EFFECT OF SOME PHYSICAL FACTORS ON LIPASE PRODUCTION BY SOME SELECTED FUNGI

Mansour, Abd allah; El-Ashhab, Fathy; El-Sheibani, Omar

doi:10.21608/jpd.2009.44725

EFFECT OF SOME PHYSICAL FACTORS ON LIPASE PRODUCTION BY SOME SELECTED FUNGI

Journal of Productivity and Development

Article 11, Volume 14, Issue 3, August 2009, Page 673-681 PDF (370.92 K)

Document Type: Original Article

DOI: 10.21608/jpd.2009.44725

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Authors

Abd allah Mansour^* ¹; Fathy El-Ashhab²; Omar El-Sheibani³

¹Plant Production Department, Agriculture Faculty, Garyounis University, Benghazi, Libya.

²Chemistry Department, Science Faculty, Garyounis University, Benghazi, Libya.

³Botany Department, Science Faculty, Garyounis University, Benghazi, Libya.

Abstract

Seven fungi have been experimentally screened to get the most active two lipase producers. The experimental conditions were Czapek’s yeast extract tributyrin emulsion (CYET) liquid medium, incubated at 30 °C &pH 5.0 for 8 days. Classical kinetic technique of enzymatic analysis has been used to study the effect of physical factors (pH, temperature &incubation period). Optimal yield of lipase produced by Aspergillus niger after 8 days of incubation at 30 °C &pH 5.0, while optimal yield of lipase produced by Rhizopus nigricans after 6 days of incubation at 30 °C &pH 6.0 .

Keywords

Some physical factors; lipase production; some selected fungi

Full Text

EFFECT OF SOME PHYSICAL FACTORS ON LIPASE PRODUCTION BY SOME SELECTED FUNGI

A.M.A. Mansour*; F.S. El-Ashhab**and O. M. El-Sheibani***

^* Plant Production Department, Agriculture Faculty, Garyounis University, Benghazi, Libya.

^** Chemistry Department, Science Faculty, Garyounis University, Benghazi, Libya.

^*** Botany Department, Science Faculty, Garyounis University, Benghazi, Libya.

ABSTRACT:

Keywords: Some physical factors, lipase production, some selected fungi

INTRODUCTION:

The importance of lipases in industrial processes has been throughly discussed in review articles (Rattary, 1984 and Yamane, 1987). Use of these enzymes in production of food flavors has been well documented (Nelson, 1972; Dwivedi, 1973; Arnold et al., 1975 and Shahani et al., 1976).

With the advent of biotechnology, fermentation processes have been employed to produce a wide variety of lipases from microbial sources (Kamimura et al., 2001; Elibol and Ozer, 2000). Molds have produced lipase, and the nature of the enzymes varies among species (Fukumoto et al., 1963; Iwai et al., 1964; Jensen, 1974; Kinsella and Hwang, 1976; Chander et al., 1980 & 1981; Fodiloglu and Erkmen, 1999 and Kader et al., 2007). Enzyme- producing microorganisms include bacteria (Kulkarni and Gadre, 2002; Babu et al., 2006), yeast (Corzo and Revah, 1999) and actinomycetes ( Sommer et al., 1997).

Lipases from microorganisms have drawn much attention especially for their potential use in biotechnology, mainly due to their availability and stability (Ghosh et al., 1996 and Wang et al., 1995).

Microbial enzymes that have been used to generate cheese flavors from milkfat have also been discussed by Jolly& Kosikowski (1973); Huang & Dooly (1976); Sood & Kosikowski (1979); Arbige et al. (1986) and Omar et al. (1986).

The objective of the present work was to potentially characterize the effect of some physical factors on lipase production by some selected fungi, and to use these organisms as natural and economical source of the enzyme.

MATERIALS AND METHODS

Organisms:

Seven fungi (Aspergillus niger, Aspergillus oryzae, Fusarium oxysporium, Penicillium italicum, Rhizopus nigricans, Rhizoctonia solani & Trichoderma viridae) obtained from National Collection of Yeast Cultures (NCYC), Agric. Res. Counsel, food Res. Inst., Colney lane Norwich. These cultures were maintained with periodic transfer on Czapek’s yeast extract – tributyrin agar slant.

Growth medium:

Fungal spores were grown in a sterilized (121°C/15 min) growth medium composed of (%): 3 sucrose; 0.3 NaNO_3; 0.1 KH₂PO_4; 0.05 MgSO₄. 7H₂O; 0.001 FeSO₄. 7H₂O; 0.1 yeast extract, and 1 ml of tributyrin emulsion.

Production of lipase:

Fungal organisms were inoculated into 40 ml of the growth liquid medium in 250 ml Erlenmyer flasks at the rate of 3 ml spore suspension. Then incubated at 30 °C for 8 days for the production of lipase. Mycelium was separated by filtration then centrifugation at 5000 x g for 20 min.

