The controlled biosynthesis of cellobiase by Aspergillus fungi

The controlled biosynthesis of cellobiase by Aspergillus fungi

Process Biochemistry, Vol. 32, No. 1, pp. 21-28, 1997 Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0032-9592/97...

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Process Biochemistry, Vol. 32, No. 1, pp. 21-28, 1997 Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0032-9592/97 $15.00 +0.00

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The controlled biosynthesis of cellobiase by Aspergillus fungi I. V. Solovyeva,* V. M. Ananjin, A. V. Boev and O. N. Okunev I astitute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, 142292, Russia (Received 26 February 1996; accepted 13 April 1996)

Abstract

Some mechanisms of cellobiase formation were studied in Aspergillus japonicus 2092 and A. heteromorphus 3010. Formation of cellobiase in both strains was found to be a non-inducible constitutive character. The delay in formation of cellobiase in batch culture with glucose was shown to be determined by catabolic repression. Aspergillus heteromorphus 3010 was more sensitive to catabolic repression than A. japonicus 2092. To remove the effect of glucose repression two modes of fed-batch processes were developed: moderate and intensive. Both modes used the double algorithm of glucose supply based on computer-aided process control. For A. heterornorphus 3010 the moderate fed-batch was optimal, while for A. japonicus 2092 intensive fed-batch was more efficient. These regimes resulted in a three-fold increase in cellobiase in comparison with batch culture and allowed the combination of growth and biosynthesis of the enzyme in one phase. Copyright © 1996 Elsevier Science Ltd

Nomenclature /k

extremely low. The most promising fungi with respect to cellobiase activity are Aspergillus ssp. 2 The low enzyme activity of cellulase producers is the main limiting factor in the organization of commercial large-scale cellulase production. There are a few approaches to the solution of this problem: 1. screening of wild strains for cellulase activity; 2. optimization of the cultivation process (composition of the media, aeration, agitation, pH, etc.); and 3. selection of highly productive mutants. A controlled process is more effective than a non-controlled one 3-5 and empirical methods are not effective for the optimization of controlled processes. 6 Studies of microbial metabolism are useful in the intensification of biotechnological processes. 7,8 This approach allows the development of the strategy of adaptive control of enzyme biosynthesis, which makes possible the maximal formation of the enzyme by the culture. The aim of the present investigation was to develop a process of controlled biosynthesis of cellobiase based on a study of the regulatory mechanisms of enzyme formation.

Cellobiase activity (U/ml) (see Materials and Methods)

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Specific cellobiase activity (U/rag) Concentration of carbon dioxide in exhaust gas (%) Concentration of glucose in the medium (g/ litre) Growth rate (h - 1) Rate of cellobiase biosynthesis (U/mg/h) Amount of glucose fed (g) Concentration of biomass (g/iitre)

i ntroduction

Bioconversion of cellulose-containing raw materials is ~n important problem of current biotechnology due to the increasing requirements for energy, food and ~hemicals. Hydrolytic decomposition of cellulose is per~ormed by an enzyme system referred to as the ,ellulase complex. One of the essential components of ~his complex is cellobiase (EC 3.2.1.21) which carries , put the final step of cellulose hydrolysis, i.e. it converts ,'ellobiose to glucose. Some of the best known cellulase i~roducers are the Trichoderma fungi ~ although the celobiase activity of this group of microorganisms is

Materials and M e t h o d s

Strains 32 strains of the Aspergillus genus from the All-Russian Culture Collection of Microorganisms and unidentified

To whom correspondence should be addressed. 21

22

/. V.Solovyeva et al.

wild strains isolated from Far East soils were used. Cultures were maintained on malt agar slants at 40C.

