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Physical and chemical properties of lipase from Torulopsis ernobii
586
SHORT COMMUNICATIONS
BBA 33075 Physical and chemical properties of lipase from Torulopsis ernobii In a previous paper 1, it was reported that the extracellular lipase of Torulopsis ernobii was isolated in a homogeneous state on moving-boundary electrophoresis over a pH range of 2.0 to 6. 9 and ultracentrifugation at pH 5.05. The isoelectric point of the lipase was defined as pH 2.95. The lipase catalyzes the hydrolysis of olive oil at pH 6.5. In this communication, the determinations of molecular weight, amino acid composition, terminal amino acid residues and optical rotatory properties are described. For determination of the sedimentation constant 2 at zero concentration, runs were made at 60 ooo rev./min, with 0.40 , o.71 , 1.4o and 1.97 % solution of the lipase in o.1 M acetate buffer (pH 5.05) using a model 'UCA-I' Hitachi analytical ultracentrifuge. All runs were made at room temperature. The extrapolation has led to a value S°2o, w of 3.4 S at zero concentration. The partial specific volume (~) of the lipase, computed from the specific volumes 3-5 of constituent amino acid residues and mannose, was calculated to be 0.72 ml/g. Viscosity was checked at eight different concentrations at 20 °. A value for intrinsic viscosity, E~I, of 0.045 dl/g was obtained. All measurements were made in o.I M acetate buffer (pH 5.05). The molecular weight of 42 200 was computed from the formula of Scheraga and Mandelkern s, with sedimentation constant, intrinsic viscosity and partial specific volume. According to YPHANTIS'Sprocedure v, the molecular weight value of 42 ooo was obtained from the run at I I 35 ° rev./min in 0.05 M phosphate buffer (pH 5.8) at 20 °. The third method for determination of the molecular weight of the lipase was the method of ANDREWSs on Sephadex G-Ioo (2 cm × 55 cm) in o.oi M KH2PO 4. The molecular weight was estimated as 43 300. These three values of the molecular weight are in good agreement. Average molecular weight of the lipase was calculated as 42 500. The elementary analysis of the lipase shows 13.7% nitrogen (85.8% as protein) determined by the micro-Kjeldahl method and 15.5% mannose (36 moles/molecule) determined by the orcinol-H2S Q method 9. The hexosamine content in the lipase was found to be zero according to the Elson-Morgan method 9. Amino acid analyses of the acid hydrolysates of the lipase were performed on columns of Amberlite IR-i2o with the use of Hitachi KLA-3 type automatic amino acid analyzer I°. The amino acid composition of three different preparations of the lipase was examined after hydrolysis in 6 M HC1 at IiO ° for 24, 48 and 72 h. Corrections for the destruction of threonine, serine and tyrosine were made by extrapolation of zero time of acid hydrolysis. Cystine and cysteine contents were determined by using performic acid prior to hydrolysisn. For determination of free - S H groups, the titration method with p-chloromercuribenzoate12, ~3 was used with the denatured enzyme. Tryptophan content was estimated by the spectroscopic method 14. Amideammonia content was determined by the method of OKADA AND HANAFUSA is. The Torulopsis lipase has the following amino acid composition calculated from an average molecular weight of 42 500 and a total of 306 residues : Arg4, His s, Lys~3, Tyr~l, Trp2, Phe22, Cysa, Ser20, Thr22, Leu22, II%D Val2s, Met2, Glu20, Aspag, Gly2~, Ala2e, Pron, amide-ammonia3a. It does not contain cysteine. Biochim. Biophys. Acta, I54 (I968) 586-588
SHORT COMMUNICATIONS
587
TABLE I SUMMARIZED PHYSICAL PROPERTIES OF THE TORULOPSIS LIPASE
Property
Method and symbol
Value
Isoelectric p o i n t S e d i m e n t a t i o n coefficient I n t r i n s i c viscosity Diffusion coefficient* Partial specific v o l u m e Molecular weight
Electrophoresis s02o,w [~l]
Sephaxtex G - i o o Ea~D ~e aÜ b0
p H 2,95 3-4 S 0.045 dl/g 6.9" 10 -7 0.720 ml/g 42 2oo 42 ooo 43 3oo -- I 291 m # 5 87
[m'1233 mu [m'719s m~,
-- 23oo 12 400
D2o, w ~
s°2o,w YPHANTIS 7
O p t icM r o t a t i o n Optical r o t a t o r y dispersion c o n s t a n t Moffitt-Yang p a r a m e t e r Ultraviolet optical r o t a t o r y dispersion Trough Peak
* The value of the diffusion coefficient of the lipase was calculated f r o m viscosity and average molecular weight.