The cell- free extract was the source of lipase enzyme. Lipase production was determined by the method of Fukumoto et al.(1963) with some modifications.

The reaction mixture contained 3 ml of the substrate (olive oil for lipase production); 1 ml of 0.1 M acetate buffer (pH 5.6); 1 ml of 0.2 M CaCl₂, and 1 ml of the enzyme solution to be tested.

The mixture was incubated at 30 °C for 150 min. with constant shaking in calibrated water bath. At the end of incubation, the reaction mixture was mixed with 10 ml ethanol (90%) in a beaker to stop the reaction, and the free fatty acids formed by the enzyme reaction was titrated with 0.05 M KOH using a potentiometer titrator.

The activities of lipase enzyme were expressed in terms of units, one unit being defined as one ml of the difference between the volume of the alkali solution consumed for titrating the test solution and the blank containing boiled enzyme.

Measurement of Growth:

The dry weight of mycelium was measured by filtration of the cell culture through Whatman N^o 1 filter paper, washing with distilled water, and drying in an oven at 90 °C for 24 hr.

RESULTS AND DISCUSSION

Screening of lipolytic activity of the fungi:

Among seven fungi, those were screened for lipase production under the same experimental conditions the most active two organisms which produce high amount of lipase were Aspergillus niger and Rhizopus nigricans followed by Aspergillus oryzae (Table 1), while the other four fungi produced small amounts of the enzyme under these conditions and medium composition.

Table 1. Screening of lipolytic microorganisms ^a

Organisms	Wt. of biomass mg mycelium/ml medium	Lipase activity ^b (µmoles FFA)
*Aspergillus niger*	120.30	1.000
*Aspergillus oryzae*	73.30	0.150
*Fusarium oxysporium*	9.45	0.125
*Penicillium italicum*	82.90	0.150
*Rhizopus nigricans*	22.13	0.350
*Rhizoctonia solani*	94.28	0.025
*Trichoderma viridae*	20.35	0.050

Average of four trials.
1. Lipase activity is expressed as µmoles of free fatty acids liberated by 1 ml of medium.

Influence of physical factors on the production of lipases:

Because of the higher lipase production Aspergillus niger and Rhizopus nigricans were selected for further physical studies.

Incubation period:

Out of five different periods, namely 2,4,6,8, and 10 days, the maximum amount of lipase was produced by Aspergillus niger and Rhizopus nigricans after 8,6 days respectively, (Table 2). Thus it is considered as optimum incubation period for these fungi were grown in Czapek’s medium in stationary culture. The obtained optimum incubation period for Aspergillus niger was 8 days, while the

Table 2. Effect of incubation period on lipase production by Aspergillus niger and Rhizopus nigricans ^a

Incubation period (days)	*Aspergillus niger*		*Rhizopus nigricans*
Incubation period (days)	Wt. of biomass mg mycelium/ ml medium	Lipase activity^b (µmoles FFA)	Wt. of biomass mg mycelium/ ml medium	Lipase activity ^b (µmoles FFA)
2	114.07	0.150	19.88	0.175
4	174.23	0.200	117.58	0.250
6	127.95	0.250	105.38	0.350
8	104.96	0.900	67.73	0.300
10	101.13	0.450	63.60	0.125

Average of four trials.
Lipase activity is expressed as µmoles of free fatty acids liberated by 1 ml of medium.

one observed by Chander et al, (1980) was 5 days for Aspergillus wentii, while Vaidehi and Jagadamba (1984) found that it was 6 days for Aspergillus flavus. The difference between these incubation periods could be due to the type of Aspergillus species (A. niger, A. wentii, A. flavus) and the composition of growth media used by different mentioned researchers.

In studying Fusarium oxysporium and Rhizoctonia solani Vaidehi and Jagadamba (1984) found that it was 8 days.

The observed optimum incubation period for Rhizopus nigricans (6 days) was in good agreement with Chander et al. (1981) observations for the same organism grown on yeast dextrose medium. Ogendero (1980) showed that Humicola grisea var. themoidea and Mucor pusillus produced the maximum amount of lipase on 6 days, while the optimum incubation period for Talaromyces thermophilus and Termoascus crustaceus was 8 days.

Incubation temperature:

Five different incubation temperatures, 20, 25, 30, 35, and 40 °C were tested under the previously mentioned optimum incubation period for each fungus. The maximum production of lipase was found at 30 °C (Table 3). These results are in excellent agreement with those obtained by Fukumoto et al.(1963) and Chander et al.(1981) for Aspergillus niger and Rhizopus nigricans, respectively.

