J. Mol. Biol. (1991) 220, 541-543
COMMUNICATIONS
Multiple Crystal Forms of Lipases from Geotrichum candidum Joseph D. Schrag, Yunge Li, Shan Wu and Miroslaw Cyglert Biotechnology Research Institute Nation& Research Council of Canada MontrCal, Que’becH4P 2R2, Canada (Received 11 March 1991; accepted 11 April
1991)
Multiple stable crystal forms of two lipases from the fungus Geotrichum candidum have been obtained. The diffraction pattern extends to beyond 2.0 A resolution. Similarity of the cell dimensions of various forms suggested similar packing of molecules in different crystals. This was confirmed by rotation function results. Four heavy-atoms derivatives have been identified. Keywords: lipase; crystallization;
Lipases are ester hydrolases acting on triacylglycerols. Their maximum catalytic activity is at the water-lipid interface. Lipases from a number of sources ranging from microorganisms to mammalian tissues have been purified and characterized (Borgstrom & Brockman, 1984). They vary greatly in molecular size and show little sequence homology. Crystallization of a number of lipases has been reported in the literature (Fukumoto et al., 1963; Tsujisaka et al., 1973; Sugiura et al., 1977; Isobe et al., 1988; Winkler et al., 1990; Brady et al., 1990). Despite their commercial utilization in food processing and detergents, and a potential application in the production of chiral compounds in the pharmaceutical industry, the knowledge of their three-dimensional structure is limited to two enzymes belonging to different classes: human pancreatic lipase (Winkler et al., 1990) and fungal (Rizomucor miehei) lipase (Brady et al., 1990), representing low and medium-size molecules. Although these enzymes are quite different, both belong to the a/p class with a central B-pleated sheet and have Asp-His&r triads in their active sites, a resemblance to serine proteases. The catalytic site is not exposed to the solvent, which suggests that interactions at the oil-water interface may result in a conformational change which exposes the catalytic site. Multiple molecular species with lipase activity have been identified in the fungus Geotrichum candidurn (Jacobsen et al., 1989; Veeraragavan et al., 1990; Sugihara et al., 1990). Two have been purified to homogeneity and partially characterized (Veeraragavan et al., 1990; Sugihara et al., 1990). 7 Author to whom all correspondence should be addressed.
Geotrichum candidurn
Although the molecular weights and pl values reported by the two groups vary slightly, both lipases have apparent molecular weights of about 60,000 (measured by SDS/polyacrylamide gel electrophoresis) and isoelectric points between 4-3 and 46. The pH optima, enzymatic stabilities and amino acid compositions reported are very similar. Lipases from G. candidum are highly reactive toward triolein and particular preference for cis-9 unsaturated fatty acids has been reported (Jensen et al., 1965). Selectivity for the 1 and 3 positions of the triglyceride has been reported (Veeraragavan et al., 1990), although other investigators have found no positional specificity (Iwai b Tsujisaka, 1984). Unlike some lipases, the lipases from G. cundidum are only weakly inhibited by DFP (DooijewaardKloosterziel $ Wouters, 1976). Low concentrations of calcium stimulate lipase activity, while Ca2+ concentrations greater than 50 mM are inhibitory Dooijewaard1990; (Veeraragavan et al., Kloosterziel $ Wouters, 1976). The stimulatory effects of Ca2+ may be related to the emulsion properties of the substrate rather than to a direct effect on the enzyme. Glycosylation of the G. candidurn lipases has been reported to the extent of -7% of the total weight (Tsujisaka et al., 1973; Sugihara et al., 1990). Two lipase genes have been identified in G. candidum. They have been cloned and sequenced and the amino acid sequences of the lipases deduced from cDNA (Shimada et al., 1989, 1990). Both genes code for proteins of 544 amino acids and the sequences show 86% identity. There are four conserved cysteine residues, likely forming two disulfide bridges, Two conserved potential glycosylation sites have also been identified and one gene contains a third possible site. 541
