Factors affecting lipase production in Mucor mucedo

Zbl. Bakt. Hyg., I. Abt. Orig. C 3, 427-431 (1982)

Institut fiir Chemie und Physik der Siiddeutschen Versuchs- und Forschungsanstalt fur Milchwirtschaft der TU Miinchen-Weihenstephan, 8050 Freising-Weihenstephan, Federal Republic of Germany

Factors Affecting Lipase Production in Mucor mucedo H. CHANDER and H. KLOSTERMEYER Received December 18, 1981

Summary Mucor mucedo showed maximum growth and lipase production at 30°C after 3 days at pH 7.0. Aeration stimulated lipase yield by 40%. No significant difference in growth and lipase production was observed with different carbohydrates. Among the nitrogen sources tested, peptone at 2% lead to maximum lipase yield. The addition of Tributyrin to the growth medium increased lipase production by 66% while the addition of other lipids caused a reduction.

Key words: Lipase production - Mucor mucedo

Introduction The synthesis of lipolytic enzymes by molds is known to vary in different species

(Fukumoto et al., 1964; Chander et al., 1981). Microbial lipases cause the breakdown of fat by hydrolysis and the products thus formed contribute to the development of desirable flavours in milk and milk products. Although studies have been conducted on lipases from Mucor species (Somkuti and Babel, 1968; Nagaoka and Yamada, 1973), little information is available regarding factors affecting lipase production by Mucor mucedo.

Material and Methods Organism Mucor mucedo was obtained from the culture collection of the Bakteriologisches Institut, Weihenstephan, Freising, West Germany. The organism was maintained with periodic transfer on yeast dextrose agar slants for 72 h at 30°C. 28 Zbl. Bakt. Hyg., I. Abt. Orig. C 3

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H. Chander and H. Klostermeyer

Growth media Mucor mucedo was grown in a medium composed of 20 g peptone (Merck), 5 g yeast extract (BAG, Germany) 2 g NaCI (pro anal., Merck), 40 g dextrose (pro anal., Merck) in 11 distilled water. The medium was inoculated with 1 % spore suspension. Production of lipase Mucor mucedo was inoculated into 100 ml of the above medium at the rate of 1 x 107 spores per ml. The culture was then incubated at 30 DC for 72 h for the production of lipase. The mycelium was removed from the medium by centrifugation at 2500 x g for 15 min and the supernatant fluid was used as the enzyme source. Lipase activity was estimated by the method of Oi et al. (1969) with some modifications. The supernatant fluid (1.0 ml) was added to 5.0 ml of 5% olive oil emulsified in 5% gum acacia in distilled water, 5.0 ml of 0.02 M Tris-HCI buffer (pH 8.0), 1.0 ml of 0.075 M CaCI. sol., 1.0 ml of 3 M NaCI sol. and 2.0 ml of glass distilled water with continuous shaking in a water bath at 37 DC for 120 minutes. The pH of the reaction mixture of the experimental set was also brought to the level of control sample by the addition of 0.01 N NaOH. Dry weight was determined after filtering the mycelium trough Schleicher and Schull's filter paper no 604 and washing with distilled water and then drying in an oven adjusted to 100 DC till attainment of constant weight.

Results and Discussion 20

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Fig. 1. Effect of temperature and incubation time on lipase production by Mucor mucedo,

Lipase Production in Mucor mucedo

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Mucor mucedo produced maximum amount of lipase (18.5 zzmoles free fatty acids) at 30°C in 72 h (Fig. 1) as also found with Penicillium roquelorti (Eitenmiller et al., 1970) and Rbizopus nigricans (Chander et al., 1981). When the mold was grown in nutrient broth adjusted to pH ranging from 3 to 9 at intervals of 1 pH unit, maximum lipase production occurred at pH 7.0 as also noted with Penicillium camemberti (Dolezalek and Minarik, 1969). However, the present findings are contrary to the earlier observations made by Chander et al. (1981) who reported maximum yield of lipase by Rhizopus nigricans at pH 6.0. To study the effect of aeration by shaking one set of inoculated flasks was continously shaken at 30°C for 3 days at 200 rpm., while the other set was kept stationary. Lipase production within 3 days at 30 °C was 40 010 higher in shake cultures than in the stationary culture although microbial growth (dry weight) was virtually the same. These results are comparable to those obtained with Penicillium roquejorti (Eitenmiller et al., 1970) and Rhizopus nigricans (Chander et al., 1981). To determine the effect of various sugars on lipase production the organism was grown in nutrient medium devoid of dextrose but supplemented separately with different carbon sources. These were sterilized separately by Seitz filtration and added into the medium to yield a final concentration of 40 gil. All sugars tested were similarly effective with respect to growth and lipase yield (Table 1) as observed in similar experiments with Rhizopus [apanicus (Aisaka and Kerada, 1979). Reduced growth and lipase yield with galactose, lactose and glucose was observed, however, in Penicillium roque/orti (Imamura and Kataoka, 1963). Table 1. Effect of carbon sources on growth and lipase production by Mucor mucedo»

a

Carbon source

Mycelial dry weight produced (mg/ml)

Free fatty acids produced (,umoles/ml)

Glucose Xylose Sucrose Maltose Galactose Mannitol Fructose

1.68 1.57 1.72 1.68 1.78 1.56 1.67

18.0 15.9 16.5 15.6 17.2 16.9 15.6

Average of three trials.

