- Email: [email protected]
Production of lipase by Candida rugosa in solid state fermentation. 1: determination of significant process variables
ProcessBiochemisrry
28 (1993)
385-389
Production of Lipase by Cundida rugosa in Solid State Fermentation. 1: Determination of Significant Process Variables P. Venkata Rae,” Kunthala Jayaraman & C. M. Lakshmanan Centre for Biotechnology Madras-600 02.5, India (Received
5 August 1992;
and Department
of Chemical Engineering,
revised version received and accepted
Alagappa
3 October
College of Technology,
Anna University,
1992)
The effect ofprocess variables in the production of Candida rugosa lipase using rice bran has been studied by solid state fermentation (SSF). The particle size and oil content of the bran and the addition of urea and maltose have a pronounced effect on the production of lipase. Immobilization qf lipase on rice bran (in situ) has been found possible.
et al.” observed two stages in the production of lipase by C. rugosa in a medium containing olive oil, in which the first stage was glycerol depletion without lipase formation and in the second stage was fatty acid consumption with the lipase production. The solid state fermentation (SSF ) technique has been used in the production of several industrial enzymes such as a-amylase,‘” cellulases and /3-glucosidase. I4 Very few reports are available on the production of lipase by SSF. Saikai et aLI5 investigated the production of lipoprotein lipase by SSF and SmF using various species of Mucor. Maximum activity of 52 U/g dry bran was observed in the case of Mucor juvanicus 1AM 6089 by SSF. In the present investigation, the production of lipase by C. rugosa using rice bran as a solid substrate was studied.
INTRODUCTION There has been a world wide interest recently in the application of commercially available lipases for the hydrolysis of oils,‘.* synthesis of esters,3,4 interesterification of oils and fats,5 production of and the resolution of racemic biosurfactant@ mixtures to produce optically active compounds.’ Candida rugosa is recognized as a potent producer of lipase.s The industrial production conditions for C. rugosa lipase by submerged fermentation (SmF ) were reported by Yamada et al.” Candida rugosa was found to produce extracellular lipase in the presence of lipids such as fats and sterols, both being present simultaneously. Cholesterol was found to be the most effective sterol in lipase induction.“’ Valero et al.” studied the fermentation behaviour of lipase production by C. rugmu in submerged fermentation. Del Rio
MATERIALS Corresponding
author: P. Venkata Rao.
*Present address: Department of Biotechnology, Leather Research Institute, Madras 600 020, India.
Microorganism and medium Candidu rugosa DSM 2031 agar slants containing 0.3%
Central 385
Process
Biochemistry
032-9592/93/$6.00
AND METHODS
0 1993 Elsevier Science Publishers Ltd, England.
was maintained on yeast extract, 0.3%
386
P. Venkata Rao, Kunthala Jayaraman, C. M. Lakshmanan
malt extract, 0.5% peptone and 1% glucose at 4°C.” Universal yeast medium of pH 7 used for prcculture had the following composition: glucose 10 g/litre, yeast extract 3 g/litre, malt extract 3 g/litre and peptone 5 g/litre. Refined edible grade rice bran oil of 0.8% FFA and saponification value of 185 was used for determining lipase activity. The preculture was grown in the universal yeast medium at 30°C on a shaker with constant agitation (200 rev/min). The preculture reached mid log phase in 16 h with a cell count of 269 X 10h/ml (1.8 o.d.) and it was centrifuged at 10 000 g for 10 rnin at 4°C in a sterile tube. The pellet resuspended in sterile distilled water was used as inoculum for solid state fermentation. Rice bran was obtained from a local rice mill and sieved to different size fractions. The sieve analysis and composition of rice bran are shown in Table 1. The oil content of rice bran was measured by extracting the oil using a soxhlet extractor. The defatted rice bran was extracted with 5% trichloroacetic acid to precipitate the proteins. The precipitate was mixed with 10 ml of ethyl ether for the removal of residual trichloroacetic acid. Then the precipitate was suspended in water and mixed well. Protein estimation was carried out using Lowry’s method.‘” For carbohydrate estimation, the defatted rice bran samples were hydrolysed with 6 N HCl for 8 h at 100°C. The total sugar content of the hydrolysate was determined by the phenolsulphuric acid method described by Dubois et al.” using glucose as standard.