0022%2836/91&5054143
,03.00/O
0 1991 Academic Press Limited
J. D. Schrag et al.
542
Table 1 Native data statistics for G. candidum lipase crystals Unit cell dimensions Crystal form A. Lipase I 1 2 3 B. Lipase II
Space gro”P
44
b(‘Q
cm
a(“)
K)
Ho)
P1
594 594 594
844 92.1 91.9
560 544 557
900 900 98-1
1001 101.1 1023
900 900 901
p2,
581
92.0
526
900
1005
900
p21
p21
Crystallization of lipase from G. candidum was reported many years ago (Tsujisaka et al., 1973). The crystals, grown by dialysis, were not very stable and required cross-linking for preparation of heavyatom derivatives (Hata et al., 1979). A low resolution (6 A; 1 A = O-1 nm) electron density map revealed global features of the enzyme (Hata et al., 1979), but no detailed high resolution structure followed. We report here the crystallization of multiple stable forms of the higher pI lipase (lipase I, described by Veeraragavan et al., 1990) from G. candidurn, their X-ray characterization and preparation of useful heavy-atom derivatives. Crystallization of lipase II has also been achieved. The extracellular lipases were obtained from G. candidurn (ATCC 34614) in a fermentor culture using oleic acid as the lipid source as described (Veeraragavan et al., 1990). The lipases were purified by ethanol precipitation, gel filtration on a Sephacryl S-200 column, and ion exchange chromatography on a Mono-Q fast protein liquid chromatography column. A final chromatofocusing step using PBE 94 resolved the two variant lipases. Recently, a second purification protocol consisting of hydrophobic interaction chromatography on phenyl-Sepharose followed by ion exchange on Q-Sepharose has resulted in a preparation that crystallizes similarly to enzyme prepared by the first method. Lipases I and II were crystallized by vapor diffusion using polyethylene glycol (PEG?) 8000 as a precipitant. Three different crystal forms have been obtained for lipase I and one form for lipase II. The space group and unit cell parameters of each are shown in Table 1. For lipase I monoclinic (P2,) crystals with one molecule per asymmetric unit are obtained in Tris buffer at pH 8.8 at PEG concentrations of 15 to 18% (w/v). This crystal form is identical to that obtained previously (Tsujisaka et al., 1973; Hata et al., 1979) under different conditions. These crystals are very fragile and dissolve easily upon transfer to a protein-free solution. They can be stabilized by lowering the pH of the medium to 6. Attempts to crystallize the protein directly at low pH have produced two other crystal forms. At the same PEG concentrations, in N-[carbamoyl7 Abbreviation used: PEG, polyethylene glycol.
Resolution (4 2.0 2-l 2.2
&,, (%) 569 4.64 377
Redundancy ((oWeA)) 328 2.78 246
>P5
methylliminodiacetic acid (ADA) buffer at pH 6, another monoclinic (P2,) crystal form and a triclinic crystal form have been obtained. The second monoclinic form has one molecule per asymmetric unit while the triclinic form has two molecules per asymmetric unit. The three crystal forms are indistinguishable upon visual inspection. They grow easily to dimensions of 06 mm x @6 mm x 0.3 mm. Each of the three crystal forms diffracts to at least 2 A resolution. Native data sets have been collected on each of the crystal forms to a resolution of 2 to 2.2 A on a SDMS area detector (see Table 1). The calculated solvent content is about 50% in each case. Crystals of lipase II have been obtained from in 51 mMPEG 8000 at pH 7.0 15% 3-[N-morpholinolpropanesulfonic acid (Mops) buffer. The similarity in the unit cell parameters of all the crystal forms suggests that the molecular packing is also similar. This has been confirmed by rotation function studies of lipase I crystals. The rotation function between the two monoclinic crystal forms shows a single strong peak which indicates a difference in orientation of only about 15”. The self rotation function of the triclinic form indicates a non-crystallographic 2-fold axis nearly parallel to the