism of fungal lignin degradation has recently been reviewed (7 ). 0097-6156/91/0460-0225$06.00/0 © 1991 American Chemical Society

Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

226

ENZYMES IN BIOMASS CONVERSION


A program for large-scale production of lignin peroxidases was initiated at the Swiss Federal Institute of Technology in 1984. It was shown that lignin peroxida se production could be enhanced in the presence of lignin or lignin-like low molecular weight compounds (8). The enzyme was also produced in agitated cultures (9) and immobilized reactors (10). Here we report the pilot-scale produc tion and purification of lignin peroxidases as well as the stability characteristics of the enzyme as an industrial product.

Experimental

Production of Lignin Peroxidase. Medium for the inoculum was rich in yeast extract (25 g/1) and glucose (25 g/1) to promote maximal growth of the mycelia. The inoculum of Phanerochaete chrysosporium ATCC 24725 was first cultivated for 3 days at 30°C in five litres of medium divided in five shake flasks. The shake flask batches were transferred to a 100 litre bioreactor and cultivated again for 3 days at 30°C. The batches were stirred and aerated to obtain maximal growth of mycelia. The medium for enzyme production was completely synthetic and limited in carbon to prevent excessive growth of mycelia. It contained glucose 2 g/1 plus ammonia nitrogen, vitamins and trace elements according to Leisola et al.(ii). The 100 1 inoculum was transferred into 800 1 of medium in a 1000 1 bioreactor equipped with 12 m^ of nylon wool sheets supported by steel wire screen (Figure 1). The medium was continuously stirred with the impeller (100-200rpm) and satu rated with pure oxygen. The temperature was maintained at 37°C. At 24 hours after inoculation, 350 g veratryl alcohol was added to induce lignin peroxidase production. The veratryl alcohol was diluted with water to a final concentration of 5 % (by weight) and pumped through a sterile filter into the bioreactor. At 123 hours the first lignin peroxidase activity peak appeared. The medium was harvested through a coarse filter to remove possible solids and the enzyme was concentrated on a PCI ultrafilter with 50 000 Da cut-off membranes. The permeate was immediately fed back to the bioreactor through a sterile filter. The whole enzyme harvesting cycle was performed within 4 hours. Veratryl alcohol was not added for the subsequent 5 harvests. After the 6th and 8th harvest more veratryl alcohol was added in an attempt to increase the enzyme production (see Table I). A new portion of glucose was dissolved in the permeate and fed into the bioreactor through sterile filter to start a new enzyme production cycle. The second lignin peroxidase peak appeared at 189 hours. The enzyme harvesting and feeding cycles were repeated until nine batches of lignin peroxidase were collected during 499 hours of cultivation (Table I). No other nutrients than glucose were added during the process.



18. POLVINEN ET AL.

Properties of Lignin Peroxidases

Precipitation: 37g ammonium sulphate • 100 ml liquid

Dissolution: 100 g precipitate 100 g 87 % glycerol Liquid lignin peroxidase product 500 -1000 U/ml

Figure 1. Pilot-scale production of lignin peroxidases


227


228


Table L Pilot Production of Lignin Peroxidase Veratryl Ήτηβ Lignin Peroxidase Concentrate Alcohol (g) (h) (Ull) at Harvest (total U) 350 0 123 280 202 000 189 160 138 000 215 187 78000 236 213 189000 261 270 216000 50 292 432 334000 332 121 93 000 160 380 336 183 000 499 154 117 000 Total collected units 1 550 000 Added immediately after the harvest at 292 and 380 h. 3