added and thoroughly specified conditions. Immobilization
mixed and incubated
under
of lipase
Immobilization of lipase was carried out by precipitation of lipase on to rice bran with acetone (1 : 2 ratio) at O”C, as suggested by Macrae18 and Wisdom et al5 The bran was filtered and dried for 8 h in a vacuum drier at 30°C. The moisture content of the lipase immobilized on rice bran (lipase IRB) was 18% w/w on dry basis. Assay of lipase activity Lipase IRB activity was assayed in a reverse phase system’” in which lipase was dispersed in a continuous organic solvent phase containing substrate at high concentrations. The advantages of a reverse phase system over a conventional emulsion assay system are good reproducibility and easy control of reaction. The assay mixture contained 4.5 ml of rice bran oil in 6 ml of isooctane and 0.5 g of lipase IRB in a 100 ml shake flask. The mixture was incubated for 1 h at 37°C and 200 rev/min. After incubation 0.4 ml of reaction mixture was transferred to a test tube containing 4.6 ml of benzene. To this solution 1-O ml of (5% w/v) cupric acetate-pyridine solution was added and the mixture was vortex mixed for 2 min. The mixture was centrifuged for 5 min at 7000 g and the organic and aqueous phases were separated. The fatty acid produced was estimated by measuring the optical density of the organic phase at 7 15 nm as suggested by Kim et aI.2”
Solid state fermentation
RESULTS
Dry rice bran powder (5 g) was mixed with distilled water (1 : 1) in petriplates, and was then sterilized for 20 min at 120°C and 1 atmosphere. After cooling the sterile bran, inoculum was
AND DISCUSSION
The effect of fermentation time, temperature, particle size, inoculum concentration, oil content, addition of carbon and nitrogen on production of
Table 1. Particle size analysis, composition and activity of rice bran immobilized ml/g bran, initial moisture 2.2 g/g bran and time 48 h) Purticle size 04
> so0 SOO-200 250-177 <177 “Mostly husk and fiber.
I%,Fractions (WIWJ 26-6 ‘I 353 28.6 9.5
Composition Oil
lipase (pH 6, temperature
(‘% w/w dry basis)
l’roteirl
Carbohydrate
Il.0 13.7 14.0
16.0 19.5 20.8
3O”C, inoculum Activity Wls bran)
-
13.0 15.1 17.6
8.6 16.9 18.1
1
Production of lipase by Candida rugosa in solid statefermentation.
lipase was investigated by varying one parameter while keeping the other variables constant in each experiment. Experiments were conducted at pH 6 and 35°C with 1 ml of inoculum per g of dry bran (initial moisture content 2.2 g/g dry bran) in sterile plates for varying fermentation times. The results shown in Fig. 1 indicated that the maximum activity of 6.4 U/g bran was obtained in about 48 h. The effect of incubation temperature on lipase is shown in Fig. 2. The optimal temperature was found to be 3O”C, which is the same as reported for C. rugusa in submerged fermentation.’ ’ The inoculum (269 X 10h cells/ml) was varied from 0.2 to 1.4 ml/g. There was no significant effect on activity beyond 1 ml/g of bran (Fig. 3). Fermentation was carried out using rice bran of different particle sizes. The results (Table 1) indicated that rice bran fractions < 2.50 pm gave high activity. This may bc due to the higher content of protein, carbohydrate and oil in these fractions. To study the effect of oil content on lipase activity the bran of particle size < 250 ,um was defatted and varied amounts of neutral rice bran
387
I
oil (in 20 ml of hexane) were added and thoroughly mixed with the bran. Hexanc was then removed by vacuum drying. The results shown in Fig. 4 indicated that oil content is an important variable and the activity increased with an increase in oil content up to about 17%, which is approximately the oil content of < 250 pm rice bran fraction (Table 1). Inorganic nitrogen sources such as ammonium chloride, ammonium nitrate, ammonium sulphate and urea (0.4% w/w) and the organic nitrogen sources, yeast extract and malt extract (2% w/w), were individually added to the medium; the results obtained are shown in Table 2. Addition of urea and ammonium nitrate increased the lipase activity. It has been reported” that urea and ammonium nitrate maintain the pH of the substrate. Malt extract has been found to be a good organic nitrogen source for lipase production. However, the cost precludes the use of malt extract for industrial fermentations.