crystallographic b-axis. Most of our effort has focused on crystal form 1 of lipase I. Soaking in solutions containing multivalent cations has often resulted in the lack of isomorphism due to considerable changes in the b dimension (82.8 to 84.7 A; Table 2). Transfer of the crystals to pH 6 resulted in only a slight change in the b dimension. To mimic the effects of the heavy-atoms, the crystals were soaked in MgC12. This has allowed collection of a data set that is more isomorphous to the heavyatom-soaked crystals (Table 1). Using these data enhances the peaks in difference Patterson maps and therefore the data from the MgCl,-soaked crystal are used as “native” for the purpose of heavy-atom refinement. Table 2 shows the statistics on some of the potential derivative data sets that have been collected. Platinum salts have provided the most isomorphous derivatives obtained to date. Soaking in platinum tetrachloride at concentrations between 2 and 5 mM for one to three weeks resulted in substitution at multiple sites. Two crystals soaked under somewhat different conditions showed differences in
Communications
543
Table 2 Heavy-atom derivative
data
Unit cell dimensions Resolution (4
44
b(‘Q
d-4
m
I$ CG
22 2.0 2.2
59.4 59-4 59.4
847 844 837
56.0 560 559
1001 loo.1 100.1
446 569 3.35
2.5 mM, 2 weeks,
2.8
594
839
559
loo.1
4.23
1477
K ,PtCI,
pH 6, ADA 5 mm, 17 days,
2.8
594
83.7
5.59
999
55.5
21.67
H&l,
pH 7, Mops 4 mM, 17 days
2.8t
59.4
82.8
56.0
999
240
37.40
%8
594
84.5
559
999
310
I.%11
Derivative
Soak conditions
Native Native M&l,
Tris, pH %8 ADA, pH 6 ADA, pH 6 5 mM, 4 days
K 2PtCl,
Pt dia&ne ethylene-
pH 7, Mops 5 mM, 7, 7 days, pH Mops
JLrge V?,,1
ADA, N-[carbamoylmethyl]iminodiacetic acid. t This derivative is isomorphous only to -5 A
the number of minor sites and the occupancies of all sites. In both cases the isomorphous difference Pat’terson maps and the anomalous difference Patterson maps had many features in common, indicating measurable presence of anomalous signal. Platinum ethylenediamine has resulted in a single site derivative. This site is also common to both K,PtCl, derivatives. Of all the mercurials tried only H&l, has caused changes in intensities. Unfortunately, this derivative suffers from the lack of isomorphism and is only useful to a resolution of about 5 il. Two sites have been modeled for this derivative. The presence of multiple mercury sites is, however, clearly recognizable in the anomalous Patterson map calculated at 2.5 A. Multiple isomorphous replacement (m.i.r.) phases based on four derivatives were calculated to a resolution of 2.8 A. The m.i.r. map showed some of the features of the enzyme. secondary structural Interpretation of this map is currently in progress. This is NR(X
publication
number
32431.
References Borgstrom, B. & Brockman, H. L. (1984). Lipases, Elsevier. Amsterdam. Brady, L., Brzozowski, A. M., Derewenda, Z. S., Dodson, E., Dodson, G., Tolley, S., Turkenburg, J. P.,
Christiansen, L., Huge-Jensen, B., Norskov, L.: Thim, L. & Menge, U. (1996). A serine protease triad forms the catalytic centre of a triacylglycerol lipase. Nature (London), 343, 767-770. Dooijewaard-Kloosterziel, A. M. P. $ Wouters, J. T. M. (1976). Some properties of the lipase of Geotrichum candidum evaluated by a fluorometric assay technique. J. Appl. Bacterial. 40, 293-299. Fukumoto, J., Iwai, M. & Tsujisaka, Y. (1963). Studies of lipase I. Purification of a lipase secreted by Aspergillus niger. .I. Gen. Appl. Microbial. 9, 353-361. Hata, Y., Matsuura, Y., Tanaka, N., Kakudo, M., Edited
Sugihara, A., Iwai, M. & Tsujisaka, Y. (1979). Low resolution crystal structure of lipase from Geotrichum candidum (ATCC 34614). J. B&hem. 86, 1821-1827. Isobe, K., Akiba, T. & Yamaguchi, R. (1988). Crystallization and characterization of lipase from Penicillium cyclqium. Agric. Biol. Chem. 52, 41-47. Iwai, M. & Tsujisaka, Y. (1984). Fungal lipase. In Lipases B. t Brockman. H. L., eds), (Borgstrom,