Cultivation The fungi were grown in 500 ml shake flasks containing 100 ml medium at 28°C and in an ANKUM-2 fermenter at a stirring rate of 500-800 rpm and aeration of 1.5-2.01itre/min. The volume of the fermentation medium was 5 litres. The medium used in the experiments consisted of (g/litre): NaNO3, 4.0; MgSOa'7H20, 2.0; KCI, 0.5; KH2PO4, 6.0; K2HPO4, 1.0; yeast extract, 2.0; Tween-80, 1% solution. Glucose at a concentration of 0.5% was used as a carbon source. The industrial medium for polysaccharase producers containing raw plant materials (in g/100ml, sugar beet pulp, 2.0; wheat straw, 0-5; malt sprouts, 1.5) and the aforementioned salts solution was also used for cultivation. The inoculum was a spore suspension (density 2 x 108-2 x 10 9 spores/ml) obtained by washing the spores off the surface of the agar slants with distilled water. Growth of the culture on the soluble substrate was measured by dry weight.

growth medium (total cellobiase). Intracellular activity was determined by subtracting the extraceilular value from the total activity. The content of glucose and cellobiase activity were measured by the glucose oxidase-peroxidase method. 9 The amount of the enzyme hydrolysing 1/zmol of cellobiose per min at pH 4.5 and 40°C was taken as 1 unit (1 U) of cellobiase activity. Activity was expressed in units per ml and per mg of dry biomass.

Fed-batch cultivation To realize the algorithm of fed-batch process control, a fermenter-computer system consisting of an ANKUM2M fermenter and a personal computer was used. Fermentation conditions were as described earlier. The pH in the medium was maintained by addition of 10% KOH or 10% HCI. The concentration of CO2 in the exhaust gas was measured using an Infralit-4 gas analyser. Results and Discussion

Screening of cellobiaseproducers ~c~n In induction experiments the washed mycelium was obtained as follows: after 18-24 h of cultivation on a synthetic medium with 0-5% glucose the fungal biomass was transferred under sterile conditions to glass filter no. 1 and washed twice with the sterile medium without the carbon source. The biomass was then introduced into flasks with the synthetic media and inducer. Gentiobiose, sophorose, cellobiose and methyl-fl-cellobioside at a concentration of 2 g/litre were used as inducers. Washed mycelium resuspended in the synthetic medium without the carbon source was employed as the control.

Screening for cellobiase production was carried out on 32 strains from the Aspergillus genus. The fungi were grown for 7 days on the salt media with glucose and on industrial media with raw plant materials. The results of screening are presented in Table 1. As a result of the screening, 11 strains with high cellobiase activity on both media were selected for further study. Of these wild strain K 3/1 with maximal cellobiase activity was identified as A. heterornorphus F3010 and used in further investigations as a cellobiase producer. Strain A. japonicus F-2092, described earlier as a cellobiase producer, ~° was also used in this work.

Type of fermentation Catabolic repression The strains were grown in shake flasks until glucose had been exhausted (18-24 h). Various concentrations of glucose (0.5, 1"0, 2.0 and 5.0 g/litre) were then fed by pulse at 2 h intervals for 12 h. The pulsed supply was intended to maintain a fixed concentration of glucose in the medium. In the control, glucose was not fed after the initial consumption. To determine whether or not glucose is a repressor of cellulase synthesis or an inhibitor of enzyme activity, cycloheximide (CHI) (100 #g/ml) was added to washed mycelium.

Enzyme activity assays Cellobiase activity was determined in the culture liquid (extracellular cellobiase) and on a homogenate obtained after disintegration of the fungal mycelium with an IBPhM press without separating it from the

The optimal mode of cultivation is very important for the intensification of enzyme biosynthesis. It is established individually for each producer. The first stage is the batch process dynamic analysis. This makes it possible to elucidate the relationship between enzyme biosynthesis and culture growth and to determine the type of fermentation process. The formation of extracellular cellobiase in both strains consists of two phases: phase I - - growth and consumption of substrate; phase II - - synthesis of the enzyme and its release into the medium (Fig. l(a) and (b)). Cellobiase activity is detected in the culture by the time the mycelium ceases to grow and glucose is exhausted. A significant decrease of CO2 concentration is associated with the stationary phase of the culture and coincides with glucose exhaustion. This makes it possible to use this parameter as a control of glucose supply in the fermenter. To provide the maximal efficiency of a

Biosynthesis of cellobiase by Aspergillus

biphasic process it is necessary to change the parameter values during fermentation.6 To determine the optimal regime of cultivation for the improvement of cellobiase biosynthesis in both strains, the following must be studied: 1. the reasons for the biphasic character of enzyme formation; 2. the influence of exogenic inducers on cellobiase biosynthesis. 1he biphasic character of cellobiase formation The biphasic character of the enzyme formation can be determined by the following mechanisms: 1. catabolic repression and 2. competition between growth and enzyme biosynthesis. Catabolic repression is the most probable mechanism involved in the regulation of celh)biase biosynthesis in both strains. As seen from Fig. 1 tae formation of the enzyme commenced when the glucose concentration decreased to some critical value ia the case of A. japonicus 2092 (Fig. 1 (a)) or was completely exhausted in A. heteromorphus 3010 (Fig. 1(b)).