‘*2
0 0
I
I
I
OC
O-8
1.2
lnoculum 48
0
Time
Fig. 1.
72
lh
(ml/g
1.6
bran)
Fig. 3. Effect of inoculum concentration (pH 6, temperature 3o”C, initial moisture 2.2 g/g bran and time 48 h).
96
)
Time course of lipase production
by SSF.
19'
17 -
15 -
l3-
II
21 25
I 30
I 35
I LO
I
I
I
a
12
16
Oil
Temperature(‘C)
Effect of incubation temperature (pH 6, inoculum Fig. 2. ml/g bran, initial moisture 2.2 g/g bran and time 48 h).
I L
1
(*A
20
w/w)
Effect of oil content (pH 6, temperature 30°C Fig. 4. inoculum 1 ml/g bran, initial moisture 2.2 g/g bran, time 48 h and particle size < 250 pm).
388
P. Venkata
Rao,
Kunthala
Table 2. Effect of additional nitrogen source (pH 6, temperature 30°C inoculum 1 ml/g bran, initial moisture 2.2 g/g bran, particle size < 250 pm and time 48 h) Nitrogen
source
Control Inorganic Ammonium Ammonium Ammonium Urea
Lipase activity (u/g bran) 18.2
chloride nitrate sulphate
Organic Yeast extract Malt extract
18.4 23.6 21.1 24.4 18.2 23.7
Table 3. Effect of additional carbon source (pH 6, temperature 3o”C, inoculum 1 ml/g bran, initial moisture 2.2 g/g bran, time 48 h, particle size < 250 ,um and urea O-4% w/w) Carbon source
Control Glucose Maltose Sucrose Starch
Lipase activity @J/g bran) 24.3 21.0 26.6 24-8 24.3
The effect of additional carbon source was studied by adding glucose, maltose, sucrose and starch (2% w/w) to rice bran. From Table 3 it may be noted that maltose had a marginal effect on lipase activity. The above results indicate that the growth limiting factor in rice bran is the available nitrogen rather than the carbon source. The results of this investigation have shown that the production of lipase is highest at a fermentation time of 48 h, a temperature of 30°C and inoculum concentration of 1 ml/g bran. The rice bran particle size < 250 pm is a good substrate. The most significant variables affecting the lipase yield are oil, urea and maltose.
ACKNOWLEDGEMENT The financial assistance provided by CSIR to Mr P. Venkata Rao is acknowledged.