pp. 443-469, Elsevier, Amsterdam. Jacobsen, T., Olsen, J., Allerman, K., Poulsen, 0. M. & Hau, J. (1989). Production, partial purification, and immunochemical characterization of multiple forms of lipase from Geotrichum can&urn. Enzyme Mierob. Technol. 11, 90-95. Jensen, R. G., Sampugna, J., Guinn, J. G., Carpenter, D. L. & Marks, T. A. (1965). Specificity of a lipaae from Geotriehum candidum for cis-octadecanoic acid. J. Amer. Chem. Sot. 42, 1029-1032. Shimada, Y., Sugihara, A., Tominaga, Y., Iizumi, T. & Tsunasawa, S. (1989). cDNA molecular cloning of Geotrichum cundidum lipase. J. B&hem. 106, 383-388. Shimada, Y., Sugihara, A., Iizumi, T. & Tominaga, Y. (1990). cDNA molecular cloning and characterization of Geokichum candidurn lipase. TT. .J. Biochem. 107. 703-707. Sugihara. A., Shimada, Y. & Tominaga, Y. (1990). Separation and characterization of two molecular forms of Geotrichum candidum lipase. J. Biochem. 107, 426-430. Sugiura, M., Oikawa, T., Hirano, K. & Inukai, T. (1977). Purification, crystallization and properties of triacylglycerol lipase from Pseudomo?uz8jhoreseens. Biochim. Biophys. Acta, 488, 353-358. Tsujisaka, Y., Iwai, M. & Tominaga, Y. (1973). Purification, crystallization and some properties of lipase from Geotrichum candidum Link. Agric. Biol. Chem. 37, 1457-1464. Veeraragavan, K., Colpitts, T. & Gibbs, B. F. (1990). Purification and characterization of two distinct lipases from Geottichum candidurn. B&him. Biophys. A&, 1044, 26-33. Winkler, F. K., D’Arcy, A. & Hunziker, W. (1990). of human pancreatic lipase. Nature Structure (London), 343, 771-774. by A. Klug
COMMUNICATIONS
Multiple Crystal Forms of Lipases from Geotrichum candidum Joseph D. Schrag, Yunge Li, Shan Wu and Miroslaw Cyglert Biotechnology Research Institute Nation& Research Council of Canada MontrCal, Que’becH4P 2R2, Canada (Received 11 March 1991; accepted 11 April
1991)
Multiple stable crystal forms of two lipases from the fungus Geotrichum candidum have been obtained. The diffraction pattern extends to beyond 2.0 A resolution. Similarity of the cell dimensions of various forms suggested similar packing of molecules in different crystals. This was confirmed by rotation function results. Four heavy-atoms derivatives have been identified. Keywords: lipase; crystallization;
Lipases are ester hydrolases acting on triacylglycerols. Their maximum catalytic activity is at the water-lipid interface. Lipases from a number of sources ranging from microorganisms to mammalian tissues have been purified and characterized (Borgstrom & Brockman, 1984). They vary greatly in molecular size and show little sequence homology. Crystallization of a number of lipases has been reported in the literature (Fukumoto et al., 1963; Tsujisaka et al., 1973; Sugiura et al., 1977; Isobe et al., 1988; Winkler et al., 1990; Brady et al., 1990). Despite their commercial utilization in food processing and detergents, and a potential application in the production of chiral compounds in the pharmaceutical industry, the knowledge of their three-dimensional structure is limited to two enzymes belonging to different classes: human pancreatic lipase (Winkler et al., 1990) and fungal (Rizomucor miehei) lipase (Brady et al., 1990), representing low and medium-size molecules. Although these enzymes are quite different, both belong to the a/p class with a central B-pleated sheet and have Asp-His&r triads in their active sites, a resemblance to serine proteases. The catalytic site is not exposed to the solvent, which suggests that interactions at the oil-water interface may result in a conformational change which exposes the catalytic site. Multiple molecular species with lipase activity have been identified in the fungus Geotrichum candidurn (Jacobsen et al., 1989; Veeraragavan et al., 1990; Sugihara et al., 1990). Two have been purified to homogeneity and partially characterized (Veeraragavan et al., 1990; Sugihara et al., 1990). 7 Author to whom all correspondence should be addressed.