a

Glucose (kg) 1.6 2.4 0.6 0.8 2.4 1.1 1.0 1.4 3.2

a

Purification Unfractionated Preparation. Each harvest of the enzyme was concentrated to 401 and diafiltered with 0.01 M sodium acetate, pH 6.0. Enzyme concentrate was bound to Q-Sepharose and eluted with 0.4 M sodium acetate, pH 6.0. Fractions with activity were pooled, sterile filtered and stored in the cold in 10 mM veratryl alcohol. Before further purification, the solution was dialyzed. Fractionation by linear Gradient It is known that lignin peroxidases can be conveniently separated by their charge properties (22). To find out how the enzymes in our preparation could be separated, analytical ion exchange chroma tography with linear gradient elution was used. 16100 U in 188 ml of dialyzed lignin peroxidase in 0.01 M sodium acetate, pH 6.0, was applied to an anion exchange column (Q-Sepharose, Pharmacia, 0 = 1.6 cm, h = 9.2 cm). It eluted as three main peaks with a linear sodium acetate gradient (0.01 to 0.40 M ; 1.5 ml/min, 300 min, fractions 1-45; Figure 2). 10 ml fractions were collected and their activity and absorbances at 280 nm and 405 nm were measured. Lignin peroxidase fractions eluted with 0.20 to 0.33 M acetate were pooled and precipitated with ammonium sulphate (37 g to 100 ml). The precipitate was dissolved by adding 87% glycerol (1:1 w/w). Volume of the enzyme solution was 7 ml. Fractionation by Stepwise Elution. Information obtained from the analytical separation was applied for a preparative purification. Lignin peroxidase concen trate was bound to a Q-Sepharose column (0= 5 cm, V = 1000 ml) after ultrafiltration and eluted stepwise with 0.08 M , 0.18 M and 0.28 M sodium acetate, pH 6.0. The fraction which was eluted with 0.28 M buffer (V= 3.91, 4400 U/l) was purified further. It was bound to Q-Sepharose and eluted with 0.18 M and 0.3 M sodium acetate. Enzyme in the latter fraction was precipitated and dissolved in glycerol as previously described. The volume was 15 ml.


18. POLVINEN ET AL.


229

Enzyme fractions obtained by linear gradient elution and stepwise elution were pooled and used later in this work.


Purity Criteria. Purity of lignin peroxidase was analyzed with analytical anion exchange chromatography using detection at 405 nm. About 4 U of lignin peroxi dase was applied to a Pharmacia Mono Q column (HR 5/5,0.25 χ 5.0 cm) and eluted with linear sodium acetate, pH 6.0, gradient (0.01 to 0.55 M , 1 ml/min, 14 min). Properties of Lignin Peroxidase. Activity Assays. The standard activity assay mixture of 3 ml contained about 0.1 U/ml lignin peroxidase, 0.4 mM veratryl alcohol (Fluka, purum > 97%) and 0.1 M sodium tartrate, pH 3.0. The reaction was started by adding 15 μ\ of 54 mM H 2 O 2 to make a final concentration of 0.28 mM in the reaction. The production of veratraldehyde was followed by recording the change of absorbance for 12 seconds at 310 nm in a cuvette which was thermostated to 37°C. The reaction was started 24 seconds before the recording. One unit of lignin peroxidase is defined as the amount of enzyme required to oxidize one μ,πιοί of veratryl alcohol to veratralde hyde in one minute. For pH and thermoinactivated samples, the conditions were equal to those in the standard assay, except that the concentration of tartrate buffer, pH 3.0, was 0.3 M . For hydrogen peroxide treated samples, the concentration of H 2 O 2 in the mixture was 0.3 to 1.8 mM and concentration of sodium tartrate was 0.3 M . Contact with H 2 O 2 causes a period (called the lag time) where no production ofveratralde hyde was observed in the assay of activity. Length of the lag time was estimated to be the 24 s + inactive period observed in the graph. After the lag time lignin peroxi dase was able to convert veratryl alcohol to veratraldehyde. The highest rate of veratraldehyde production occured immediately after the onset of the reaction and was defined to be the residual activity. For veratryl alcohol treated samples, the concentration of veratryl alcohol in the activity assay mixture was 1-11 mM, the concentration of tartrate was 0.01, 0.02 or 0.3 M and the enzyme activity at the beginning was 0.2 U/ml. The samples were at 30°C during the reaction. For determination of the Michaelis constant, the activity of purified lignin per oxidase was measured by using the standard activity assay method except that the concentration of veratryl alcohol was varied between 7 μΜ and 2.67 mM. Stability Studies. For the inactivation treatments the buffers were made by mixing 0.2 M Na2HPU4 and 0.1 M tartaric acid to produce the pH values between 3.0 and 7.0. To study the effect of pH and temperature, 0.25 ml of the lignin peroxidase dilu ted with water was mixed with 0.75 ml of buffer, pH 3.0 - 7.0 and incubated at temperatures of30 - 70°C. The protein concentration of the incubation mixture was 50ju,g/ml. After various incubation times (0 - 27 h) the inactivation was stopped by adding 9 ml of cold 0.33 M tartrate buffer, pH 3.0.



