J. Biochem. 107, 426-430 (1990)
Separation and Characterization of Two Molecular Forms of Geotrichum candidum Lipase Akio Sugihara, Yuji Shimada, and Yoshio Tominaga Osaka Municipal Technical Research Institute, Morinomiya, Joto-ku, Osaka, Osaka 536 Received for publication, November 14, 1989
Southern blot analysis of the Geotrichum candidum genome with a cloned lipase cDNA as the probe indicated the existence of two genes on the chromosome of the fungus which are homologous to the cDNA. As expected, two forms of lipase (lipases I and II) were actually isolated by hydrophobic interaction chromatography after a multistep procedure including ammonium sulfate fractionation, anion exchange chromatography, and gel filtration of the culture filtrate. Lipase I, the first eluted fraction, was the predominant form, and more than 80% of the total activity was attributed to this form. Amino acid sequence analysis of the amino and carboxyl termini of these two enzyme preparations indicated that lipase I was the product of the lipase gene whose cDNA had previously been cloned and sequenced [Shimada et al. (1989) J. Biochem. 106, 383-388]. Lipase II, on the other hand, had similar amino acid composition, but different terminal sequences which were not found in the primary structure of lipase I deduced from the cDNA sequence. These results gave lines of evidence for the expression of truely different lipase genes and ruled out the possibility that the observed multiple forms are caused by proteolytic digestion. The molecular mass estimated by SDS-PAGE and the isoelectric point of lipase I were 64 kDa and 4.3, while those of lipase II were 66 kDa and 4.3, respectively. The two lipases had essentially the same specific activities, substrate specificities, pH stabilities, and optimal temperatures, but different pH optima and thermal stabilities.
Lipases are enzymes that catalyze the hydrolysis of triacylglycerols and are widely distributed in various animals, plants and microorganisms (1-8). These enzymes, especially those from microorganisms, have recently received increased attention after they were shown to be active even in nearly anhydrous water-immiscible organic solvents (9, 10) and able to be used for transesterification (11, 12), synthesis of esters (13-15) and peptides (16-18), and resolution of racemic mixtures into optically active alcohols or acids (19, 20). We previously reported the purification and characterization of Geotrichum candidum lipase (21). The purified enzyme preparation was judged homogeneous from discPAGE, SDS-PAGE, and isoelectric focusing; it was easily crystallized by simply concentrating the lipase solution up to 3% protein. Subsequently, we have succeeded in cloning and sequencing of the lipase cDNA (22). The cloned cDNA coded for a protein composed of 544 amino acids and a hydrophobic signal peptide of 19 amino acids. Genomic Southern blot analysis with the cDNA as the probe suggested the existence of two different genes homologous to the cDNA. Thus, production of two isozymes was implied. Multiple forms of exocellular Upases have been described for fungi (23-26), and reports of this kind are rapidly increasing with the recent advances in purification techniques. This phenomenon is due to both post-transcriptional processings like partial proteolysis and deglycosylation, and synthesis of truely different lipases. These findings led us to reinvestigate the molecular homogeneity of the previously purified enzyme preparation. We now report the isolation of two isozymes by further purification 426
and characterization of G. candidum lipase, as well as the existence of another gene homologous to the lipase cDNA detected by Southern blot analysis. MATERIALS AND METHODS Southern Blot Analysis—The chromosomal DNA was prepared from G. candidum cells cultivated on YPD medium [1% yeast extract, 2% polypeptone, and 2% glucose (pH 5.8)] for 40 h at 27'C according to the method of Hereford et al. (27). Transfer of DNA from an agarose gel to a nylon filter (Hybond-N, Amersham) and DNA-DNA hybidrization were performed by the methods of Southern (28) and of Jeffreys and Fravell (29), respectively. "P- labeled probe was prepared using the nick translation kit of Takara Shuzo according to the protocol from the supplier. The other methods were as described previously (22). Enzyme Assay—Lipase activity was assayed essentially as described previously (21), but on a half scale in this study. In the standard assay conditions, the assay mixture contained 1 ml of olive oil, 4.5 ml of 50 mM acetate (pH 5.6), 0.5 ml of 100 mM CaCl2, and 5-50//I of enzyme solution. The mixture was incubated for 30 min at 30'C with stirring at 500 rpm, and the lipase reaction was stopped by adding 20 ml of ethanol. The amount of fatty acids released during the incubation was determined by titrating the mixture with 50 mM KOH using an APB-117 titrator (Kyoto Electronics). One unit of lipase activity is defined as the activity which liberates 1 //mol of fatty acids under the specified conditions. Substrate specificity was J. Biochem.