Geotrichum candidurn
Although the molecular weights and pl values reported by the two groups vary slightly, both lipases have apparent molecular weights of about 60,000 (measured by SDS/polyacrylamide gel electrophoresis) and isoelectric points between 4-3 and 46. The pH optima, enzymatic stabilities and amino acid compositions reported are very similar. Lipases from G. candidum are highly reactive toward triolein and particular preference for cis-9 unsaturated fatty acids has been reported (Jensen et al., 1965). Selectivity for the 1 and 3 positions of the triglyceride has been reported (Veeraragavan et al., 1990), although other investigators have found no positional specificity (Iwai b Tsujisaka, 1984). Unlike some lipases, the lipases from G. cundidum are only weakly inhibited by DFP (DooijewaardKloosterziel $ Wouters, 1976). Low concentrations of calcium stimulate lipase activity, while Ca2+ concentrations greater than 50 mM are inhibitory Dooijewaard1990; (Veeraragavan et al., Kloosterziel $ Wouters, 1976). The stimulatory effects of Ca2+ may be related to the emulsion properties of the substrate rather than to a direct effect on the enzyme. Glycosylation of the G. candidurn lipases has been reported to the extent of -7% of the total weight (Tsujisaka et al., 1973; Sugihara et al., 1990). Two lipase genes have been identified in G. candidum. They have been cloned and sequenced and the amino acid sequences of the lipases deduced from cDNA (Shimada et al., 1989, 1990). Both genes code for proteins of 544 amino acids and the sequences show 86% identity. There are four conserved cysteine residues, likely forming two disulfide bridges, Two conserved potential glycosylation sites have also been identified and one gene contains a third possible site. 541
0022%2836/91&5054143
,03.00/O
0 1991 Academic Press Limited
J. D. Schrag et al.
542
Table 1 Native data statistics for G. candidum lipase crystals Unit cell dimensions Crystal form A. Lipase I 1 2 3 B. Lipase II
Space gro”P
44
b(‘Q
cm
a(“)
K)
Ho)
P1
594 594 594
844 92.1 91.9
560 544 557
900 900 98-1
1001 101.1 1023
900 900 901
p2,
581
92.0
526
900
1005
900
p21
p21
Crystallization of lipase from G. candidum was reported many years ago (Tsujisaka et al., 1973). The crystals, grown by dialysis, were not very stable and required cross-linking for preparation of heavyatom derivatives (Hata et al., 1979). A low resolution (6 A; 1 A = O-1 nm) electron density map revealed global features of the enzyme (Hata et al., 1979), but no detailed high resolution structure followed. We report here the crystallization of multiple stable forms of the higher pI lipase (lipase I, described by Veeraragavan et al., 1990) from G. candidurn, their X-ray characterization and preparation of useful heavy-atom derivatives. Crystallization of lipase II has also been achieved. The extracellular lipases were obtained from G. candidurn (ATCC 34614) in a fermentor culture using oleic acid as the lipid source as described (Veeraragavan et al., 1990). The lipases were purified by ethanol precipitation, gel filtration on a Sephacryl S-200 column, and ion exchange chromatography on a Mono-Q fast protein liquid chromatography column. A final chromatofocusing step using PBE 94 resolved the two variant lipases. Recently, a second purification protocol consisting of hydrophobic interaction chromatography on phenyl-Sepharose followed by ion exchange on Q-Sepharose has resulted in a preparation that crystallizes similarly to enzyme prepared by the first method. Lipases I and II were crystallized by vapor diffusion using polyethylene glycol (PEG?) 8000 as a precipitant. Three different crystal forms have been obtained for lipase I and one form for lipase II. The space group and unit cell parameters of each are shown in Table 1. For lipase I monoclinic (P2,) crystals with one molecule per asymmetric unit are obtained in Tris buffer at pH 8.8 at PEG concentrations of 15 to 18% (w/v). This crystal form is identical to that obtained previously (Tsujisaka et al., 1973; Hata et al., 1979) under different conditions. These crystals are very fragile and dissolve easily upon transfer to a protein-free solution. They can be stabilized by lowering the pH of the medium to 6. Attempts to crystallize the protein directly at low pH have produced two other crystal forms. At the same PEG concentrations, in N-[carbamoyl7 Abbreviation used: PEG, polyethylene glycol.