Confirmation can be obtained by Chander et al. (1980), Eitenmeller et al. (1970), and Chander et al.(1977) for Aspergillus wentii, Penicillium roqueforti, and Penicillium chrysogenum respectively, with different incubation periods. Nevertheless, maximum amount of lipase production at 30-45 °C were also

Table 3. Effect of incubation temperature on lipase production by Aspergillus niger and Rhizopus nigricans ^a

Incubation temperature (°C)	*Aspergillus niger*		*Rhizopus nigricans*
Incubation temperature (°C)	Wt. of biomass mg mycelium/ ml medium	Lipase activity ^b (µmoles FFA)	Wt. of biomass mg mycelium/ ml medium	Lipase activity ^b (µmoles FFA)
20	103.32	0.175	95.55	0.200
25	115.83	0.350	123.13	0.250
30	119.10	0.875	94.33	0.350
35	136.15	0.300	91.80	0.150
40	138.58	0.300	92.09	0.100

Average of four trials.
Lipase activity is expressed as µmoles of free fatty acids liberated by 1 ml of medium.

observed by Liu et al.(1972) and Somkuti & Bable (1969) for other fungi, Humicola lanuginose and Mucor pusillus, respectively.

Starting pH values:

The growth medium was adjusted to pH 4, 5, 6, 7 and 8. Maximum lipase was produced at pH 5.0 by Aspergillus niger and 6.0 by Rhizopus nigricans (Table 4).

Table 4. Effect of starting pH on lipase production by Aspergillus niger and Rhizopus nigricans ^a

Starting pH	*Aspergillus niger*		*Rhizopus nigricans*
Starting pH	Wt. of biomass mg mycelium/ ml medium	Lipase activity^b (µmoles FFA)	Wt. of biomass mg mycelium/ ml medium	Lipase activity ^b (µmoles FFA)
4	118.78	0.225	78.00	0.300
5	119.10	0.875	94.33	0.350
6	118.23	0.375	141.95	0.550
7	109.38	0.175	118.23	0.450
8	105.10	0.150	114.93	0.150

Average of four trials.
Lipase activity is expressed as µmoles of free fatty acids liberated by 1 ml of medium.

These results agree with findings of Fukumoto et al. (1963) with Aspergillus niger and with findings of Chander et al.(1977, 1980 and 1981) and Hosono et al.(1973)who demonstrated maximum yield of lipase by Rhizopus nigricans, Aspergillus wentii, Penicillium chrysogenum and Candida muscorum, respectively at pH 6.0 . On contrary, higher pH optima was noted in Penicillium camemberti and Gleosporium olvarium (Dolezalek and Minarik, 1969 and Grasas, 1973).

Conclusively, from this study, it could be concluded that the optimal yield of lipase was produced by Aspergillus niger at pH 5 and 30 °C for 8 days incubation, while the optimal yield of lipase produced by Rhizopus nigricans at pH 6 and 30 °C for 6 days incubation.

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تأثیر بعض العوامل الفیزیائیة على انتاج بعض الفطریات لانزیم اللیبیز

عبدالله منصور^* ، فتحی الأشهب^** و عمر الشیبانی^***

^*قسم الإنتاج النباتی - کلیة الزراعة – جامعة قاریونس – بنغازی – لیبیا.

^** قسم الکیمیاء - کلیة العلوم – جامعة قاریونس – بنغازی – لیبیا.

*** قسم النبات - کلیة العلوم – جامعة قاریونس – بنغازی – لیبیا.

تم انتخاب سبعة فطریات مختلفة لتحدید افضلها انتاجا لإنزیم اللیبیز باستخدام بیئة تشابک مع مستخلص الخمیرة ومستحلب الترایبیوترین السائلة کبیئة لتنمیة هذه الفطریات والتی حضنت عند 30 ^°م ودرجة pH 5.0 ولمدة 8 ایام.

وجد ان اعلى انتاج لانزیم اللیبیز کان عن طریق فطر Aspergillus niger یلیه فطر Rhizopus nigricans ثم فطر Aspergillus oryzae حیث کان اقلها انتاجا للانزیم. لذلک فقد استخدمت فطریات Aspergillus niger ، Rhizopus nigricans لدراسة العوامل الفیزیائیة مثل درجة حرارة التحضین (20 ، 25 ، 30 ، 35 ، 40 °م) و pH البیئة المبدئی عند درجات (4 ، 5 ، 6 ، 7 ، 8 ) وکذلک مدة التحضین (2 ، 4 ، 6 ، 8 ، 10 ایام) بدون رج.

تم الحصول على افضل انتاج لانزیم اللیبیز بواسطة فطر Aspergillus niger بعد ثمانیة ایام تحضین عند درجة حرارة 30 ^°م ودرجة pH مبدئی للبیئة عند 5.

أما امثل إنتاج لإنزیم اللیبیز من فطر Rhizopus nigricansفقد کان بعد ستة ایام من التحضین عند درجة حرارة 30 ^°م وفی بیئة ضبطت درجة ال pH المبدئیة لها عند 6.

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