Jayaraman,
C. M. Lakshmanan
REFERENCES 1. Linfield, W. M., O’Brien, S., Serota, S. & Barauskas, R. A., Lipid-lipase interactions. 1. Fat splitting with lipase from Candida rwosa. J. Am. Oil Chem. Sot.. / 61 i_1984) 1067-71. .‘ 2. Hoq, M. M.. Yamane, T., Shimizu, S., Funada, T. & Ishida, S., Continuous hydrolysis of olive oil by lipase in microporous hydrophobic membrane bioreactor. J. Am. OilChem. Soc.,62 (1985) 1016-21. 3. Hoq, M. M., Yamane, T., Shimuzu, S., Funada, T. & Ishida. S., Continuous synthesis of glycerides by lipase in a microporous membrane reactor. J. Am. Oil Chem. Sot., 61( 1984) 776-81. 4. Patterson, J. D. E., Blain, J. A., Shaw, L. E. L., Todd, R. & Bell, G., Synthesis of glycerides and esters by fungal cell bound enzymes in continuous reactor systems. Biotechnol. L&t.. 1 ( 1979) 2 J I- 16. _5 Wisdom, R. A.; Dunnill, P. & Lilly, M. D., Enzymatic interesterification of fats: laboratory and pilot scale studies with immobilized lipase from Rhizopus arrhizus. Biorechnol. Bioengng., 29 (1987) 1081-5. T., Nishitani, T. & Inamasu. S., 6. Seino, H., Uchibori, Enzymatic synthesis of carbohydrate esters of fatty acid. ( 1) Esterification of sucrose, glucose, fructose and sorbitol. J. Am. Oil Chem. SW., 61( 1984) 176 l-5. 7. Cambou, B. & Klibanov, A. M., Comparison of different strategies for the lipase catalyzed preparative resolution of racemic acids and alcohols: asymmetric hydrolysis, esterification and interesterification. Biotechnol. Bioengng., 27 (1984) 1449-54. H. & Jensen, R. G., Lipases. In Lipolytic 8. Brockcrhof, enzymes. Academic Press, New York, 1974, pp. 25-75. 9. Yamacla, K., Machida, H., Hiaashi, T., Koide, A. & Ueda, K., Production of lipas: from microorganisms i 111) medium composition of C. cylindracea. Agric. Biof. C‘hem.. 37 ( 1963)-645. 10. Ota, Y., Miyairi, S. & Yamada, K.. Sterol requirement for the lioase oroduction bv C. ncaosa. Aaric. Biol. Chrm.. 32(1968)i476-8. ’ 11. Valero, F.. Avats. E. Looez-santin. J. & Poch, M.. Lipase production by C. &q&a: fermentation behavior. kiotechnol. Le/t., 10 (1988) 741-4. 12. Del Rio, J. L., Serra, P.. Valero, F., Poch, M. & Sula, C., Reaction scheme of lioase production bv C. rwosa growing on olive oil. B&tech&. Lett., 12 (1990) 835-9. 13. Ramcsh, M. V. & Lonsane, B. K., Critical importance of moisture content of the medium in alpha amylose proM 27 in a solid state duction by Bacillus licheniformis fermentation system. Appi. Microbial. Riotechnol., 33 ( 1990) 501-5. 14. Madamwar, D., Pate], S. & Parikh, H., Solid state fermentation for cellulascs and beta glucosidase production by Aspergillus niger. J. Ferment. and Bioengng., 67 (1989) 424-6. K., Tamura, G. & 15. Saiki, T., Narasaki, T., Aramaki, Arima, K., Studies on the lipoprotein lipases of microBiol. Chem.. 32 11968) organisms. Part III. Apric. <, 14-58-63. 16. I.owrv. 0. H.. Rosebrounh. N. J.. Farr. A. L. & Randall. R. L.: Protein measurement with the folin phenol reagent. J. Biol. Chem.. 193 (19.51) 265-75. ‘ am&on, J. K., Robers, P. A. 17. Dubois, M., Gilles, K. A., H & Smith, F., Calorimetric method for determination of sugars and related substances. Anal. Chem., 28 (1956) 350-6. of 18. Macrae, A. R., Lipase catalyzed interesterification oils and fats. J. Am. Oil Chem. Sot., 60 (1983) 291-4.
Production of lipase by Candida rugosa in solid state fermentation. 19. Kang, S. T. & Thee, J. S., Characteristics of immobilized lipase-catalyzed hydrolysis of olive oil of high concentration in reverse phase system. Biutrchnol. Bioengng., 33 (1989) 1469-76.
20. 21.
1
389
Kim, K. H., Kwon, D. Y. & Rhee, J. S., Effect of organic solvents on lipase for fat splitting. Lip&, 19 (1984) 975-7. Ilasseltine, C. W., Biotechnology report solid state fermentation. Biotechnol. Bioengng., 14 (1972) 517-32.