Resolution (4 2.0 2-l 2.2
&,, (%) 569 4.64 377
Redundancy ((oWeA)) 328 2.78 246
>P5
methylliminodiacetic acid (ADA) buffer at pH 6, another monoclinic (P2,) crystal form and a triclinic crystal form have been obtained. The second monoclinic form has one molecule per asymmetric unit while the triclinic form has two molecules per asymmetric unit. The three crystal forms are indistinguishable upon visual inspection. They grow easily to dimensions of 06 mm x @6 mm x 0.3 mm. Each of the three crystal forms diffracts to at least 2 A resolution. Native data sets have been collected on each of the crystal forms to a resolution of 2 to 2.2 A on a SDMS area detector (see Table 1). The calculated solvent content is about 50% in each case. Crystals of lipase II have been obtained from in 51 mMPEG 8000 at pH 7.0 15% 3-[N-morpholinolpropanesulfonic acid (Mops) buffer. The similarity in the unit cell parameters of all the crystal forms suggests that the molecular packing is also similar. This has been confirmed by rotation function studies of lipase I crystals. The rotation function between the two monoclinic crystal forms shows a single strong peak which indicates a difference in orientation of only about 15”. The self rotation function of the triclinic form indicates a non-crystallographic 2-fold axis nearly parallel to the crystallographic b-axis. Most of our effort has focused on crystal form 1 of lipase I. Soaking in solutions containing multivalent cations has often resulted in the lack of isomorphism due to considerable changes in the b dimension (82.8 to 84.7 A; Table 2). Transfer of the crystals to pH 6 resulted in only a slight change in the b dimension. To mimic the effects of the heavy-atoms, the crystals were soaked in MgC12. This has allowed collection of a data set that is more isomorphous to the heavyatom-soaked crystals (Table 1). Using these data enhances the peaks in difference Patterson maps and therefore the data from the MgCl,-soaked crystal are used as “native” for the purpose of heavy-atom refinement. Table 2 shows the statistics on some of the potential derivative data sets that have been collected. Platinum salts have provided the most isomorphous derivatives obtained to date. Soaking in platinum tetrachloride at concentrations between 2 and 5 mM for one to three weeks resulted in substitution at multiple sites. Two crystals soaked under somewhat different conditions showed differences in
Communications
543
Table 2 Heavy-atom derivative
data
Unit cell dimensions Resolution (4
44
b(‘Q
d-4
m
I$ CG
22 2.0 2.2
59.4 59-4 59.4
847 844 837
56.0 560 559
1001 loo.1 100.1
446 569 3.35
2.5 mM, 2 weeks,
2.8
594
839
559
loo.1
4.23
1477
K ,PtCI,
pH 6, ADA 5 mm, 17 days,
2.8
594
83.7
5.59
999
55.5
21.67
H&l,
pH 7, Mops 4 mM, 17 days
2.8t
59.4
82.8
56.0
999
240
37.40
%8
594
84.5
559
999
310
I.%11
Derivative
Soak conditions
Native Native M&l,
Tris, pH %8 ADA, pH 6 ADA, pH 6 5 mM, 4 days
K 2PtCl,
Pt dia&ne ethylene-
pH 7, Mops 5 mM, 7, 7 days, pH Mops
JLrge V?,,1
ADA, N-[carbamoylmethyl]iminodiacetic acid. t This derivative is isomorphous only to -5 A
the number of minor sites and the occupancies of all sites. In both cases the isomorphous difference Pat’terson maps and the anomalous difference Patterson maps had many features in common, indicating measurable presence of anomalous signal. Platinum ethylenediamine has resulted in a single site derivative. This site is also common to both K,PtCl, derivatives. Of all the mercurials tried only H&l, has caused changes in intensities. Unfortunately, this derivative suffers from the lack of isomorphism and is only useful to a resolution of about 5 il. Two sites have been modeled for this derivative. The presence of multiple mercury sites is, however, clearly recognizable in the anomalous Patterson map calculated at 2.5 A. Multiple isomorphous replacement (m.i.r.) phases based on four derivatives were calculated to a resolution of 2.8 A. The m.i.r. map showed some of the features of the enzyme. secondary structural Interpretation of this map is currently in progress. This is NR(X
publication
number
32431.