Table 1. ExtraceUular cellobiase activity of Aspergillus strains -n industrial (A) and mineral media with glucose (B) ,',;train ,t. niger 21119 • t. niger 34 1. niger 22093 ,1. niger 35 I t. niger 36 ,t. niger 40 ,t. niger 22259 . t. niger 801 t. foenicis 2084 t. japonicus 2145 ~. foetidus 2083 4. awamori 437 i. carbonarius 21 Unidentified strains K 3/1 i 1116 1117 1118 1121 ~59a 510 682 K 7/2 511 570 509 609 556 508 223a 588

Cellobiase activity (U/ml) A 16"6 7.8 24.8 6"2 6"2 6"5 14.3 3"3 15-6 6"5 4.2 9'5 6"0

B 5"50 2.70 5'50 5"00 2"70 6"10 1-40 13"00 1'85 1"85 3"00 1"85

48.5 11"7 10-4 13"0 9"7 4"4 8"3 5"5 0 3"2 5"8 0 4"5 0 0 0

17"60 3-40 1"40 7'40 1-85 3"70 4"90 4"40 1"10 0 1"20 1'85 0 1"85 2-40 1-50 0 0 0

23

Proceeding from the assumption that catabolic repression is involved in the regulation of cellobiase biosynthesis in both strains, a series of experiments to elucidate the possible repressive action of glucose was carried out. Different concentrations of glucose (0.5-5 g/litre) were added to the medium in the second phase by pulse (Fig. 2(a) and (b)). The repression of cellobiase formation was observed in both strains. The increase of the intracellular cellobiase activity was followed by the period when the enzyme activity was constant or not detected in A. japonicus 2092 and A. heteromorphus 3010, respectively. The duration of this period depended on the concentration of fed glucose (Fig. 2(a) and (b)). The same dependence was obtained for the total and extracellular ceUobiase activity. The inhibitor of translation cycloheximide (CHI) was used to examine the possibility that glucose caused the repression of cellulase biosynthesis. Addition of cycloheximide to the washed mycelium resulted in the cessation of cellobiase formation in comparison with the control. This suggested that cellobiase was synthesised 'de novo' and glucose repressed the biosynthesis of the enzyme in both strains (Fig. 3(a) and (b)). Previous investigations showed different sensitivities of the strains to catabolic repression. 11 The concentration of glucose which completely repressed cellobiase biosynthesis was 1 and 2 g/litre for A. heteromorphus 3010 and A. japonicus 2092 strains, respectively. Under conditions of batch culture with glucose (Fig. l(a) and (b)) the maximal values of specific growth rate were observed in the period from 8 to 12 h and reached 0.18 and 0-25 h-1 for A. japonicus 2092 and A. heterornorphus 3010, respectively. The maximal values of specific cellobiase biosynthesis rate were reached at the period from 16 to 18 h (36 U/g~) and from 32 to 36 h (219 U/g/h) in A. japonicus 2092 and A. heteromorphus 3010, respectively. In the experiments with glucose addition in the fed-batch regime when the biosynthesis of cellobiase was derepressed, formation of the enzyme and growth of the culture were observed simultaneously in both strains (Fig. 4(a) and (b)). The biphasic character of cellobiase formation in the batch culture with glucose is therefore determined by catabolic repression. The algorithm of the process control should therefore determine the rate of glucose addition to the fermenter. Induction Synthesis of most fungal cellulases is controlled by the induction-repression mechanism. 12,~3 True cellulase inducers are considered to be low-molecular-weight products of cellulose degradation (cellobiose) and products of its transglycosylation (sophorose and gentiobiose). To study the probable induction mechanism of cellobiase formation a number of known cellulolytic enzyme inducers were used (Tables 2 and 3).