230


To study the effect of hydrogen peroxide, the lignin peroxidase was incubated in buffer, either pH 3.0 or 5.0, at the temperature of either 0°C or 10°C. Protein concentration was 30/ig/ml and the concentration of H 2 O 2 was 0.2 -11.6 mM. The incubation time varied between 0 and 295 min. After incubation 0.4 ml of the enzyme sample was pipetted directly into the activity assay mixture. To study the effect of veratryl alcohol, purified lignin peroxidase or unfractionated enzyme preparation was incubated with buffer, pH 3.0 or 5.0. The concen tration of veratryl alcohol in the incubation mixture was 0, 10 or 100 mM. Incubation times were 38 days at 20°C and 40 days at 4°C. The protein con centration of purified enzyme was 80/*g/ml and of unfractionated preparation 180 /ig/ml. The incubation mixtures were sterile filtered to prevent microbial growth. Mathematical Treatment of the Results. In almost all experiments inactivation followed first order kinetics with a high correlation. The half-lives of lignin peroxidase were calculated from the following equation, where k is a rate constant of inactivation: ti/ = ln2lk

(1)

2

The Arrhenius equation: k—A exp (-E /RT) a

(2)

was used in this work to express the correlation between temperature and half-life of the enzyme at different pH's. A computer program, RS/1, (13) was used in performing calculations and plotting graphs. The half-life of enzyme activity was determined by plotting the logarithm of residual activity (U/l) versus incubation time. For all the fitted lines in the inactivation experiment concerning temperature and pH the F-value (14,15) was determined by the RS/1 program. The F-value is the single most useful measure of the significance of the fit as a whole. Also the number of points in a graph is taken into consideration in its calculation. F-value increases with increasing significance of the fit. The points in Arrhenius plots, which represent half-lives, were weighted with the related F-value when fitting the lines (Figure 3). On the basis of the equations of these fitted lines in Arrhenius plots, corrected half-lives were calculated, and these corrected values were used when the half-lives of lignin peroxidase were plotted as a function of pH at diffe rent temperatures (Figure 4). Results Production. The inoculum grew vigorously in the rich yeast extract containing media and produced a thick viscous dispersion in the stirred tank bioreactor. No lignin peroxidase activity could be detected at this stage. When transferred to the 1000 1 production bioreactor, the mycelium of Rchrysosporium attached com pletely to the nylon wool sheets within a few hours after inoculation and the medium remained completely clear throughout the cultivation. The enzyme had to be harvested immediately after the maximal activity level was reached due to its



231


POLVINEN ET AL.

2.9 1000

3 χ 1/T

3.1

3.2

(T i s t e m p e r a t u r e

Figure 3. Arrhenius plots. Q pH 3.0; Ο pH 3.5; • pH 5.0; • pH 6.0.

3.3

i n degrees

Kelvin)

φ pH 4.0; • pH 4.5;



232


6.5 PH Figure 4. Half-lives of lignin peroxidase as a function of pH at different tempera tures. Calculated half-lives: ^ 30°C; Ο 40°C; Δ 50 C; • 60°C. Experimentally obtained half-lives: • 30°C; · 40°C; • 50°C; χ 60°C. e


18. POLVINEN ET AL.


233


rapid inactivation. A total of 1.5 million units of lignin peroxidase was collected (Table I). After the first purification step with Q-Sepharose, the yield was 1.12 million units. The cultivation had to be discontinued when spaces between the sheets grew full of mycelia. At this stage both glucose, veratryl alcohol and nutrient addition failed to induce more enzyme production. Purification. One main peak was observed with detection at 405 nm, when puri fied lignin peroxidase was analyzed by Mono Q chromatography. The retention time of the main peak was 11.8 min and its area was 98.9% of the total peak area. Possibly the enzyme solution contains, however, two isoenzymes which have very similar properties. The K of the purified lignin peroxidase for veratryl alcohol was 139 μΜ on the basis of Eadie-Hofstee plot. m

