Improvement of extracellular recombinant glucose oxidase production ...

Biotechnology Letters, Vol 20, No 3, March 1998, pp. 319–323

Improvement of extracellular recombinant glucose oxidase production in fed-batch culture of Saccharomyces cerevisiae: Effect of different feeding strategies Arnab Kapat, Joon-Ki Jung* and Young-Hoon Park Biopilot Plant, Korea Research Institute of Bioscience and Biotechnology, KIST, P.O.Box 115, Yusong, Taejon 305-600, Republic of Korea. Out of four different feeding strategies tested for the production of extracellular recombinant glucose oxidase from Saccharomyces cerevisiae, constant feeding of galactose on the exhaustion of initial glucose, gave the highest yield154 U/ml which was 62% above the yield achieved in batch operation (95 U/ml).

Introduction Glucose oxidase (GOD; b-D-glucose:O2 1-oxidoreductase, E.C. 1.1.3.4) catalyzes the oxidation of b-D-glucose to Dglucono-d-lactone and hydrogen peroxide using molecular oxygen as an electron acceptor. In the subsequent step, Dglucono-d-lactone is hydrolyzed nonenzymatically to Dgluconic acid and the reduced FADH2-enzyme is reoxidized by O2. The enzyme is of considerable commercial importance in food industry (Crueger and Cruger, 1990) and is also widely used for many clinical analyses and in biosensors (Richter, 1987; Pandey et al., 1992). However, some of these applications are impaired by the high price of the commercial preparations and by the presence of catalase as contaminant. These difficulties are primarily due to the fact that in the native source, Aspergillus niger, GOD has been found to be located intracellularly (Ishimori et al., 1982) and when it is to be secreted, the problem of simultaneous secretion of other hydrolytic enzymes could not be avoided (Witteveen et al., 1992; 1993). To circumvent this problem, the GOD gene from Aspergillus niger has been transformed in Saccharomyces cerevisiae using various expression vectors with different promoters (Frederick, et al., 1990; Whittington, et al., 1990). It resulted in 80% extracellular secretion of GOD (Baetselier et al., 1991). The present study aims at the improvement of extracellular GOD production in fed batch culture of recombinant Saccharomyces cerevisiae. Several nutrient feeding strategies have been explored to control the cell growth in order to * Fax: 82-42-860-4516, E-mail: [email protected] © 1998 Chapman & Hall

facilitate enzyme production. Non-feed back controlled fed batch culture was employed where the feeding rate was controlled manually according to a predetermined constant. The results of this fed batch culture technique were compared with those of batch culture and with the previously published reports. Materials and methods Microorganism An expression vector (pGAL-GO2) containing GAL10 promoter of S. cerevisiae, a-amylase signal sequence of Aspergillus oryzae, GOD structural gene of Aspergillus niger and GAL7 terminator of S. cerevisiae was constructed (unpublished data). The expression vector was transformed into S. cerevisiae KCTC 2805 using the auxotroph method (Boeke, et al., 1984). The organism was grown in a 250 ml Erlenmeyer flask containing 20 ml of the selective medium [g/l: YNB (Difco), 6.7; Casamino Acids (Difco), 5; glucose, 20]. The flask was incubated at 30°C for 24 h on a rotary shaker at 150 rpm. The culture was again transferred to 500 ml Erlenmeyer flask containing 180 ml of the same selective medium. The flask was incubated at 30°C for 24 h at 150 rpm. It was further used as the inoculum for the batch and fed-batch experiments. Cultivation in bioreactor Batch and fed-batch cultivation were performed in a 5 l bioreactor (Korea Fermentor Co., Korea) with a working volume of 2 l to 2.75 l. For batch cultures, 200 ml of inoculum was transferred to a 5 l bioreactor containing 1.8 l of enzyme production medium [g/l: yeast extract, 40; Biotechnology Letters ⋅ Vol 20 ⋅ No 3 ⋅ 1998