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Fig. 1. Dynamics of cellobiase activity of (a) A. japonicus 2092 and (b) A. heteromorphus 3010 in simple batch culture with glucose. --, Concentration of CO2; % concentration of glucose; a, concentration of biomass; o, total activity; a, extracellular activity. The addition of the inducers to washed mycelium after 18-24 h of growth caused no increase in intraand extracellular cellobiase activity in either strain in comparison with the control. These results indicate that cellobiase formation in the strains studied is regulated without the involvement of the induction

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Biosynthesis of cellobiase by Aspergillus

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mechanism. Hence, the synthesis of cellobiase in both rases is constitutive and it is not necessary to supply the exogenic inducer to the medium.

Development of the controlled process of cellobiase t,iosynthesis 3ellobiase synthesis in A. japonicus 2092 and A. heteromorphus 3010 is constitutive and repressed by glucose. The glucose concentration is therefore an important parameter in the development of controlled cellobiase biosynthesis. The dependence of the specific cellobiase ~ynthesis rate (Vs) on the glucose feed concentration !G) has the form shown in Scheme 1. Thus for optimal cellobiase synthesis it is important ~o maintain the glucose concentration at an optimal lixed level. This condition may be satisfied by the controlled fed-batch process with glucose feeding input control. Based on these principles a scheme of controlled fed-batch cultivation is proposed (Scheme 2). The control algorithm developed earlier TM determines the glucose input rate depending on its

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Scheme 2. Scheme of controlled fed-batch cultivation of the strains A. japonicus 2092 and A. heteromorphus 3010.

concentration in the medium, and 3which is estimated by an indirect method measuring the CO2 concentration in the exhaust gas. A significant decrease in CO2 concentration correlates with glucose exhaustion from the medium (see Fig. 1). Since it is impossible to determine exactly the glucose concentration related to the maximum rate of cellobiase synthesis, the pulse algorithm of glucose feeding in the fermenter was used. The amount of glucose in each pulse was determined by the volume of alkaline titrant providing the pH control in the culture. The ratio of glucose supply and alkaline titrant consumption was calculated empirically to be 0-14 g/ml of 10% KOH. Such an algorithm makes it possible to avoid the accumulation of glucose in the medium and to perform cellobiase biosynthesis under derepressed conditions. From the kinetics of changes in CO2 concentration observed, the fed-batch modes were divided into two types: 1. moderate fed-batch, which maintains the concentration of CO2 at a level not lower than the preset value (Fig. 5(a)); 2. intensive fed-batch at which the difference between the neighbouring peaks of the varying CO2 values should be no less than the preset value

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Table 2. Effect of cellulolytic enzyme inducers on cellobiase formation by A. japonicus 2092 Inducer 4 Gentiobiose Sophorose Cellobiose Lactose Methyl-fl-o-cellobioside Control

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(Fig. 5(b)). 1°'14 In the first case the rate of glucose feeding is constant, while in the case of intensive fedbatch it increases exponentially and this results in the accumulation of a higher residual glucose concentration in the medium. Since the fungi used showed different sensitivities to catabolic repression, it was necessary to apply fedbatch regimes with different intensities. For A.

japonicus 2092, which was less sensitive to catabolic repression, the intensive fed-batch mode was more efficient (Fig. 6(a) and (b)), while for the more sensitive A. heteromorphus 3010 strain moderate fed-batch mode was optimal (Fig. 7(a) and (b)). In both cases a good correlation of the kinetics of CO2 concentration, amount of glucose fed, biomass concentration and total, extracellular and specific cellobiase activities

Table 3. Effect of cellulolytic enzyme inducers on cellobiase formation byA. heteromorphus 3010 Inducer 4 Cellobiose Gentiobiose Sophorose Methyl-fl-o-ceUobioside Control

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Biosynthesis of cellobiase by Aspergillus

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were observed. This indicates derepression of cellobiase biosynthesis and combination of growth and enzyme biosynthesis in one phase. Application of these modes of cultivation makes it possible to increase cellobiase activity three times in comparison with batch t ultures.