28 (1993)
385-389
Production of Lipase by Cundida rugosa in Solid State Fermentation. 1: Determination of Significant Process Variables P. Venkata Rae,” Kunthala Jayaraman & C. M. Lakshmanan Centre for Biotechnology Madras-600 02.5, India (Received
5 August 1992;
and Department
of Chemical Engineering,
revised version received and accepted
Alagappa
3 October
College of Technology,
Anna University,
1992)
The effect ofprocess variables in the production of Candida rugosa lipase using rice bran has been studied by solid state fermentation (SSF). The particle size and oil content of the bran and the addition of urea and maltose have a pronounced effect on the production of lipase. Immobilization qf lipase on rice bran (in situ) has been found possible.
et al.” observed two stages in the production of lipase by C. rugosa in a medium containing olive oil, in which the first stage was glycerol depletion without lipase formation and in the second stage was fatty acid consumption with the lipase production. The solid state fermentation (SSF ) technique has been used in the production of several industrial enzymes such as a-amylase,‘” cellulases and /3-glucosidase. I4 Very few reports are available on the production of lipase by SSF. Saikai et aLI5 investigated the production of lipoprotein lipase by SSF and SmF using various species of Mucor. Maximum activity of 52 U/g dry bran was observed in the case of Mucor juvanicus 1AM 6089 by SSF. In the present investigation, the production of lipase by C. rugosa using rice bran as a solid substrate was studied.
INTRODUCTION There has been a world wide interest recently in the application of commercially available lipases for the hydrolysis of oils,‘.* synthesis of esters,3,4 interesterification of oils and fats,5 production of and the resolution of racemic biosurfactant@ mixtures to produce optically active compounds.’ Candida rugosa is recognized as a potent producer of lipase.s The industrial production conditions for C. rugosa lipase by submerged fermentation (SmF ) were reported by Yamada et al.” Candida rugosa was found to produce extracellular lipase in the presence of lipids such as fats and sterols, both being present simultaneously. Cholesterol was found to be the most effective sterol in lipase induction.“’ Valero et al.” studied the fermentation behaviour of lipase production by C. rugmu in submerged fermentation. Del Rio
MATERIALS Corresponding
author: P. Venkata Rao.
*Present address: Department of Biotechnology, Leather Research Institute, Madras 600 020, India.
Microorganism and medium Candidu rugosa DSM 2031 agar slants containing 0.3%
Central 385
Process
Biochemistry
032-9592/93/$6.00
AND METHODS
0 1993 Elsevier Science Publishers Ltd, England.
was maintained on yeast extract, 0.3%
386
P. Venkata Rao, Kunthala Jayaraman, C. M. Lakshmanan
malt extract, 0.5% peptone and 1% glucose at 4°C.” Universal yeast medium of pH 7 used for prcculture had the following composition: glucose 10 g/litre, yeast extract 3 g/litre, malt extract 3 g/litre and peptone 5 g/litre. Refined edible grade rice bran oil of 0.8% FFA and saponification value of 185 was used for determining lipase activity. The preculture was grown in the universal yeast medium at 30°C on a shaker with constant agitation (200 rev/min). The preculture reached mid log phase in 16 h with a cell count of 269 X 10h/ml (1.8 o.d.) and it was centrifuged at 10 000 g for 10 rnin at 4°C in a sterile tube. The pellet resuspended in sterile distilled water was used as inoculum for solid state fermentation. Rice bran was obtained from a local rice mill and sieved to different size fractions. The sieve analysis and composition of rice bran are shown in Table 1. The oil content of rice bran was measured by extracting the oil using a soxhlet extractor. The defatted rice bran was extracted with 5% trichloroacetic acid to precipitate the proteins. The precipitate was mixed with 10 ml of ethyl ether for the removal of residual trichloroacetic acid. Then the precipitate was suspended in water and mixed well. Protein estimation was carried out using Lowry’s method.‘” For carbohydrate estimation, the defatted rice bran samples were hydrolysed with 6 N HCl for 8 h at 100°C. The total sugar content of the hydrolysate was determined by the phenolsulphuric acid method described by Dubois et al.” using glucose as standard.