For the detection of N-terminal amino acid residues, the dinitrophenol method of SANGER16,17was used. DNP- derivatives were determined by two-dimensional paper chromatography, while the aqueous fraction was examined b y one-dimensional paper chromatography as described in the previous paper 17. I t was found that there was 0.76 mole of DNP-glutamic acid per molecule of the lipase in the N-terminal. For confirmation of glutamic acid, the DNP-amino acid was heated with 28% NH4OH and the glutamic acid liberated was identified by paper chromatography 17. The rate studies of the liberation of amino acids from the lipase by carboxypeptidase A (Sigma Chem. Co., U.S.A., DFP-treated preparation) at p H 8.0 have permitted identification of C-terminal amino acid. The liberation of amino acids by the action of carboxypeptidase A on the lipase (E :S = 1:300) was estimated with the automatic amino acid analyzer 17. It was found that the C-terminal group was 0.67 mole of threonine, while the second amino acid seemed to be leucine. Thus, the molecule of the lipase seemed to be a single polypeptide chain including one N-terminal of glutamic acid (or glutamine) and one C-terminal of threonine. In spite of these findings, the possibility of a double chain structure has not been completely excluded. To clarify the internal structure of the lipase in aqueous solution, the optical rotatory dispersion technique is was used. The optical rotatory dispersion measurements were made with a J a p a n Spectroscopic model ORD/UV-5 recording polarimeter at room temperature. The detailed procedures were followed as previously described 19 21: In the 32o-6oo-m/, wavelength region, the concentration of the lipase used was 0.3% in 0.05 M phosphate buffer at p H 6.1. In the ultraviolet region, the protein concentration was o.i % in 0.0005 M phosphate buffer (pH 6.1) at 2IO-25o-m# spectral zone, and it was 0.o5% to 0.00625% at 195-21o m/~. Further dilutions of the initial solution were made with H20. For the native lipase, optical rotation of ~a~D2. = - - I , optical rotatory dispersion constant of 2e = 291, the values of --ao -- 5 and --bo = 87 in the Moffitt-Yang parameters were determined. On treatment with 8 M urea at Biochim. Biophys. $cta, 154 (1968) 586-588
588
SHORT COMMUNICATIONS
pH 6. 9 for I6 h or with o.x M NaOH for 3 h at room temperature, the --ao value became negative without indicating a change in the bo value. The --ao values obtained with the above-mentioned denaturations were 334 and 305, respectively. The optical rotatory dispersion curve of the lipase in the deep ultraviolet region resembled the other curves of the partially a-helical proteins ~-25, which have a minimum at 233 m/~ and a high positive maximum at 198 m#. The a-helix content in the molecule was calculated to be about I6~o. The Torulopsis lipase has a minimum at 233 m# with Em'l : --2300 and a maximum at 198 m# with Im'] ~ +12400. The ultraviolet optical rotatory dispersion curve of denatured enzyme with o.I M NaOH in the presence of 8 M urea is confirmed to be identical to the corresponding curve of the randomly coiled form of peptide chain and proteins 22 26. According to these findings, it seems that the peptide chain of the native lipase is folded. The summarized physical data of the lipase are shown in Table I.