References Borgstrom, B. & Brockman, H. L. (1984). Lipases, Elsevier. Amsterdam. Brady, L., Brzozowski, A. M., Derewenda, Z. S., Dodson, E., Dodson, G., Tolley, S., Turkenburg, J. P.,
Christiansen, L., Huge-Jensen, B., Norskov, L.: Thim, L. & Menge, U. (1996). A serine protease triad forms the catalytic centre of a triacylglycerol lipase. Nature (London), 343, 767-770. Dooijewaard-Kloosterziel, A. M. P. $ Wouters, J. T. M. (1976). Some properties of the lipase of Geotrichum candidum evaluated by a fluorometric assay technique. J. Appl. Bacterial. 40, 293-299. Fukumoto, J., Iwai, M. & Tsujisaka, Y. (1963). Studies of lipase I. Purification of a lipase secreted by Aspergillus niger. .I. Gen. Appl. Microbial. 9, 353-361. Hata, Y., Matsuura, Y., Tanaka, N., Kakudo, M., Edited
Sugihara, A., Iwai, M. & Tsujisaka, Y. (1979). Low resolution crystal structure of lipase from Geotrichum candidum (ATCC 34614). J. B&hem. 86, 1821-1827. Isobe, K., Akiba, T. & Yamaguchi, R. (1988). Crystallization and characterization of lipase from Penicillium cyclqium. Agric. Biol. Chem. 52, 41-47. Iwai, M. & Tsujisaka, Y. (1984). Fungal lipase. In Lipases B. t Brockman. H. L., eds), (Borgstrom,
pp. 443-469, Elsevier, Amsterdam. Jacobsen, T., Olsen, J., Allerman, K., Poulsen, 0. M. & Hau, J. (1989). Production, partial purification, and immunochemical characterization of multiple forms of lipase from Geotrichum can&urn. Enzyme Mierob. Technol. 11, 90-95. Jensen, R. G., Sampugna, J., Guinn, J. G., Carpenter, D. L. & Marks, T. A. (1965). Specificity of a lipaae from Geotriehum candidum for cis-octadecanoic acid. J. Amer. Chem. Sot. 42, 1029-1032. Shimada, Y., Sugihara, A., Tominaga, Y., Iizumi, T. & Tsunasawa, S. (1989). cDNA molecular cloning of Geotrichum cundidum lipase. J. B&hem. 106, 383-388. Shimada, Y., Sugihara, A., Iizumi, T. & Tominaga, Y. (1990). cDNA molecular cloning and characterization of Geokichum candidurn lipase. TT. .J. Biochem. 107. 703-707. Sugihara. A., Shimada, Y. & Tominaga, Y. (1990). Separation and characterization of two molecular forms of Geotrichum candidum lipase. J. Biochem. 107, 426-430. Sugiura, M., Oikawa, T., Hirano, K. & Inukai, T. (1977). Purification, crystallization and properties of triacylglycerol lipase from Pseudomo?uz8jhoreseens. Biochim. Biophys. Acta, 488, 353-358. Tsujisaka, Y., Iwai, M. & Tominaga, Y. (1973). Purification, crystallization and some properties of lipase from Geotrichum candidum Link. Agric. Biol. Chem. 37, 1457-1464. Veeraragavan, K., Colpitts, T. & Gibbs, B. F. (1990). Purification and characterization of two distinct lipases from Geottichum candidurn. B&him. Biophys. A&, 1044, 26-33. Winkler, F. K., D’Arcy, A. & Hunziker, W. (1990). of human pancreatic lipase. Nature Structure (London), 343, 771-774. by A. Klug