added and thoroughly specified conditions. Immobilization
mixed and incubated
under
of lipase
Immobilization of lipase was carried out by precipitation of lipase on to rice bran with acetone (1 : 2 ratio) at O”C, as suggested by Macrae18 and Wisdom et al5 The bran was filtered and dried for 8 h in a vacuum drier at 30°C. The moisture content of the lipase immobilized on rice bran (lipase IRB) was 18% w/w on dry basis. Assay of lipase activity Lipase IRB activity was assayed in a reverse phase system’” in which lipase was dispersed in a continuous organic solvent phase containing substrate at high concentrations. The advantages of a reverse phase system over a conventional emulsion assay system are good reproducibility and easy control of reaction. The assay mixture contained 4.5 ml of rice bran oil in 6 ml of isooctane and 0.5 g of lipase IRB in a 100 ml shake flask. The mixture was incubated for 1 h at 37°C and 200 rev/min. After incubation 0.4 ml of reaction mixture was transferred to a test tube containing 4.6 ml of benzene. To this solution 1-O ml of (5% w/v) cupric acetate-pyridine solution was added and the mixture was vortex mixed for 2 min. The mixture was centrifuged for 5 min at 7000 g and the organic and aqueous phases were separated. The fatty acid produced was estimated by measuring the optical density of the organic phase at 7 15 nm as suggested by Kim et aI.2”
Solid state fermentation
RESULTS
Dry rice bran powder (5 g) was mixed with distilled water (1 : 1) in petriplates, and was then sterilized for 20 min at 120°C and 1 atmosphere. After cooling the sterile bran, inoculum was
AND DISCUSSION
The effect of fermentation time, temperature, particle size, inoculum concentration, oil content, addition of carbon and nitrogen on production of
Table 1. Particle size analysis, composition and activity of rice bran immobilized ml/g bran, initial moisture 2.2 g/g bran and time 48 h) Purticle size 04
> so0 SOO-200 250-177 <177 “Mostly husk and fiber.
I%,Fractions (WIWJ 26-6 ‘I 353 28.6 9.5
Composition Oil
lipase (pH 6, temperature
(‘% w/w dry basis)
l’roteirl
Carbohydrate
Il.0 13.7 14.0
16.0 19.5 20.8
3O”C, inoculum Activity Wls bran)
-
13.0 15.1 17.6
8.6 16.9 18.1
1
Production of lipase by Candida rugosa in solid statefermentation.
lipase was investigated by varying one parameter while keeping the other variables constant in each experiment. Experiments were conducted at pH 6 and 35°C with 1 ml of inoculum per g of dry bran (initial moisture content 2.2 g/g dry bran) in sterile plates for varying fermentation times. The results shown in Fig. 1 indicated that the maximum activity of 6.4 U/g bran was obtained in about 48 h. The effect of incubation temperature on lipase is shown in Fig. 2. The optimal temperature was found to be 3O”C, which is the same as reported for C. rugusa in submerged fermentation.’ ’ The inoculum (269 X 10h cells/ml) was varied from 0.2 to 1.4 ml/g. There was no significant effect on activity beyond 1 ml/g of bran (Fig. 3). Fermentation was carried out using rice bran of different particle sizes. The results (Table 1) indicated that rice bran fractions < 2.50 pm gave high activity. This may bc due to the higher content of protein, carbohydrate and oil in these fractions. To study the effect of oil content on lipase activity the bran of particle size < 250 ,um was defatted and varied amounts of neutral rice bran
387
I
oil (in 20 ml of hexane) were added and thoroughly mixed with the bran. Hexanc was then removed by vacuum drying. The results shown in Fig. 4 indicated that oil content is an important variable and the activity increased with an increase in oil content up to about 17%, which is approximately the oil content of < 250 pm rice bran fraction (Table 1). Inorganic nitrogen sources such as ammonium chloride, ammonium nitrate, ammonium sulphate and urea (0.4% w/w) and the organic nitrogen sources, yeast extract and malt extract (2% w/w), were individually added to the medium; the results obtained are shown in Table 2. Addition of urea and ammonium nitrate increased the lipase activity. It has been reported” that urea and ammonium nitrate maintain the pH of the substrate. Malt extract has been found to be a good organic nitrogen source for lipase production. However, the cost precludes the use of malt extract for industrial fermentations.