Enzymology Laboratory, Central Research Institute, Kikkoman Shoyu Co., Ltd., Noda, Chiba-prefecture (Japan) i 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
FUMIHIKO YOSHIDA HIROSHI MOTAI
EIjI ICHISHIMA*
H. MOTAI, E. ICHISHIMA AND V. YOSHIDA, Nature, 21o (1966) 3o8. H. If. SCHACHMAN, Ultracentrifugation in Biochemistry, Academic Press, New York, 1859. T. L. MCMEEKIN, M. L. GROVES AND N. J. HIPP, J. Am. Chem. Soc., 71 (1948) 3298. T. ISEMURA AND S. FUJITA, J. Biochem., Tokyo 44 (1957) 797E. J. Col-IN, J. T. EDSALL, Proteins, Amino Acids and Peptides, Reinhold, New York, 1943, p. 37 o. H. K. SCHACHMAN, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 4, Academic Press, New York, 1957, p. 32. D. A. YPHANTIS, Ann. N . Y . Sci., 88 (I96O) 586. P. ANDREWS, Biochem. J., 91 (I964) 222. 1~. J. WINTER, in D. GLICK, Methods of Biochemical Analysis, Vol. 2, Interscience, New York, 1955, p. 290. D. H. SPACKMAN, W. n . STEIN AND S. MOORE, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. 6, Academic Press, New York, 1963, p. 819. S. SCHRAM, S. MOORE AND E. J. BIGWOOD, Biochem. J., 57 (1954) 33. R. BENESCH AND lD. BENESCH, Methods of Biochemical Analysis, Vol. io, Interscience, 1962, P. 54. P. D. BOYER, J. Am. Chem. Soc., 76 (1954) 4331. T. W. GOODWlN AND R. A. MORTON, Biochem. J., 4 ° (1946) 628. Y. OKADA AND H. HANAFUSA, Bull. Chem. Soc. Japan, 27 (1954) 478. F. SANGER, Biochem. J., 45 (1949) 563 • E. ICHISlMA AND F. YOSHIDA, J. Biochem. Tokyo, 59 (1966) 183. G. D. FASMAN, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. 6, Academic Press, New York, 1963, ). 928. E. ICHISHIMA AND F. YOSHIDA Biochim. Biophys. Acta, 128 (1966) 13o. E. ICHISHIMA AND F. YOSHIDA Agr. Biol. Chem. Tokyo, 31 (1967) 507 . E. ICHISHIMA AND V. YOSHIDA Biochim. Biophys. Acta, 147 (1967) 341. B. JIRGENSONS, J. Biol. Chem. 24 ° (1965) lO64 . B. JIRGENSONS, J. Biol. Chem. 241 (1966) 147. B. JIRGENSONS, J. Biol. Chem. 241 (1966) 4855 . B. JIRGENSONS, J. Biol. Chem. 242 (1967) 912. A. ROSENBERG, J. Biol. Chem. 241 (1966) 5126.
Received December I2th, 1967 * R e q u e s t s for reprints should be addressed to E. ICHISHIMA.
Biochim. Biophys. Acta, 154 (1968) 586-588
SHORT COMMUNICATIONS
BBA 33075 Physical and chemical properties of lipase from Torulopsis ernobii In a previous paper 1, it was reported that the extracellular lipase of Torulopsis ernobii was isolated in a homogeneous state on moving-boundary electrophoresis over a pH range of 2.0 to 6. 9 and ultracentrifugation at pH 5.05. The isoelectric point of the lipase was defined as pH 2.95. The lipase catalyzes the hydrolysis of olive oil at pH 6.5. In this communication, the determinations of molecular weight, amino acid composition, terminal amino acid residues and optical rotatory properties are described. For determination of the sedimentation constant 2 at zero concentration, runs were made at 60 ooo rev./min, with 0.40 , o.71 , 1.4o and 1.97 % solution of the lipase in o.1 M acetate buffer (pH 5.05) using a model 'UCA-I' Hitachi analytical ultracentrifuge. All runs were made at room temperature. The extrapolation has led to a value S°2o, w of 3.4 S at zero concentration. The partial specific volume (~) of the lipase, computed from the specific volumes 3-5 of constituent amino acid residues and mannose, was calculated to be 0.72 ml/g. Viscosity was checked at eight different concentrations at 20 °. A value for intrinsic viscosity, E~I, of 0.045 dl/g was obtained. All measurements were made in o.I M acetate buffer (pH 5.05). The molecular weight of 42 200 was computed from the formula of Scheraga and Mandelkern s, with sedimentation constant, intrinsic viscosity and partial specific volume. According to YPHANTIS'Sprocedure v, the molecular weight value of 42 ooo was obtained from the run at I I 35 ° rev./min in 0.05 M phosphate buffer (pH 5.8) at 20 °. The third method for determination of the molecular weight of the lipase was the method of ANDREWSs on Sephadex G-Ioo (2 cm × 55 cm) in o.oi M KH2PO 4. The molecular weight was estimated as 43 300. These three values of the molecular weight are in good agreement. Average molecular weight of the lipase was calculated as 42 500. The elementary analysis of the lipase shows 13.7% nitrogen (85.8% as protein) determined by the micro-Kjeldahl method and 15.5% mannose (36 moles/molecule) determined by the orcinol-H2S Q method 9. The hexosamine content in the lipase was found to be zero according to the Elson-Morgan method 9. Amino acid analyses of the acid hydrolysates of the lipase were performed on columns of Amberlite IR-i2o with the use of Hitachi KLA-3 type automatic amino acid analyzer I°. The amino acid composition of three different preparations of the lipase was examined after hydrolysis in 6 M HC1 at IiO ° for 24, 48 and 72 h. Corrections for the destruction of threonine, serine and tyrosine were made