World Journal of Microbiology & Biotechnology 12, 399-401

Thermotolerant strain of Bacillus licheniformis producing lipase H. Khyami-Horani A thermotolerant variant of Bacillus licheniformis strain H1 (isolated from Jordan valley soil) was highly active in degrading macromolecules, and possessed a lipase activity with a half life of 30 min at 70°C. This activity was produced during exponential growth. The extracellular crude lipase showed maximal activity at pH 10, and retained 65% of its stability at pH 12. Key words: Alkaline enzyme, Bacillus licheniformis, thermostable enzyme.

Bacterial lipases (triacylglycerol acylhydrolases EC 3.1.1.3) catalyse the hydrolysis of triglycerides into diglycerides, monoglycerides, glycerol and fatty acids, and under certain conditions catalyse the reverse reaction, esterification, forming glycerides from glycerol and fatty acids (Jaeger et al. 1994). The main reason for the steadily growing interest in lipases, reflected by the increase in the number of bacterial lipases which have been purified and characterized and whose genes have been sequenced, is their potential application in various industrial processes (Bjorkling et al. I991). Alkaline thermostable lipases are currently used as additives in detergents, with promising applications in the detergent industry (Newmark 1988). A number of thermopbilic lipase-producing Bacillus species have been described (Gowland et al. 1987; Emanuilova et al. I993; Janssen et al. 1994). Some mesophilic Bacillus species also showed the ability to produce thermostable lipases (Brune & Gotz 1992). In a previous study, we isolated and characterized the flora of thermophilic and thermotolerant bacilli isolated from desert soil and other solar heated sites in Jordan (Khyami-Horani & Priest 1994). In the search for alkaline thermostable lipases, the isolates were screened for their ability to produce lipases. The properties of lipolytic Bacillus strain with preliminary characterization of its lipase activity is described here.

Materials and Methods Microbial Strains

In a previous study one hundred and sixty-six Bacillus strains were isolated from soil, mud, water and compost from heated (4055°C) and unheated environments in Jordan (Khyami-Horani & Priest 1994). These isolated strains were tested for the ability to hydrolyse Tween 80 and Tween 20. Media and Culture Conditions

The medium for lipase-producing strains contained (g/l): peptone, 10.0; NaCl, 5.0; CaC12, 0.1; i0 ml Tween 80/1 and 15 g agar/1, pH 8.0. Hydrolysis of Tween 80 was detected by observing the opacity around and under the colonies. Colonies showing the highest activity were chosen for further studies. Lipase-produclng bacteria were grown aerobically for 16 to 18 h at 50°C, pH 9.0 in 300-m] Erlenmeyer flasks on a rotary shaker (200 rpm), each containing 50 ml medium containing (g/l): Lab beef extract (Oxoid), 1.0; yeast extract (Difco), 2.0; peptone (Difco), I0.0; NaCl, 5.0; substrate, 1.0; after autoclaving, CaClz was added from a separately sterilized stock to 0.2 raM. The culture medium was inoculated with 0.I ml from 10 ml precultures. Crude Enzyme Preparation

Cells were pelleted by centrifugation at 6000 x g for 20 rain. The lipase activity of the culture supernatant was assayed. The supernatant was then precipitated with ammonium sulphate (60% w/v at 4°C). The precipitate was solubilized in 40 ml 0.1 M Tris HCI buffer pH 9.0 and stored at 4°C. Enzyme Assays

The author is with the Department of Biological Sciences, University of Jordan, P.O. Box 2686, Amman 11181, Jordan; fax: (00962-6) 684366.

Lipase activity was assayed titrimetically at pH 9.0 as described previously (Gilbert et al. 1991). The reaction mixture contained

@ 1996 Rapid Science Publishers

WorId]ournalof Microbiology& Biotechnology,Vol /2, I996

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H. Khyami-Horani Table 1. Taxonomic characteristics of Bacillus licheniformis strain H1.

5 ml olive oil emulsion (25 ml olive oil, 75 ml 2% polyvinyl alcohol), 4 ml Tris/HC1 buffer (0.2 M), 110 mM CaClz and I ml enzyme solution. The mixture was incubated with continuous agitation at 50 or 55°C for 15 min. Enzyme activity was stopped by the addition of a 20 ml acetone: ethanol mixture (1:1 v/v) and the liberated fatty acids were titrated with 0.05 M NaOH. The activity which liberated 1/2mole of fatty acid from olive oil per minute was defined as one unit.