‘*2
0 0
I
I
I
OC
O-8
1.2
lnoculum 48
0
Time
Fig. 1.
72
lh
(ml/g
1.6
bran)
Fig. 3. Effect of inoculum concentration (pH 6, temperature 3o”C, initial moisture 2.2 g/g bran and time 48 h).
96
)
Time course of lipase production
by SSF.
19'
17 -
15 -
l3-
II
21 25
I 30
I 35
I LO
I
I
I
a
12
16
Oil
Temperature(‘C)
Effect of incubation temperature (pH 6, inoculum Fig. 2. ml/g bran, initial moisture 2.2 g/g bran and time 48 h).
I L
1
(*A
20
w/w)
Effect of oil content (pH 6, temperature 30°C Fig. 4. inoculum 1 ml/g bran, initial moisture 2.2 g/g bran, time 48 h and particle size < 250 pm).
388
P. Venkata
Rao,
Kunthala
Table 2. Effect of additional nitrogen source (pH 6, temperature 30°C inoculum 1 ml/g bran, initial moisture 2.2 g/g bran, particle size < 250 pm and time 48 h) Nitrogen
source
Control Inorganic Ammonium Ammonium Ammonium Urea
Lipase activity (u/g bran) 18.2
chloride nitrate sulphate
Organic Yeast extract Malt extract
18.4 23.6 21.1 24.4 18.2 23.7
Table 3. Effect of additional carbon source (pH 6, temperature 3o”C, inoculum 1 ml/g bran, initial moisture 2.2 g/g bran, time 48 h, particle size < 250 ,um and urea O-4% w/w) Carbon source
Control Glucose Maltose Sucrose Starch
Lipase activity @J/g bran) 24.3 21.0 26.6 24-8 24.3
The effect of additional carbon source was studied by adding glucose, maltose, sucrose and starch (2% w/w) to rice bran. From Table 3 it may be noted that maltose had a marginal effect on lipase activity. The above results indicate that the growth limiting factor in rice bran is the available nitrogen rather than the carbon source. The results of this investigation have shown that the production of lipase is highest at a fermentation time of 48 h, a temperature of 30°C and inoculum concentration of 1 ml/g bran. The rice bran particle size < 250 pm is a good substrate. The most significant variables affecting the lipase yield are oil, urea and maltose.
ACKNOWLEDGEMENT The financial assistance provided by CSIR to Mr P. Venkata Rao is acknowledged.
