Crystallographic data and circular dichroism spectrum of lipase from Geotrichum candidum link

J. Mol. Biol. (1975) 99, 807-808

LETTERS TO THE EDITOR

Crystallographic Data and Circular Dichroism Spectrum of Lipase from Geotrichum candidum Link Single crystals of lipase from Geot~richurn candidum Link have been obtained for a high resolution structural analysis. The crystals are monoclinic, space group P21, with unit cell dimensions e r a ----59.3 A, b = 83.8 A, c = 55.7 A,/~ = 100-1% An asymmetric unit contains one protein molecule. Lipases are widely distributed in animals, plants and micro-organisms. I n particular, those from bacteria, yeasts and fungi have received much attention in the last decade because of their applications in industry and medicine. Most of them are discharged through the external membrane into the culture medium (exoceUnlar lipases). Geotrichum candidum Link lipase has been isolated and characterized previously (Tsujisaka et al., 1973a). G. candidum Link produces lipase inducibly only in the presence of the specific substance such as vegetable oil or f a t t y acid in the culture medium (Tsujisaka ct al., 19735; Iwai et al., 1973). This strain is different from G. candidum in t h a t the latter does not require such specific substances for lipase production. G. candidum Link lipase has molecular weight of 53,000-55,000 and isoclectric point of 4.33. ~mino acid analysis does not detect the presence of any sulphurcontaining amino acid. Further, the crystalline preparation contains 7 ~/o carbohydrate and a very small amount of lipid. I n order to estimate roughly the helical content, I

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FzG. 1. Circular diohroism spectrum of Geotrichum candidum Link lipase. The protein dissolved in 0.01 M-sodium aoetate buffer (pH 5.6, 17°C). 807

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circular dichroism measurements were made with a Jasco J20 spectropolarimeter. Figure 1 shows the spectrum expressed in terms of mean residue ellipticity in units of deg cm2/decimol. The amount of m-helix calculated from the equation developed by Chen et al. (1972) is 0.27, Large single crystals suitable for X-ray analysis were obtained in the following manner. A 0.4°/o solution (spec. act. 300 or above) was concentrated in a collodion bag under reduced pressure at room temperature. Fine crystals surrounded by an amorphous precipitate appeared when the protein concentration reached 1 ~/o. Concentration was continued until the volume was reduced to one-third of its original and the supernatant was stored at 8°C. After several days, well-shaped transparent crystals grew to a size of 0.5 mm × 0.3 m m × 0"1 ram. They were stored in ammonium sulphate solution at 70~/o saturation (pH 6.0). The crystals withstand X-ray exposure reasonably well below 20°C, but are unstable at higher temperatures. The monocl~nle crystals belong to the space group P21 with unit cell dimensions of a = 59.3 A, b ---- 83.8 A, c -~ 55.7 A, fl = 100.1°. The density of the crystals measured in a potassium tartrate solution is 1.26 g/cm a. In conjunction with a unit cell volume of 272,500 j~a, this value indicates that the asymmetric unit contains one protein molecule. This corresponds to a crystal volume per unit of protein molecular weight, VM, of 2"57, which is within 1.68 to 3.53 AS/daltons range found by i~Iatthews (1968) for over 100 crystalline proteins. Then the crystal contains 49% solvent by weight. Crystals soaked in platinum and uranium compounds exhibit appreciable and lsomorphous changes from the native diffraction pattern. Intensity measurements from the native protein crystals at 5 A resolution have been carried out with a fourcircle diffractometer. Osaka Municipal Technical Research Institute Kita-ogimachi 38 Kita-ku, Osaka 530, Japan

AKIO SUGT~AI~A YOSHIO TSUJISAlr A MIEKO I W ~

¥ o s ~ o TO~AOA Svsum~uOxu-mrm~ Institute for Protein Research Osaka University, Suita Osaka 565, Japan

Yos1~11rl" ~ A T S u u-~A

NoBuo TAWA~A i~ASAO I~'AXUD0

Received 10 June 1975 REFERENCES Chen, Y-H., Tang, J. T. & Martinez, H. (1972). Biochemistry, 11, 4120-4131. Iwai, M., Tsujisaka, Y., Okamoto, Y. & Fukumoto, J. (1973). Agr. Biol. Chem. 37, 929-931. Matthews, B. W. (1968). J. Mel. Biol. 33, 491-497. Tsujisaka, Y., Iwai, M. & Tominaga, Y. (1973a). Agr. Biol. Ghem. 37, 1457-1464. Tsujisaka, Y., Iwai, M., Fukumoto, J. & Okamoto, Y. (19735). Agr. Biol. Chem. 37, 837-842.