427
Multiple Forms of Geotrichum candidum Lipase measured using various simple triglycerides. Protein Determination—Protein was determined as described by Lowry et al. (35) with bovine albumin as the standard. Purification of Lipase—The three initial purification steps of G. candidum lipase (ammonium sulfate fractionation, anion exchange chromatography on DEAE-Sephadex A-50, and size exclusion on Sephadex G-100) were followed as described (21). Further purification was accomplished by hydrophobic interaction chromatography on a TSK Butyl Toyopearl 650M (Tosoh) column. The ultrafiltration concentrate from the Sephadex G-100 chromatography containing 375 mg of the protein was brought to 25% saturation with ammonium sulfate and put on a Butyl Toyopearl 650M column (3.4x29 cm) equilibrated with 10 mM acetate (pH 5.6) containing 25% saturated ammonium sulfate. The column was washed with 3 column volumes of the same buffer, and the material was eluted with a decreasing linear gradient of 25-0% saturated ammonium sulfate in the same buffer at a flow rate of 20 ml/h. SDS-PAGE—The discontinuous polyacrylamide SDS gel electrophoresis was done in accordance with Laemmli (36) on a 0.5-mm-thick vertical slab gel. The gel system included a resolving gel (7.5% acrylamide) and a stacking gel (3% acrylamide). The gel was stained with 0.15% Coomassie Brilliant Blue after electrophoresis. Macroglobulin (oxidized: 340 kDa, reduced: 170 kDa), phosphorylase b (97.4 kDa), glutamate dehydrogenase (55.4 kDa), and lactate dehydrogenase (36.5 kDa) were used as reference markers and purchased from Boehringer Mannheim Biochemicals. Isoelectric Focusing—Isoelectric focusing was carried out on a 0.4 mm-thick acrylamide gel using a model 111 cell (Bio-Rad). The gel was made with 5% acrylamide and 2% Bio-Lyte (pH3-5, Bio-Rad). Isogel pi markers (FMC Bioproducts) were used as isoelectric point standards. Amino Acid Analysis—Amino acid analysis was as described previously (22). The carbohydrate content in the enzyme was determined by the phenol-sulfuric acid method (30). Amino (N-) Terminal Sequence Analysis—A 7.5-mg amount of the protein was reduced and carboxymethylated according to Hire (31). After extensive dialysis, the carboxymethylated protein was treated with 4 units of pyroglutamate aminopeptidase (Boehringer Mannheim Biochemicals) according to Podell (32), followed by separation of the resulting pyroglutamic acid and the subsequent
deblocked protein on an SP-Sephadex A-50 column. The deblocked protein was subjected to manual Edman degradation, as reported by Sato et al. (33). Carboxyl (C-) Terminal Sequence Analysis—The method of Hayashi (34) was employed to determine the carboxyl terminal sequence of the enzyme. A 2.8-mg amount of the enzyme was dissolved in 600//I of 100 mM pyridineacetate (pH 5.5) containing 0.5% SDS and boiled for 2 min. To the protein solution was added carboxypeptidase Y (Takara Shuzo) at a molar ratio of 1 : 100, and digestion was done at 30*C. Aliquots (50^1) were removed after 0.25, 0.5, 1, 2, 4, and 8 h, and 9 //I of double-distilled HC1 was added to each aliquot to stop the digesiton. The supernatants were subjected to amino acid analysis. RESULTS Southern Blot Analysis—G. candidum chromosomal DNA was first digested with .EcoRI or Hindm, and then subjected to agarose gel electrophoresis followed by Southern blot analysis (Fig. 1) with 32P-labeled pGCLl, a plasmid harboring the lipase cDNA (22), as the probe. Two hybridization bands were detected, at 10 kb and 4 kb, in the .EcoRI-digested DNA (lane 1), and one dark band at 2.6 kb and two light bands at 1.8 kb and 0.8 kb in the HindJIldigested DNA (lane 2). In light of the facts that the lipase cDNA has no £coRI or Hindin sites and its molecular size is 1,857 bp, either band of the .EcoRI digests and the dark 2.6 kb band of the HindUl digests were judged to involve the lipase gene corresponding to the cDNA integrated in pGCLl. The other band of the EcoRI digests and the two
S
50
100
150
200
Fraction nimber (5ml/tiije) 1
Fig. 2. Butyl Toyopearl 650M column chromatography of Geotrichum candidum lipase. The enzyme preparation obtained by ammonium sulfate fractionation of the culture nitrate followed by DEAE-Sephadex A-50 ion exchange chromatography and gel filtration on Sephadex G-100 was further subjected to Butyl Toyopearl 650M chromatography as described in the text. O, absorbance at 280 nm; • , enzyme activity.