Jayaraman,
C. M. Lakshmanan
REFERENCES 1. Linfield, W. M., O’Brien, S., Serota, S. & Barauskas, R. A., Lipid-lipase interactions. 1. Fat splitting with lipase from Candida rwosa. J. Am. Oil Chem. Sot.. / 61 i_1984) 1067-71. .‘ 2. Hoq, M. M.. Yamane, T., Shimizu, S., Funada, T. & Ishida, S., Continuous hydrolysis of olive oil by lipase in microporous hydrophobic membrane bioreactor. J. Am. OilChem. Soc.,62 (1985) 1016-21. 3. Hoq, M. M., Yamane, T., Shimuzu, S., Funada, T. & Ishida. S., Continuous synthesis of glycerides by lipase in a microporous membrane reactor. J. Am. Oil Chem. Sot., 61( 1984) 776-81. 4. Patterson, J. D. E., Blain, J. A., Shaw, L. E. L., Todd, R. & Bell, G., Synthesis of glycerides and esters by fungal cell bound enzymes in continuous reactor systems. Biotechnol. L&t.. 1 ( 1979) 2 J I- 16. _5 Wisdom, R. A.; Dunnill, P. & Lilly, M. D., Enzymatic interesterification of fats: laboratory and pilot scale studies with immobilized lipase from Rhizopus arrhizus. Biorechnol. Bioengng., 29 (1987) 1081-5. T., Nishitani, T. & Inamasu. S., 6. Seino, H., Uchibori, Enzymatic synthesis of carbohydrate esters of fatty acid. ( 1) Esterification of sucrose, glucose, fructose and sorbitol. J. Am. Oil Chem. SW., 61( 1984) 176 l-5. 7. Cambou, B. & Klibanov, A. M., Comparison of different strategies for the lipase catalyzed preparative resolution of racemic acids and alcohols: asymmetric hydrolysis, esterification and interesterification. Biotechnol. Bioengng., 27 (1984) 1449-54. H. & Jensen, R. G., Lipases. In Lipolytic 8. Brockcrhof, enzymes. Academic Press, New York, 1974, pp. 25-75. 9. Yamacla, K., Machida, H., Hiaashi, T., Koide, A. & Ueda, K., Production of lipas: from microorganisms i 111) medium composition of C. cylindracea. Agric. Biof. C‘hem.. 37 ( 1963)-645. 10. Ota, Y., Miyairi, S. & Yamada, K.. Sterol requirement for the lioase oroduction bv C. ncaosa. Aaric. Biol. Chrm.. 32(1968)i476-8. ’ 11. Valero, F.. Avats. E. Looez-santin. J. & Poch, M.. Lipase production by C. &q&a: fermentation behavior. kiotechnol. Le/t., 10 (1988) 741-4. 12. Del Rio, J. L., Serra, P.. Valero, F., Poch, M. & Sula, C., Reaction scheme of lioase production bv C. rwosa growing on olive oil. B&tech&. Lett., 12 (1990) 835-9. 13. Ramcsh, M. V. & Lonsane, B. K., Critical importance of moisture content of the medium in alpha amylose proM 27 in a solid state duction by Bacillus licheniformis fermentation system. Appi. Microbial. Riotechnol., 33 ( 1990) 501-5. 14. Madamwar, D., Pate], S. & Parikh, H., Solid state fermentation for cellulascs and beta glucosidase production by Aspergillus niger. J. Ferment. and Bioengng., 67 (1989) 424-6. K., Tamura, G. & 15. Saiki, T., Narasaki, T., Aramaki, Arima, K., Studies on the lipoprotein lipases of microBiol. Chem.. 32 11968) organisms. Part III. Apric. <, 14-58-63. 16. I.owrv. 0. H.. Rosebrounh. N. J.. Farr. A. L. & Randall. R. L.: Protein measurement with the folin phenol reagent. J. Biol. Chem.. 193 (19.51) 265-75. ‘ am&on, J. K., Robers, P. A. 17. Dubois, M., Gilles, K. A., H & Smith, F., Calorimetric method for determination of sugars and related substances. Anal. Chem., 28 (1956) 350-6. of 18. Macrae, A. R., Lipase catalyzed interesterification oils and fats. J. Am. Oil Chem. Sot., 60 (1983) 291-4.
Production of lipase by Candida rugosa in solid state fermentation. 19. Kang, S. T. & Thee, J. S., Characteristics of immobilized lipase-catalyzed hydrolysis of olive oil of high concentration in reverse phase system. Biutrchnol. Bioengng., 33 (1989) 1469-76.
20. 21.
1
389
Kim, K. H., Kwon, D. Y. & Rhee, J. S., Effect of organic solvents on lipase for fat splitting. Lip&, 19 (1984) 975-7. Ilasseltine, C. W., Biotechnology report solid state fermentation. Biotechnol. Bioengng., 14 (1972) 517-32.