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A. Kapat et al. Hycase Amine (Sheffield, USA), 5; glucose, 40; galactose (Sigma, USA), 60]. The initial pH of the medium was adjusted to 5.5 by 1 M HCl. Subsequent pH control during the course of cultivation was done by using 2.5 N NaOH and 1 N HCl. For fed-batch culture experiments, different carbon sources were fed after the exhaustion of initial glucose. The batch and fed-batch cultivation was carried out for 96 h at 30°C 6 2°C. The agitation was 420 rpm and the aeration was fixed at 0.4 vvm. The foam was controlled with 30% w/v Neorin 302 (Polyol, Korea). Determination of feed rate for fed-batch cultivation The feed rate for the fed-batch cultivation was calculated on the basis of the volumetric glucose consumption rate (2.5 g/h.l) in batch cultivation. Feeding was performed using a precalibrated peristaltic pump (Spectra Chrom Microflow Pump, USA). The feed rate was fixed at 0.14 ml/min for the feed concentration of 300 g/l. The rate of carbon sources addition was approximately equal to the rate of carbon source consumption. Analytical methods The GOD acitivity in the cell free culture medium was analyzed using 2,2’-azino-di-[3-ethylbenzthiazolinesulphonate] (ABTS, Sigma, USA) (Hellmuth et al., 1995). The exocellular GOD titre was expressed in terms of U/ml. One Unit is defined as the amount of enzyme that catalyzes the release of 1 mmol of product per second. Aspergillus niger GOD (Sigma, USA) was used as standard and a correction factor was determined on the basis of the unit activity defined by Sigma (using o-dianisidine) and the unit activity obtained using above mentioned method. The enzyme activity was finally expressed in terms of Sigma standard GOD activity [1 U GOD (o-dianisidine) 5 1.66 U GOD (ABTS)]. The specific GOD activities were obtained by dividing exocellular GOD titre by the corresponding weight of dry cell mass at a particular time.

consumption rate, yield coefficient for biomass on the substrate (YX/S) and volumetric carbon consumption rate were 0.39 h–1, 0.67 h–1 and 0.18 and 2.5 g/l.h respectively. Glucose was exhausted at 8 h of the cultivation. The microorganism was then grown batchwise on both glucose and galactose. The results of the batch culture are shown in Figure 1. Since, the initial concentrations of glucose and galactose were very high, there was substantial production of ethanol in the culture medium which was considered responsible for slower growth of the organism. The accumulated ethanol was later utilized as a carbon source after the reducing sugars were totally exhausted and the growth reached its staionary phase. The highest GOD level (95 U/ ml) was obtained at 72 h after the inhibitory effect of ethanol was eliminated. Fed-batch culture for the improvement of GOD production In order to reduce the repressive effect of glucose on the induction of GAL10 promoter, the cells were first grown in a glucose-containing growth medium in a 5 l stirred tank bioreactor and galactose was then added following four

Cell mass was expressed in terms of gram dry cel mass (DCM) per milliliter of culture medium. The ethanol concentration was determined by gas chromatography. Glucose was estimated using a glucose analyzer (YSI 2700 Select, Yellow Spring Instruments, USA) and galactose by a galactose kit (Boehringer Manheim, Germany). Results and discussion Batch culture for the production of GOD in S. cerevisiae The recombinant S. cerevisiae was first grown with glucose in a 5 l batch bioreactor to estimate the minimum concentration of glucose needed to maintain its growth. The maximum specific growth rate, specific substrate

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Figure 1 Profiles of dry cell mass, glucose, galactose and ethanol concentrations (a), dissolved oxygen (b), glucose oxidase titre and specific activity of GOD (c) in batch cultivation of recombinant S. cerevisiae containing the plasmid pGAL-GO2.