2
kb 23.7 — _
9.5 — 6.7 —
2.3 2.0
Fig. 1. Southern blot analysis of Geotrichum candidum chromosomal DNA. The chromosomal DNA was prepared from Geotrichum candidum cells as described in the text. The DNA digested with EcoRI (lane 1) or HindUl (lane 2) was run on a 1% agarose gel, transferred onto a nylon filter, and hybridized with "P-labeled pGCLl, a plasmid carrying lipase cDNA. Lambda DNA fragments digested with HindUl were used as size markers.
Vol. 107, No. 3, 1990
TABLE I. Purification of Geotrichum candidum Upases I and II by Butyl Toyopearl 650M chromatography. Total protein (mg)
Applied Butyl-Toyopearl 650M Lipase I Lipase II
375 288 44
Specific Total Yield activity activity (units) (units/mg)
173,250 140,330 20,790
462 487 473
100 81 12
428
A. Sugihara et ai. TABLE HI. N- and C-terminal amino acid sequences of Geotrichum candidum Upases I and II, and those deduced from the nucleotide sequence of pGCLl.
2 -
97.4 — - —
55.4
C-terminus -Leu-Phe-Gly -Leu-Tyr-Gly -Leu-Phe-Gly
N-terminus Lipase I
Separation and Characterization of Two Molecular Forms of Geotrichum candidum Lipase Akio Sugihara, Yuji Shimada, and Yoshio Tominaga Osaka Municipal Technical Research Institute, Morinomiya, Joto-ku, Osaka, Osaka 536 Received for publication, November 14, 1989
Southern blot analysis of the Geotrichum candidum genome with a cloned lipase cDNA as the probe indicated the existence of two genes on the chromosome of the fungus which are homologous to the cDNA. As expected, two forms of lipase (lipases I and II) were actually isolated by hydrophobic interaction chromatography after a multistep procedure including ammonium sulfate fractionation, anion exchange chromatography, and gel filtration of the culture filtrate. Lipase I, the first eluted fraction, was the predominant form, and more than 80% of the total activity was attributed to this form. Amino acid sequence analysis of the amino and carboxyl termini of these two enzyme preparations indicated that lipase I was the product of the lipase gene whose cDNA had previously been cloned and sequenced [Shimada et al. (1989) J. Biochem. 106, 383-388]. Lipase II, on the other hand, had similar amino acid composition, but different terminal sequences which were not found in the primary structure of lipase I deduced from the cDNA sequence. These results gave lines of evidence for the expression of truely different lipase genes and ruled out the possibility that the observed multiple forms are caused by proteolytic digestion. The molecular mass estimated by SDS-PAGE and the isoelectric point of lipase I were 64 kDa and 4.3, while those of lipase II were 66 kDa and 4.3, respectively. The two lipases had essentially the same specific activities, substrate specificities, pH stabilities, and optimal temperatures, but different pH optima and thermal stabilities.