Different nitrogen sources (20 gil) were separately added to the nutrient medium devoid of nitrogen source. The highest lipase yields were obtained with peptone (Table 2). Other nitrogen sources showed less lipase production while urea (20 %) was the least suitable source. The growth of the mold was not substantially affected. Peptone was also found to be the best nitrogen source for lipase production with Candida mycoderma (Hosono and Tokita, 1970) and Rhizopus nigricans (Chander et al., 1981). Instead of urea, sodium nitrate was found to be the

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H.Chander and H.Klostermeyer

Table 2. Effect of nitrogen sources" on growth and lipase production by Mucor mucedo»

a b

Nitrogen source

Mycelial dry weight produced (mg/ml)

Free fatty acids produced (Ilmoles/ml)

Peptone Casein hydrolysate Milk casein Potassium nitrate Ammonium nitrate Ammonium chloride Ammonium sulphate Sodium nitrate Urea

1.65 1.72 1.48 1.52 1.58 1.41 1.58 1.38 1.12

18.0 13.5 12.8 13.8 13.0 9.9 14.4 11.4 3.6

Average of three trials. Nitrogen sources (20 gil) were separately added to the nitrogen free medium.

Table 3. Effect of different lipid sources on growth and lipase production by Mucor mucedo»

a

Lipid source.

Mycelia dry weight produced (mgjrnl)

Free fatty acids produced (Ilmoles/ml)

Olive oil Mustard oil Butter oil Corn oil Sunflower oil Tributyrin Tiolein Control

1.42 1.44 1.52 1.49 1.48 1.74 1.44 1.65

11.2 12.0 11.2 10.5 12.5 30.0 7.5 18.3

Average of three trials.

poorest nitrogen source for the growth and lipase production of Penicillium roqueforti (Eitenmiller et aI., 1970). Among various sterilized oils added to the basal medium in 0.10f0 concentration, tributyrin increased lipase production by 66 Ofo without causing substantial stimulation of the growth (Table 3) while triolein suppressed enzyme production by 58 {}/o. Lipase production by Mucor mucedo did not appear to be adaptive in the true sense of the term as most of the oils were slightly inhibitory to lipase production. Tributyrin also enhanced lipase production in Penicillium roquelorti (Imamura and Kataoka, 1963) whereas tryglycerides with long-chain unsaturated fatty acids reduced lipase synthesis as these were not properly utilized by the organism. Smith and Alford (1966) also assumed that inhibition of lipase synthesis by lard in Pseudomonas fragi was presumably due to accumulation of unsaturated

Lipase Production in Mucor mucedo

431

free fatty acids following hydrolysis of fat. A reduction in lipase production by

Penicillium roqueforti in the presence of butter oil, corn oil and olive oil was also observed by Eitenmiller et al. (1970). These observations may be valuable in harnessing lipase for useful processing applications.

References

Aisaka, K., Kerada, 0.: Production of lipoprotein lipase and lipase by Rhizopus iapanicus. Agr. bioI. Chern. 43, 2125-2129 (1979) Chander, H., Batisb, V.K., Ghodekar, D.R., Srinivasan, R.A.: Factors affecting lipase production in Rhizopus nigricans. J. Dairy Sci. 64, 193-196 (1981) Dolezalek, ]., Minarik, B.: Effect of pH of the medium and of rennet on enzymatic activity of the mold strain Penicillium camemberti. Sk. Chern. technol. Praze E 20, 67-77 (1969) Eitenmiller, R. R., Vakil,]. R., Shabani, K. M.: Production and properties of Penicillium roqueforti lipases. J. Food Sci. 35, 130-133 (1970) Fukumoto, ]., luiai, M., Tsujisaka, Y.: Studies on lipase. IV. Purification and properties of lipase secreted by Rhizopus delemar. J. gen. appl. Microbiol. 10, 257-265 (1964) Hosono, A., Tokita, F.: The lipolytic properties of Candida mycoderma and Debaryomyces klockeri isolated from Limburger cheese and some properties of lipase produced by these yeasts. Jap. J. Zootech. Sci. 41, 519-527 (1970) Imamura, T., Kataoka, A.: Biochemical studies on the manufacturing of Roqueforti type cheese. I. Lipase producing abilities of Penicillium roqueforti. jap. J. Zootech. Sci. 34, 344-348 (1963) Nagaoka, K., Yamada, Y.: Purification of Mucor lipases and their properties. Agr. BioI. Chern. 37, 2791-2796 (1973) Ci, S., Sawada, A., Satomura, Y.: Purification and properties of fungal lipase preparation used for milk flavouring. Agr. BioI. Chern. 33, 729-738 (1969) Smith, ].L., Alford, ].A.: Inhibition of microbiallipases by fatty acids. Appl. Microbiol. 14, 699-705 (1966) Somkuti, G. A., Babel, F.].: Lipase activity of Mucor pusillus. Appl. Microbiol. 16,617-619 (1968) Professor Dr. H.Klostermeyer, Institut fiir Chemie und Physik der Siiddtsch. Versuchsund Forschungsanstalt fur Mi1chwirtschaft, Vottingerstr.Ai, D-8050 Freising-Weihenstephan