by extrapolation of zero time of acid hydrolysis. Cystine and cysteine contents were determined by using performic acid prior to hydrolysisn. For determination of free - S H groups, the titration method with p-chloromercuribenzoate12, ~3 was used with the denatured enzyme. Tryptophan content was estimated by the spectroscopic method 14. Amideammonia content was determined by the method of OKADA AND HANAFUSA is. The Torulopsis lipase has the following amino acid composition calculated from an average molecular weight of 42 500 and a total of 306 residues : Arg4, His s, Lys~3, Tyr~l, Trp2, Phe22, Cysa, Ser20, Thr22, Leu22, II%D Val2s, Met2, Glu20, Aspag, Gly2~, Ala2e, Pron, amide-ammonia3a. It does not contain cysteine. Biochim. Biophys. Acta, I54 (I968) 586-588
SHORT COMMUNICATIONS
587
TABLE I SUMMARIZED PHYSICAL PROPERTIES OF THE TORULOPSIS LIPASE
Property
Method and symbol
Value
Isoelectric p o i n t S e d i m e n t a t i o n coefficient I n t r i n s i c viscosity Diffusion coefficient* Partial specific v o l u m e Molecular weight
Electrophoresis s02o,w [~l]
Sephaxtex G - i o o Ea~D ~e aÜ b0
p H 2,95 3-4 S 0.045 dl/g 6.9" 10 -7 0.720 ml/g 42 2oo 42 ooo 43 3oo -- I 291 m # 5 87
[m'1233 mu [m'719s m~,
-- 23oo 12 400
D2o, w ~
s°2o,w YPHANTIS 7
O p t icM r o t a t i o n Optical r o t a t o r y dispersion c o n s t a n t Moffitt-Yang p a r a m e t e r Ultraviolet optical r o t a t o r y dispersion Trough Peak
* The value of the diffusion coefficient of the lipase was calculated f r o m viscosity and average molecular weight.
For the detection of N-terminal amino acid residues, the dinitrophenol method of SANGER16,17was used. DNP- derivatives were determined by two-dimensional paper chromatography, while the aqueous fraction was examined b y one-dimensional paper chromatography as described in the previous paper 17. I t was found that there was 0.76 mole of DNP-glutamic acid per molecule of the lipase in the N-terminal. For confirmation of glutamic acid, the DNP-amino acid was heated with 28% NH4OH and the glutamic acid liberated was identified by paper chromatography 17. The rate studies of the liberation of amino acids from the lipase by carboxypeptidase A (Sigma Chem. Co., U.S.A., DFP-treated preparation) at p H 8.0 have permitted identification of C-terminal amino acid. The liberation of amino acids by the action of carboxypeptidase A on the lipase (E :S = 1:300) was estimated with the automatic amino acid analyzer 17. It was found that the C-terminal group was 0.67 mole of threonine, while the second amino acid seemed to be leucine. Thus, the molecule of the lipase seemed to be a single polypeptide chain including one N-terminal of glutamic acid (or glutamine) and one C-terminal of threonine. In spite of these findings, the possibility of a double chain structure has not been completely excluded. To clarify the internal structure of the lipase in aqueous solution, the optical rotatory dispersion technique is was used. The optical rotatory dispersion measurements were made with a J a p a n Spectroscopic model ORD/UV-5 recording polarimeter at room temperature. The detailed procedures were followed as previously described 19 21: In the 32o-6oo-m/, wavelength region, the concentration of the lipase used was 0.3% in 0.05 M phosphate buffer at p H 6.1. In the ultraviolet region, the protein concentration was o.i % in 0.0005 M phosphate buffer (pH 6.1) at 2IO-25o-m# spectral zone, and it was 0.o5% to 0.00625% at 195-21o m/~. Further dilutions of the initial solution were made with H20. For the native lipase, optical rotation of ~a~D2. = - - I , optical rotatory dispersion constant of 2e = 291, the values of --ao -- 5 and --bo = 87 in the Moffitt-Yang parameters were determined. On treatment with 8 M urea at Biochim. Biophys. $cta, 154 (1968) 586-588
588
SHORT COMMUNICATIONS
pH 6. 9 for I6 h or with o.x M NaOH for 3 h at room temperature, the --ao value became negative without indicating a change in the bo value. The --ao values obtained with the above-mentioned denaturations were 334 and 305, respectively. The optical rotatory dispersion curve of the lipase in the deep ultraviolet region resembled the other curves of the partially a-helical proteins ~-25, which have a minimum at 233 m/~ and a high positive maximum at 198 m#. The a-helix content in the molecule was calculated to be about I6~o. The Torulopsis lipase has a minimum at 233 m# with Em'l : --2300 and a maximum at 198 m# with Im'] ~ +12400. The ultraviolet optical rotatory dispersion curve of denatured enzyme with o.I M NaOH in the presence of 8 M urea is confirmed to be identical to the corresponding curve of the randomly coiled form of peptide chain and proteins 22 26. According to these findings, it seems that the peptide chain of the native lipase is folded. The summarized physical data of the lipase are shown in Table I.