Lipases are enzymes that catalyze the hydrolysis of triacylglycerols and are widely distributed in various animals, plants and microorganisms (1-8). These enzymes, especially those from microorganisms, have recently received increased attention after they were shown to be active even in nearly anhydrous water-immiscible organic solvents (9, 10) and able to be used for transesterification (11, 12), synthesis of esters (13-15) and peptides (16-18), and resolution of racemic mixtures into optically active alcohols or acids (19, 20). We previously reported the purification and characterization of Geotrichum candidum lipase (21). The purified enzyme preparation was judged homogeneous from discPAGE, SDS-PAGE, and isoelectric focusing; it was easily crystallized by simply concentrating the lipase solution up to 3% protein. Subsequently, we have succeeded in cloning and sequencing of the lipase cDNA (22). The cloned cDNA coded for a protein composed of 544 amino acids and a hydrophobic signal peptide of 19 amino acids. Genomic Southern blot analysis with the cDNA as the probe suggested the existence of two different genes homologous to the cDNA. Thus, production of two isozymes was implied. Multiple forms of exocellular Upases have been described for fungi (23-26), and reports of this kind are rapidly increasing with the recent advances in purification techniques. This phenomenon is due to both post-transcriptional processings like partial proteolysis and deglycosylation, and synthesis of truely different lipases. These findings led us to reinvestigate the molecular homogeneity of the previously purified enzyme preparation. We now report the isolation of two isozymes by further purification 426
and characterization of G. candidum lipase, as well as the existence of another gene homologous to the lipase cDNA detected by Southern blot analysis. MATERIALS AND METHODS Southern Blot Analysis—The chromosomal DNA was prepared from G. candidum cells cultivated on YPD medium [1% yeast extract, 2% polypeptone, and 2% glucose (pH 5.8)] for 40 h at 27'C according to the method of Hereford et al. (27). Transfer of DNA from an agarose gel to a nylon filter (Hybond-N, Amersham) and DNA-DNA hybidrization were performed by the methods of Southern (28) and of Jeffreys and Fravell (29), respectively. "P- labeled probe was prepared using the nick translation kit of Takara Shuzo according to the protocol from the supplier. The other methods were as described previously (22). Enzyme Assay—Lipase activity was assayed essentially as described previously (21), but on a half scale in this study. In the standard assay conditions, the assay mixture contained 1 ml of olive oil, 4.5 ml of 50 mM acetate (pH 5.6), 0.5 ml of 100 mM CaCl2, and 5-50//I of enzyme solution. The mixture was incubated for 30 min at 30'C with stirring at 500 rpm, and the lipase reaction was stopped by adding 20 ml of ethanol. The amount of fatty acids released during the incubation was determined by titrating the mixture with 50 mM KOH using an APB-117 titrator (Kyoto Electronics). One unit of lipase activity is defined as the activity which liberates 1 //mol of fatty acids under the specified conditions. Substrate specificity was J. Biochem.
427
Multiple Forms of Geotrichum candidum Lipase measured using various simple triglycerides. Protein Determination—Protein was determined as described by Lowry et al. (35) with bovine albumin as the standard. Purification of Lipase—The three initial purification steps of G. candidum lipase (ammonium sulfate fractionation, anion exchange chromatography on DEAE-Sephadex A-50, and size exclusion on Sephadex G-100) were followed as described (21). Further purification was accomplished by hydrophobic interaction chromatography on a TSK Butyl Toyopearl 650M (Tosoh) column. The ultrafiltration concentrate from the Sephadex G-100 chromatography containing 375 mg of the protein was brought to 25% saturation with ammonium sulfate and put on a Butyl Toyopearl 650M column (3.4x29 cm) equilibrated with 10 mM acetate (pH 5.6) containing 25% saturated ammonium sulfate. The column was washed with 3 column volumes of the same buffer, and the material was eluted with a decreasing linear gradient of 25-0% saturated ammonium sulfate in the same buffer at a flow rate of 20 ml/h. SDS-PAGE—The discontinuous polyacrylamide