Morphology

Rods, 3.0-4.0,um; motile; Gram-positive; central oval spores

Growth Temperature pH NaCl

Aerobic and anaerobic conditions Optimum 55°C, range 37-75°C range 8-10 2-10%

Acid from

Arabinose, cellobiose, fructose, glucose, inositol, maltose, mannose, raffinose, rhamnose, sucrose, trehalose, xylose

Results and Discussion

Utilization of

Citrate, propionate, succinate

Production of

Catalase, oxidase, indole, nitrate, acetoin

Sixty six per cent of the Bacillus strains were able to hydrolyze Tween 80 and Tween 20 at 55°C. The strain which showed the highest activity at 55°C and pH 9.0 was chosen for subsequent study. The taxonomic characteristics of this strain are summarized in Table 1. This strain, designated H1, has been described as a thermotolerant variant of Bacillus licheniformis (Khyami-Horani & Priest 1994).

Soil; Ghor Safi area; Jordan valley

Source

2 1.8 --~'

10 E

"~ 1.4 "~ 1.2 ..~ 1.6

1

u~ OJ

"o

""

o.8

0.1

0.6 ~

0.4 K

~J

0,2 ~ 0

0.01 8 10 12 14 16 18 2 0

0 2 4 6

Time (h) Figure 1. Bacillus s t r a i n H1 w a s g r o w n on s t a n d a r d c o m p l e t e medium at 55°C and pH 9.0, and the lipase activity in the supernatant was assayed at intervals. Cell growth (iX); lipase activity

(,) Table 2. Effect ot temperature on enzyme activity and stability.

Temperature °C 37

45

50

55

60

65

Relative activity (%)

60

84

98

100

80

40

Relative stability (%)

96

98

100

100

95

75

70

80

61

20

Table 3. Effect of oH on enzyme activity and stability. pH 6

7

8

9

10

11

Activity (%)

40

44

62

78

100

77

Stability (%)

54

81

94

100

100

84

400

World]ournal of Microbiology & Bio~echnology, go112, 1996

Taxonomic Characteristics

Cultural Conditions The activity of extracellular lipase produced by Bacillus licheniformis HI was measured during growth in a standard complete medium. As with other Bacillus strains (Gowland et al. 1987; Gilbert et al. 199i; Janssen et al. I994), the activity was maximal during late exponential growth after 10 h cultivation (about 2.0 U ml- 1), and decreased rapidly during the stationary phase (Figure 1). The production of lipase during the exponential phase has been observed with other Bacillus species, but the incubation time was shorter (Gowland et al. 1987; Janssen et al. 1994). The amount of lipase produced was dependent on the composition of carbon sources and presence of lipids, and the conditions for optimal lipase production differed for each bacterial strain (Brune & Gotz I992). In the present study it was found that the addition of triglycerides (olive oil), detergents (Tween 80 & 20), or glycerol to the complete medium had no significant effect on the level of lipase production, as previously observed in Bacillus subtilis (Lesuisse et al. 1993). On the other hand, Emanuilova et al. (1993) reported the induction of lipase by Tween 80 in Bacillus MC7. Glycogen and casamino acids stimulated enzyme production in B. Iicheniformis. However, it was observed that the level of enzyme activity was higher in the presence of glucose, whereas glucose had no effect on the enzyme activity of B. subtilis 168 (Lesuisse et al. 1993). Accordingly, glucose was adopted as the main carbon source,

Properties of Crude Lipase Optimum Temperature and Thermostability. The enzyme was assayed at different temperatures in Tris/HC1 buffer (pH 9) and showed maximal activity at 55°C, retaining 80% of its activity at 60°C, after which the activity decreased sharply.