.'onclusions

As a result of screening, A. japonicus 2092 and A. J~eteromorphus 3010 were selected for detailed study. Some mechanisms of cellobiase formation were studied tor both strains. Formation of cellobiase in both cases was found to be of non-inducible constitutive character. Formation of cellobiase in batch culture with glucose consists of two phases: growth (I) and enzyme biosynthesis (II). The biphasic nature of cellobiase forrnation was shown to be determined by catabolic repression. Strain A. heteromorphus 3010 was more .~ensitive to catabolic repression than A. japonicus 2092. 1"o decrease the catabolic repression effect fed-batch iermentation was used for both strains. Two modes of ~ed-batch cultivation were developed - - moderate and

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intensive. Both modes used the double algorithm of glucose supply based on the process control by the preset profile of CO2 concentration in the exhaust gas and the consumption of the pH stabilizing titrant, For A. japonicus 2092 the intensive fed-batch mode was more efficient, while for A. heteromorphus 3010 the moderate fed-batch mode was optimal. Application of these modes of fed-batch cultivation resulted in a three-fold increase in cellobiase activity in comparison with batch culture in both strains, and allowed the combination of growth of mycelium and enzyme biosynthesis in one phase. Thus, the control of substrate feeding in these processes was adapted to the requirements of the cultures. References

1. Ennary, T.-M. and Markkanen, P., Production of cellulolytic enzymes in fungi. Adv. Biochem. Eng., 1977, 5, 3-24. 2. Sternberg, D., Yijayakumar, P. and Reese, E. F., fl-Glucosidase: microbial production and effect on enzymatic hydrolysis of cellulose. Canadian Journal of Microbiology, 1977, 23, 139-147.

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I.V. Solovyeva et al.

3. Gottvaldova, M., Kucera, J. and Podrasky, V., Enhancement of cellulase production by Trichoderma viride using carbon-nitrogen double fed-batch. Biotechnology Letters, 1982, 4, 229-232. 4. Hendy, N., Wilke, C. and Blanch, H., Enhanced cellulase production using Solca Floc in fed-batch fermentation. Biotechnology Letters, 1982, 4, 785-788. 5. Watson, T. G., Nelligan, T. and Lessing, L., Cellulase production by Trichoderma reesei (Rut-C30) in fed-batch culture. Biotechnology Letters, 1984, 6, 667-672. 6. Kantere, V. M., Theoretical Foundations of Technology of Microbial Productions. Agropromizdat, Moscow, 1990, pp. 39-63. 7. Hahn-Hagerdal, B., Scoog, K. and LochmeierVogel, E., Mapping of microbial metabolism for improved performance in biotechnical processes. Third European Congress on Biotechnology, 1984, 1, 129-131. 8. Pirt, S. J., Microbial physiology in the Penicillium fermentation. Trends in Biotechnology, 1987, 250, 69-72. 9. Berezin, I. V., Rabinovich, M. L. and Sinitsin, A.

10.

11.

12. 13. 14.

P., Study of the possibilities of the kinetic spectrophotometric method of the glucose assay. Biokhirniya (in Russian), 1977, 42, 1631-1636. Castellanos, P. S. and Okunev, O. N., Screening of cellobiase producers and optimization of nutrient medium for its biosynthesis by the strains Aspergillus niger. Prikl. Biokhim. Mikrobiol. (in Russian), 1986, 22, 80-85. Ananjin, V. M., Boyev, A. V., Solovyeva, I. V. and Okunev, O. N., Adaptive control of the biosynthesis of enzymes with different sensitivity to catabolic repression. Prikl. Biokhim. Mikrobiol. (in Russian), 1991, 27, 862-865. Sternberg, D. and Mandels, G. R., Induction of ceUulolytic enzymes in Trichoderma reesei by sophorose. Journal of Bacteriology, 1979, 139, 761-769. Eriksson, K.-E., Microbial Polysaccharides and Polysaccharases. Academic Press, New York, 1979, p. 285. Ananjin, V. M., Komkov, A. S., Solovjeva, I. V., Boyev, A. V. and Okunev, O. N., Some approaches to adaptive control of fed-batch culture of the cellobiase producer Aspergillus japonicus. Acta Biotechnology, 1991, 11, 121-128.