Effect of Temperature. The rate of inactivation obeys the Arrhenius equation well within the experimental range (Figure 3) and the plots do not intersect each other, although the slopes differ slightly in a regular manner. If the fitted lines are extrapolated, they intersect at high temperature (about 90°C), and inactivation of lignin peroxidase seems to become independent of pH. The half-lives calculated from the Arrhenius plots are close to the observed values between one and 1250 minutes. When the experimentally obtained half-life is long, the enzyme is slightly inactivated during incubation, and the determination of the half-life is very inaccurate. Effect of pH. The pH vs. stability curves of Figure 4 are plotted using the half-lives calculated from the Arrhenius plots (Figure 3). Stability is expressed as log ti/ to make possible to visualize the curves at the temperature range (30-60°C). Maxi mum stability was achieved at pH 4.5 - 5.0. The stability of the enzyme at pH 3.0 which was used for the activity assay was much lower than the stability at pH 4.5. 2

Effect of Hydrogen Peroxide. Hydrogen peroxide had two different effects on lignin peroxidase. Firstly it caused a lag time in the assay of activity. The length of the lag time before the onset of the reaction varied depending on the hydrogen peroxide concentration, pH and contact time. After the lag time the reaction star-

Table Π. Effect of Hydrogen Peroxide on Stability of Lignin Peroxidase Concentration of Half-life of Lignin Peroxidase Activity (min) HjQ (mM) pH3.0 pH5.0 0°C 1ŒC 0°C îœc 0 Years Years Years Years 0.2 153 325 10 133 286 56 29 44 5.0 92 21 193 11.6 53 141 Esthnation 7

3

3

3

3

a


234



ted rapidly with the maximal rate. The rate was dependent on the contact time and conditions before the activity assay. The lowest concentration used (0.2 mM) reduced the half-life to a few hundred minutes. Further increase of hydrogen peroxide, up to 11.6 mM, shortened the half-life relatively little (Table II). Lignin peroxidase was inactivated more rapidly in the presence of H 2 O 2 at pH 3.0 than at pH 5.0. At the latter pH, the half-lives at 0°C and 10°C were about two times longer. The half-lives at 0°C were nearly four times longer than at 10°C. Effect of Veratryl Alcohol. At pH 5.0 the purified lignin peroxidase was not inactivated under the conditions tested. At pH 3.0 the enzyme lost its activity when incubated at 20°C for 38 days, and the presence of veratryl alcohol could not stabilize it. 100 mM veratryl alcohol even inactivated the enzyme to some extent. Ionic strength did not significantly affect the activities. The effect of veratryl alcohol was the same when unfractionated enzyme was used. This time the ionic strength in the activity assay mixture affected the activities, probably because one enzyme in the unfractionated preparation is sensitive to high ionic strength (72). Discussion In this study we have shown that the production of lignin peroxidases by P.chrysosporium can be scaled-up by using the ability of the fungus to grow on solid surfaces. More than 1500 000 units where produced during one month's produc tion period in a 1 bioreactor involving a total of nine enzyme recovery cycles. The enzyme was conveniently concentrated by ultrafiltration and further purified by ion exchange chromatography. A detailed scientific study on the properties of the five major isozymic forms of the lignin peroxidases produced in our pilot reactor has recently been published (22). Our purified enzyme in this study is composed of two isozymes having isoelectric points of 3.85 and 3.80 and molecular masses of 42 000. In this study we have characterized the enzyme's stability as an industrial product. Lignin peroxidases are very stable when stored at pH 4.5 - 5.0 and low tempera tures ( < 30°C). Their pH stability optimum differs from the activity optimum which is close to 2.5 for most of the isozymes (72). However, the pH-optimum for lignin degradation is the same as the stability optimum for the enzyme, indicating that lignin peroxidase is not the rate limiting factor in lignin degradation. Furthermore some interesting reactions of lignin peroxidase like ester formation from veratryl alcohol methyl ether also proceed optimally at pH 4.5 (16). Usually the kinetics of a chemical reaction follows the Arrhenius equation so that straight lines can be drawn over a wide temperature range. In this work it has been assumed that dependencies are linear, although the extreme points differ from the lines. Aitken and Irvine (77) have recently reported on the stability characteristics of lignin peroxidases. They also have shown that the enzyme was most stable at pH 4.5, although higher pH values were not tested. The stability was dependent on protein concentration and veratryl alcohol had a stabilizing effect. The latter result was contrary to our experience.