Improvement of extracellular recombinant glucose oxidase production in fed-batch culture of Saccharomyces cerevisiae different feeding strategies after the initial glucose was exhausted. The result of intermittent galactose feeding is shown in Figure 2. The maximum titre of extracellular GOD (130 U/ml) was achieved at 72 h of cultivation. The specific activity of GOD followed the same trend revealing the fact that the GOD production was associated with the cell growth.The improvement of the GOD production was noted to be 37% over that obtained in the batch culture. Glucose solution (300 g/l) was fed constantly after the addition of galactose at the point of exhaustion of initial glucose (i.e. at 8 h). The result is shown in Figure 3.

increase in cell mass. The improvement was 17% over the batch cultivation. This low activity of GOD would be attributed to partial repression of the promoter in presence of residual glucose. A fed batch experiment was conducted with combined feeding of glucose and galactose. The result is shown in Figure 4. The utilizaion of glucose and galactose resulted in higher ethanol production which could not be consumed as a carbon source due to steady supply of easily assimilable reducing sugars. But despite the presence of ethanol, the extracellular GOD level was higher at 80 h of cultivation and marked 56 % improvement over the batch mode. The results of galactose feeding are shown in Figure 5.

This culture was characterized by heavy cell growth and high ethanol production. The maximum titre of extracellular GOD (111 U/ml) was obtained at 96 h (Fig 3c). The production of extracellular GOD was comparatively less. The specific GOD activity surpassed the extracellular GOD titre at 32 h and it decreased at the later phase of cultivation due to overall decrease in GOD production and

Due to the feeding of galactose as a sole carbon source, the ethanol concentration was at the lowest amongst all the feeding method employed and though the cell growth was relatively less, the expression of GOD gene was the highest which resulted 62% improvement of the production over batch cultivation. A similar phenomenon was observed in

Figure 2 Profiles of dry cell mass, glucose, galactose and ethanol concentrations (a), dissolved oxygen (b), glucose oxidase titre and specific activity of GOD (c) in fed batch cultivation of recombinant S. cerevisiae (containing the plasmid pGAL-GO2) with intermittent addition of galactose.

Figure 3 Profiles of dry cell mass , glucose, galactose and ethanol concentrations (a), dissolved oxygen (b), glucose oxidase titre and specific activity of GOD (c) in fed batch cultivation of recombinant S. cerevisiae (containing the plasmid pGAL-GO2) with constant feeding of glucose after the intermittent addition of galactose. Biotechnology Letters ⋅ Vol 20 ⋅ No 3 ⋅ 1998

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A. Kapat et al. shake flask culture of S. cerevisiae expressing IFN-g from GPD (G) hybrid promoter (Fieschko et al., 1987). It was observed that the activity of extracellular GOD in the culture medium was not entirely dependent on the cell growth. Though constant feeding of glucose resulted in the highest production of cell mass, the extracellular GOD was

relatively low. As be seen in Table 1, the constant feeding of galactose on the exhaustion point of glucose at a required rate resulted in the highest titre of the enzyme. This indicates the two stage culture, the cell growth and gene expression stages, could give improved production of GOD as reported in case of other recombinant organism systems (Cheng et al., 1997; Yang et al., 1997).

Figure 4 Profiles of dry cell mass , glucose, galactose and ethanol concentrations (a), dissolved oxygen (b), glucose oxidase titre and specific activity of GOD (c) in fed batch cultivation of recombinant S. cerevisiae (containing the plasmid pGAL-GO2) with combined constant feeding of glucose and galactose.

Figure 5 Profiles of dry cell mass , glucose, galactose and ethanol concentrations (a), dissolved oxygen (b), GOD titre and specific activity of GOD (c) in fed batch cultivation of recombinant S. cerevisiae (containing the plasmid pGAL-GO2) with constant feeding of galactose.

Table 1 A summary of cultivation results at various mode in 5 l bioreactor. Mode of operation

Feeding strategy

Batch Fed batch

Max. DCM (g/l)

Max. EtOH (g/l)

Max. titre of GOD (U/ml)

Sp. Activity of GOD (U/g DCM)

% improvement in GOD titre

30 ± 1.4

39 ± 2.2

95 ± 3.7

3.1

100

Intermittent feeding of galactose

30 ± 2.5

30 ± 2.8

130 ± 4.8

4.3

137

Constant feeding of glucose

38 ± 0.4

47 ± 3.2

111 ± 2.7

2.8

117

Constant feeding of glucose and galactose

35 ± 1.6

38 ± 0.3

149 ± 2.8

4.2

156

Constant feeding of galactose

34 ± 3.3

35 ± 2.2

154 ± 2.2

4.4

162

Max: Maximum; DCM: Dry cell mass; EtOH: Ethanol; GOD: Glucose oxidase. The results are the averages of triplicate experiments with standard error.

