Biotechnology Letters 21: 897–899, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands. 897 A novel alkaline, thermostable, protease-free lipase from Pseudomonas sp. Neelima Kulkarni & R.V. Gadre ∗ Chemical Engineering Division, National Chemical Laboratory, Pune 411008, India ∗ Author for correspondence (Fax: 91-020 589 3041; E-mail: gadre@che.ncl.res.in Received 23 June 1999; Revisions requested 25 June 1999/23 July 1999; Revisions received 21 July 1999/16 August 1999; Accepted 16 August 1999 Key words: alkali-tolerant, lipase, protease-free, Pseudomonas, thermostable Abstract An extracellular, alkali-tolerant, thermostable lipase was from a Pseudomonas sp. It had optimal activity at 65 ◦ C and retained 75% of its activity at 65 ◦ C for 90 min. The pH optimum was 9.6 and it retained more than 70% activity between pH 5 and 9 for 2 h. The culture broth was free of protease and, at 30 ◦ C, the culture filtrate retained all the activity for at least 7 days, without any stabilizer. In shake flask culture, addition of groundnut oil (3 g l −1 ) towards the end of growth phase increased the activity from 4 U ml −1 to 8 U ml −1 . Introduction Alkali tolerance and thermostability of lipases are desirable characteristics for their commercial exploita- tion. Although production of such lipases has been reported, published literature on their preparation is rather scanty (Berto et al. 1997). Lipases are often secreted in late stages of growth (Tan & Gill 1985). Proteases secreted concomitantly can change the char- acteristics of lipases and, at times, degrade them (Kumura et al. 1991, Cordenons et al. 1996). Oil can be used as sole carbon source (Tan & Gill 1985) or as an inducer in complex media for the production of lipase (Dharmsthiti & Kuhasuntisuk 1998). We report here a Pseudomonas sp. that secrets alkali-tolerant, protease-free, thermostable lipase and effect of the oil addition schedule on lipase production. Materials and methods Chemicals The media ingredients were procured from HiMe- dia (Mumbai, India). AR grade salts, gum Aca- cia and iso-propanol were from S.D. fine chemicals (Boisar, India). Hammarsten casein was from Sisco Research Laboratories (Mumbai, India) while butyric acid, caproic acid, pNPP, tributyrin and triolein were from Sigma. Cottonseed meal and refined groundnut oil were purchased locally. Enzyme production and characterization An isolate was selected that secreted protease-free li- pase and was identified as Pseudomonas sp. according to Palleroni (1984). The isolate was grown in shake flask in a medium containing 10 g soya peptone, 10 g cotton seed meal, 3.0 g groundnut oil, 1.0 g gum Acacia, 8.63 g Na 2 HPO 4 , 6.08 g NaH 2 PO 4 · 2H 2 O, 1.0 g mgSO 4 · 7H 2 O, 1.0 g NaCl, 0.5 g CaCl 2 · 2H 2 O, per liter H 2 O. In another experiment, separately ster- ilized groundnut oil was added (3 g l −1 ) to different flasks at 0, 8, 12, 16 and 20 h. Lipase activity was determined till 60 h. The enzyme from cell-free broth was precipitated by ammonium sulfate at 30% satura- tion and the precipitate was dialyzed against distilled water. The pH optimum of the crude enzyme prepa- ration was studied in the range 3 to 10.5. The pH stability was determined by incubating the enzyme in buffers of different pH in the range of 3 to 10.5 for 2 h, at 30 ◦ C, followed by activity estimation at pH 9.6. The temperature optimum was studied in the range of 30 to 80 ◦ C. Temperature stability was determined by holding the enzyme preparation at 65 and 75 ◦ C for 90 min followed by activity determination. The cell-