Extracellular Production of Active Rhizopus oryzae Lipase by Saccharomyces cerevisiae
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概要
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The gene encoding Rhizopus oryzae lipase(ROL) was successfully expressed, and extracellularly active ROL was produced by Saccharomyces cerevisiae. The genes encoding the mature region(mROL)and the mROL having the pro sequence(ProROL)were fused to the gene encoding the pre- or prepro-α-factor leader region, and expressed under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis(UPR-ICL), which is strongly functional in S.cerevisiae. Efficient lipase activity was observed in the culture medium when the gene encoding ProROL was expressed. The recombinant lipase(rROL)produced by S.cerevisiae was purified by ethanol precipitation, butyl-Toyopearl 650M chromatography, and Sephacryl S-100 HR gel filtration to a single band by native-PAGE. However, the single band was found to consist of two proteins with different molecular masses(35 kDa and 46 kDa)on SDS-PAGE. The N-terminal analysis showed that 35 kDa protein was processed in the prosequence, and attached 28 amino acids of the C-terminal part of the prosequence, named rPro28ROL, while the 46 kDa protein had the entire pro-region, named rProROL. The preference of rROL for a series of triglycerides was determined using the titrimetric assay. rROL showed higher activity on saturated substrates having C_8-, C_<10>-, C_<12>-, and C_<14>- fatty acid chains in triglycerides. Extracellular lipase activity reached 2880 U/l at 120 h of cultivation in YPD medium.
- 社団法人日本生物工学会の論文
- 1998-08-25
著者
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UEDA MITSUYOSHI
Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemist
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TANAKA ATSUO
Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemist
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Tanaka A
Department Of Etiology And Pathophysiology National Cardiovascular Center Research Institute
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Atomi H
Department Of Synthetic Chemistry And Biological Chemistry Graduate School Of Engineering Kyoto Univ
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Atomi Haruyuki
Laboratory Of Applied Biological Chemistry Department Of Synthetic Chemistry And Biological Chemistr
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Tanaka A
京都大学大学院工学研究科
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UEDA Mitsuyoshi
京都大学大学院工学研究科
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Takahashi Shouji
Laboratory Of Applied Biological Chemistry Department Of Synthetic Chemistry And Biological Chemistr
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Atomi Haruyuki
Dept. Synth. Chem. Biol. Chem. Grad. Sch. Eng. Kyoto Univ.:crest Imanaka Project
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Tanaka A
Mie Univ. Mie Jpn
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SCHMID Rolf
Institut fur Technische Biochemie, Universitat Stuttgart
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Tanaka A
Kao Corp. Wakayama Jpn
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Schmid R
Univ. Stuttgart Stuttgart Deu
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Takagi Atsuko
Department Of Etiology And Pathophysiology National Cardiovascular Center Research Institute
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Schmid Rolf
Institut Fur Technische Biochemie Universitat Stuttgart
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BEER HANS
Max-Planck-Institute for Biochemistry
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BORNSCHEUER UWE
Institute for Technical Biochemistry, University of Stuttgart
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Bornscheuer Uwe
Institute For Technical Biochemistry University Of Stuttgart
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Tanaka Atsuo
Laboratory Of Applied Biological Chemistry Department Of Synthetic Chemistry And Biological Chamistr
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Ueda Mitsuyoshi
Laboratory Of Applied Biological Chemistry Department Of Synthetic Chemistry And Biological Chemistr
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Ueda Mitsuyoshi
Laboratory Of Applied Biological Chemistry Department Of Synthetic Chemistry And Biological Chemistr
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Tanaka Atsuo
Laboratory Of Industrial Biochemistry Department Of Industrial Chemistry Faculty Of Engineering Kyot
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SCHMID Rolf
Institut für Technische Biochemie, Universität Stuttgart
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