18. POLVINEN ET AL.


235


Lignin peroxidases are rapidly inactivated in the presence of H 2 O 2 . The mechanism has been studied in detail by Wariishi and Gold (18). The enzyme is overoxidized in the presence of excess H 2 O 2 into an inactive compound III form which can be regenerated e.g. with veratryl alcohol. This was also shown in our experiments. Compound III can further be irreversibly inactivated when exposed to H 2 O 2 for longer times (18) which explains why only a small part of the activity was recovered in the presence of veratryl alcohol. Due to oxygen radicals and substrate radicals (especially phenols), lignin peroxidases are also inactivated during their reaction cycle even when excess H 2 O 2 is not present (19). literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Tien, M.; Kirk, T. K. Science 1983, 221, 661-663. Glenn, J. K.; Morgan, Μ. Α.; Mayfield, M . B.; Kuwahara, M.; Gold, M . H. Biochem.Biophys. Res.Comm. 1983, 114, 1077-1083. Milgrom, L. New Scientist 1985, 16 may, 16-17. Kuila, D.; Tien, M.; Fee, J. Α.; Ondrias, M . R. Biochemistry 1985, 24, 3394-3397. Haemmerli, S. D.; Leisola, M . S. Α.; Fiechter, A.FEMS Microbiol.Lett. 1986, 35, 33-36. Kirk, T. K.; Farrell, R. L. Ann.Rev.Microbiol. 1987, 41, 465-505. Schoemaker, Η. E.; Leisola, M . S. A. J.Biotechnol. 1990, 13, 101-109. Leisola, M . S. Α.; Meussdoerffer, F.; Waldner, R.; Fiechter, A. J.Biotechnol. 1985, 2, 379-382. Leisola, M.; Fiechter, A. FEMS Microbiol.Lett.1985, 29, 33-36. Linko, Y-Y.; Leisola, M . S. Α.; Lindholm, N.; Troller, J.; Linko, P.; Fiechter, A. J.Biotechnol 1986, 4, 283-291. Leisola, M.; Thanei-Wyss, U.; Fiechter, A. J.Biotechnol. 1985, 3, 97-107. Glumoff, T.; Harvey, P. J.; Molinari, S.; Goble, M.; Frank, G.; Palmer, J.; Smit, J. D. G.; Leisola, M . S. A. Eur.J.Biochem. 1990, 187, 515-520. RS/1 User's quide; Bolt Beranek and Newman Inc. Cambridge, MA, 1984; Books 1-3. RS/1 User's quide; Bolt Beranek and Newman Inc. Cambridge, MA, 1984; Book 2; pp 168-169. Box, G. E. P.; Hunter, W. G.; Hunter, J. S. Statistics for Experimenters; John Wiley and sons: New York, 1978; pp 460-461. Schmidt, H.; Haemmerli, S.; Schoemaker, H . E.; Leisola, M . S. A. Biochemistry 1989, 28, 1776-1783. Aitken, M . D.; Irvine, R. L. Biotech.Boeng. 1989, 34, 1251-1260. Wariishi, H.; Gold, M . H . J.Biol. Chem. 1990, 265, 2070-2077. Harvey, P. J.; Palmer, J. M . J.Biotechnol. 1990, 13, 169-180.

RECEIVED August 16, 1990


Pilot-Scale Production and Properties of Lignin Peroxidases - ACS

Recommend Documents