Lipase from thermotolerant B. licheniformis The enzyme was stable up to 60°C and retained 60% of its activity at 70°C with a half-life of 30 min (Table 2). Our enzyme was relatively thermostable, since it maintained 100% of its activity after 15 rain at 70°C. Other thermostable alkaline lipases have been reported in mesophilic strains of Bacillus (Emanuilova et al. 1993). Optimum pH for Activity and pH Stability. The enzyme was active between pH 8 and pH 11, with optimal activity at pH 10. The activity decreased above pH 11 and below pH 8 after 60 min incubation. The enzyme was stable at an alkaline pH between 9 to 11 retaining 65% of its activity at pH 12 after 30 rain at 4°C (Table 3). Alkaline lipases have also been reported in some Bacillus strains (Moller et al. 1991; Lesuisse et al. 1993), whereas other strains showed optimum activity at or around neutral pH (Kennedy & Lennarz 1979; Gowland et al. 1987). Effect of cations. The lipase activity was enhanced to 120% by addition of Ca 2+ (10 mM) to the reaction mixture. The positive effect of Ca z + is postulated to be based on the formation of Ca 2+ salts of long-chain fatty acids, thus removing the hydrolysed products from the reaction equilibrium (Brune & Gotz 1992). Addition of Cu 2+ or Fe 3+ reduced the activity by 55%. Lessuise and co-workers also reported such inhibitory effects on B. subtilis (Lesuisse et al. 1993), which could involve the catalytic site directly (Kennedy & Lennarz 1979). The lipase produced by B. Iicheniformis H1 shares some common features with B. pumilus and B. subtilis lipases (Moller et al. 1991; Lesuisse et al. 1993). Further characterization of this enzyme is in progress.

References

Acknowledgements

Bjorkling, F., Gotfredesen, S.E. & Kirk, O. 1991 The future impact of industrial lipases. Trends in Biotechnology 9, 360--363. Brune, K.A. & Gotz, F. 1992 Degradation of lipids by bacterial lipases. In Microbial Degradation of Natural Products, ed Winkelmann, G. pp. 243-266. Weinheim: VCH. Emanuilova, E., Kambourova, M., Dekosvka, M. & Manolov, R. 1993 Thermoalkalophilic lipase producing Bacillus selected by continuous cultivation. FEMS Microbiology Letters 108, 247-250. Gilbert, E.J., Drodzd, J.W. & Jones, C.W. 1991 Physiological regulation and optimization of lipase activity in Pseudomonas aeroginosa EF2. Journal of General Microbiology 137, 2215-2221. Gowland, P., Kemick, M. & Sundaram, T.K. I987 Thermophilic bacterial isolates producing lipases. FEMS Microbiology Letters 48, 339-343. Jaeger, K.E.,Ransac, S., Dijkstr, B.W., Colson, C., Va, M. & Misset, O. 1994 Bacterial lipases. FEMS Microbiology Reviews 15, 29-63. Janssen, P.H., Monk, C.R. & Morgan, H.W. 1994 A thermophilic, lipolytic Bacillus sp. and continuous assay of its p-nitrophenylpalmitate esterase activity. FEMS Microbiology Letters 120, 195-200. Kennedy, M.B. & Lennarz, W.J. I979 Characterization of the extracellular lipase of Bacillus subtilis and its relationship to a membrane-bound lipase found in a mutant strain. Journal of Biological Chemistry 254, 1080-1089. Khyami-Horani, H. & Priest, F.G. 1994 Thermotolerant varieties of Bacillus licheniformis isolated from desert environments. Journal of Applied Bacteriology 77, 392-400. Lesuisse, E., Schanck, K. & Colson, C. 1993 Purification and preliminary characterization of the extracellular tipase of Bacillus subtilis 168, an extremely basic pH tolerant enzyme. European Journal of Biochemistry 216, 155-160. Moiler, B., Vetter, R., Wilke, D. & Fowllois, B. 1991 Alkaline Bacillus lipases coding DNA sequences therefor and bacilli which produce these lipases. Patent application Wo 91/16422. Newmark, P. 1988 Two European companies market lipases. Bio/Technology 369, 134-138.

The author wishes to thank Samer Rida and Oraib Arabiat for their technical assistance.

(Received in revised form 24 February 1996; accepted I6 March 1996)

World ]ournal of Microbiology & Biotechnologg, go112, 1996

401

Thermotolerant strain of Bacillus licheniformis producing lipase.

A thermotolerant variant of Bacillus licheniformis strain H1 (isolated from Jordan valley soil), was highly active in degrading macromolecules, and po...
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