Enzymology Laboratory, Central Research Institute, Kikkoman Shoyu Co., Ltd., Noda, Chiba-prefecture (Japan) i 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
FUMIHIKO YOSHIDA HIROSHI MOTAI
EIjI ICHISHIMA*
H. MOTAI, E. ICHISHIMA AND V. YOSHIDA, Nature, 21o (1966) 3o8. H. If. SCHACHMAN, Ultracentrifugation in Biochemistry, Academic Press, New York, 1859. T. L. MCMEEKIN, M. L. GROVES AND N. J. HIPP, J. Am. Chem. Soc., 71 (1948) 3298. T. ISEMURA AND S. FUJITA, J. Biochem., Tokyo 44 (1957) 797E. J. Col-IN, J. T. EDSALL, Proteins, Amino Acids and Peptides, Reinhold, New York, 1943, p. 37 o. H. K. SCHACHMAN, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. 4, Academic Press, New York, 1957, p. 32. D. A. YPHANTIS, Ann. N . Y . Sci., 88 (I96O) 586. P. ANDREWS, Biochem. J., 91 (I964) 222. 1~. J. WINTER, in D. GLICK, Methods of Biochemical Analysis, Vol. 2, Interscience, New York, 1955, p. 290. D. H. SPACKMAN, W. n . STEIN AND S. MOORE, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. 6, Academic Press, New York, 1963, p. 819. S. SCHRAM, S. MOORE AND E. J. BIGWOOD, Biochem. J., 57 (1954) 33. R. BENESCH AND lD. BENESCH, Methods of Biochemical Analysis, Vol. io, Interscience, 1962, P. 54. P. D. BOYER, J. Am. Chem. Soc., 76 (1954) 4331. T. W. GOODWlN AND R. A. MORTON, Biochem. J., 4 ° (1946) 628. Y. OKADA AND H. HANAFUSA, Bull. Chem. Soc. Japan, 27 (1954) 478. F. SANGER, Biochem. J., 45 (1949) 563 • E. ICHISlMA AND F. YOSHIDA, J. Biochem. Tokyo, 59 (1966) 183. G. D. FASMAN, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. 6, Academic Press, New York, 1963, ). 928. E. ICHISHIMA AND F. YOSHIDA Biochim. Biophys. Acta, 128 (1966) 13o. E. ICHISHIMA AND F. YOSHIDA Agr. Biol. Chem. Tokyo, 31 (1967) 507 . E. ICHISHIMA AND V. YOSHIDA Biochim. Biophys. Acta, 147 (1967) 341. B. JIRGENSONS, J. Biol. Chem. 24 ° (1965) lO64 . B. JIRGENSONS, J. Biol. Chem. 241 (1966) 147. B. JIRGENSONS, J. Biol. Chem. 241 (1966) 4855 . B. JIRGENSONS, J. Biol. Chem. 242 (1967) 912. A. ROSENBERG, J. Biol. Chem. 241 (1966) 5126.
Received December I2th, 1967 * R e q u e s t s for reprints should be addressed to E. ICHISHIMA.
Biochim. Biophys. Acta, 154 (1968) 586-588