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Improvement of extracellular recombinant glucose oxidase production in fed-batch culture of Saccharomyces cerevisiae Transformation of S. cerevisiae with GOD gene from A. niger has been reported (Fredrick et al., 1990) cloned the GOD gene in S. cerevisiae. The cultivation of this recombinant S. cerevisiae was conducted in 100 l scale with sucrose as the carbon source (Baetselier et al., 1991). After 192 h of cultivation, 240 U/ml of total GOD was obtained of which 80% was the extracellular fraction. At 96 h of cultivation, however, only 80 U/ml of extracellular GOD was obtained. The results of the present study shows the advantage of using galactose regulated GAL10 promoter and the merit of proposed feeding strategy because of the shorter cultivation time and higher production of extracellular GOD. Since, the aim of this study was to determine the degree of improvement in extracellular enzyme production in manual fed batch culture over that of batch one and to get an insight of the pattern of carbon source utilization, a more sophisticated feed back controlled fed batch system was not used. However, the result of this study will be compared with that of feed back controlled fed batch cultivation which is being undertaken in the present laboratory. An optimum method of cultivation will be adopted after thorough cost benefit analysis in order to make the process technically and economically feasible for industrial application. References Baetselier, A.D., Vasavada, A., Dohet, P., Ha-Thi, V., Beukelaer, T, Erpicum, T., Clerck, L.D., Hanotier, J. and Resenberg, S. (1991). Bio/Technology, 9: 559–561

Boeke, J.D., Lacroute, F. and Fink, G.R. (1984). Mol. Gen. Genet. 197: 345–346 Cheng, C., Huang, Y.L., and Yang, S.T.( 1997). Biotechnol. Bioeng. 56: 23–31 Crueger, A. and Crueger, W. (1990). Glucose transforming enzymes. In: Microbial Enzymes and Biotechnology, WM Fogarty and CE Kelly, eds 2nd edn. p 177–227 , London, New York: Elsevier Fieschko, J.C., Egan, K.M., Ritch, T., Koski, R.A., Jones, M. and Bitter, G.H. (1987). Biotechnol. Bioeng. 29: 1113–1121 Frederick, K.R., Tung, J., Emerick, R.S., Masiarz, F.R., Chamberlain, S.H., Vasavada, A., Rosenberg, S., Chakraborty, S., Schopter, L.M. and Massey, V. (1990). J. Biol. Chem. 265: 3793–3802 Hellmuth, K., Pluschkell, S., Jung, J. K., Ruttkowski, E. and Rinas, U. (1995). Appl. Micobiol. Biotechnol. 43: 978–984 Ishimori, Y., Karube, I. and Suzuki, S. (1982). Enzyme Microb. Technol. 4: 85–88 Pandey, P.C., Kayastha, A.M. and Pandey, V. (1992). Appl. Biochem. Biotechnol. 33: 139–144 Richter, G. (1987). Glucose oxidase: In: Industrial Enzymology: The Application of Enzymes in Industry, T Golfrey and JR Reichelt, eds pp 428–436, New York:The Nature Press Whittington, H., Kerry-Williams, S., Bidgood, K., Dodsworth, N., Peberdy, J., Dobson, M., Hinchliffe, E. and Ballance, D.J. (1990). Curr. Genet. 18: 531–536 Witteveen, C.F.B., Veenhuis, M., Visser, J. (1992). Appl. Environ. Microbiol. 58: 1190–1194 Witteveen, C.F.B., van de Vondervoort, P. J.I., van den Broeck, H.C., van Engelenburg, F.A.C., de Graaff, L.H., Hillebrand, M.H.B.C., Schaap, P.J., Visser, J. (1993). Curr. Genet. 24: 408–416 Yang, D.S., Bae, C.S., and Lee, J. (1997). Biotechnol. Lett. 19: 655–659

Received: 18 December Revisions requested: 1998/21 January Revisions received: 20 January 1998/9 February Accepted: 10 February

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