SDS gel electrophoresis was done in accordance with Laemmli (36) on a 0.5-mm-thick vertical slab gel. The gel system included a resolving gel (7.5% acrylamide) and a stacking gel (3% acrylamide). The gel was stained with 0.15% Coomassie Brilliant Blue after electrophoresis. Macroglobulin (oxidized: 340 kDa, reduced: 170 kDa), phosphorylase b (97.4 kDa), glutamate dehydrogenase (55.4 kDa), and lactate dehydrogenase (36.5 kDa) were used as reference markers and purchased from Boehringer Mannheim Biochemicals. Isoelectric Focusing—Isoelectric focusing was carried out on a 0.4 mm-thick acrylamide gel using a model 111 cell (Bio-Rad). The gel was made with 5% acrylamide and 2% Bio-Lyte (pH3-5, Bio-Rad). Isogel pi markers (FMC Bioproducts) were used as isoelectric point standards. Amino Acid Analysis—Amino acid analysis was as described previously (22). The carbohydrate content in the enzyme was determined by the phenol-sulfuric acid method (30). Amino (N-) Terminal Sequence Analysis—A 7.5-mg amount of the protein was reduced and carboxymethylated according to Hire (31). After extensive dialysis, the carboxymethylated protein was treated with 4 units of pyroglutamate aminopeptidase (Boehringer Mannheim Biochemicals) according to Podell (32), followed by separation of the resulting pyroglutamic acid and the subsequent
deblocked protein on an SP-Sephadex A-50 column. The deblocked protein was subjected to manual Edman degradation, as reported by Sato et al. (33). Carboxyl (C-) Terminal Sequence Analysis—The method of Hayashi (34) was employed to determine the carboxyl terminal sequence of the enzyme. A 2.8-mg amount of the enzyme was dissolved in 600//I of 100 mM pyridineacetate (pH 5.5) containing 0.5% SDS and boiled for 2 min. To the protein solution was added carboxypeptidase Y (Takara Shuzo) at a molar ratio of 1 : 100, and digestion was done at 30*C. Aliquots (50^1) were removed after 0.25, 0.5, 1, 2, 4, and 8 h, and 9 //I of double-distilled HC1 was added to each aliquot to stop the digesiton. The supernatants were subjected to amino acid analysis. RESULTS Southern Blot Analysis—G. candidum chromosomal DNA was first digested with .EcoRI or Hindm, and then subjected to agarose gel electrophoresis followed by Southern blot analysis (Fig. 1) with 32P-labeled pGCLl, a plasmid harboring the lipase cDNA (22), as the probe. Two hybridization bands were detected, at 10 kb and 4 kb, in the .EcoRI-digested DNA (lane 1), and one dark band at 2.6 kb and two light bands at 1.8 kb and 0.8 kb in the HindJIldigested DNA (lane 2). In light of the facts that the lipase cDNA has no £coRI or Hindin sites and its molecular size is 1,857 bp, either band of the .EcoRI digests and the dark 2.6 kb band of the HindUl digests were judged to involve the lipase gene corresponding to the cDNA integrated in pGCLl. The other band of the EcoRI digests and the two
S
50
100
150
200
Fraction nimber (5ml/tiije) 1
Fig. 2. Butyl Toyopearl 650M column chromatography of Geotrichum candidum lipase. The enzyme preparation obtained by ammonium sulfate fractionation of the culture nitrate followed by DEAE-Sephadex A-50 ion exchange chromatography and gel filtration on Sephadex G-100 was further subjected to Butyl Toyopearl 650M chromatography as described in the text. O, absorbance at 280 nm; • , enzyme activity.
2
kb 23.7 — _
9.5 — 6.7 —
2.3 2.0
Fig. 1. Southern blot analysis of Geotrichum candidum chromosomal DNA. The chromosomal DNA was prepared from Geotrichum candidum cells as described in the text. The DNA digested with EcoRI (lane 1) or HindUl (lane 2) was run on a 1% agarose gel, transferred onto a nylon filter, and hybridized with "P-labeled pGCLl, a plasmid carrying lipase cDNA. Lambda DNA fragments digested with HindUl were used as size markers.
Vol. 107, No. 3, 1990
TABLE I. Purification of Geotrichum candidum Upases I and II by Butyl Toyopearl 650M chromatography. Total protein (mg)
Applied Butyl-Toyopearl 650M Lipase I Lipase II
375 288 44
Specific Total Yield activity activity (units) (units/mg)
173,250 140,330 20,790
462 487 473
100 81 12
428
A. Sugihara et ai. TABLE HI. N- and C-terminal amino acid sequences of Geotrichum candidum Upases I and II, and those deduced from the nucleotide sequence of pGCLl.
2 -
97.4 — - —
55.4
C-terminus -Leu-Phe-Gly -Leu-Tyr-Gly -Leu-Phe-Gly
N-terminus Lipase I
