High-level expression of a lipase from Bacillus thermocatenulatus BTL2 in Pichia pastoris and some properties of the recombinant lipase

The BTL2 lipase gene from Bacillus thermocatenulatus was subcloned into the pPICZαA vector and integrated further into the genome of Pichia pastoris GS115. One of the best transformants harboring the linearized plasmid pPα-BTL2 integrating into the P. pastoris genomic DNA was cultivated in a 5-L bio...

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Bibliographic Details
Published in:Protein expression and purification 2003-03, Vol.28 (1), p.102-110
Main Authors: Quyen, Dinh Thi, Schmidt-Dannert, Claudia, Schmid, Rolf D
Format: Article
Language:English
Subjects:
Bacillus - enzymology
Bacillus - genetics
Bacillus thermocatenulatus
Detergents - pharmacology
Enzyme Stability
Expression
Gene Expression
Hydrogen-Ion Concentration
Lipase
Lipase - biosynthesis
Lipase - genetics
Lipase - isolation & purification
Lipase - metabolism
Pichia - genetics
Pichia pastoris
Plasmids - genetics
Properties
Recombinant Proteins - biosynthesis
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Sequence Analysis, DNA
Solvents - pharmacology
Substrate Specificity
Temperature
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Summary:The BTL2 lipase gene from Bacillus thermocatenulatus was subcloned into the pPICZαA vector and integrated further into the genome of Pichia pastoris GS115. One of the best transformants harboring the linearized plasmid pPα-BTL2 integrating into the P. pastoris genomic DNA was cultivated in a 5-L bioreactor filled with 4 L of the culture medium BMMY. The BTL2 lipase was produced as an extracellular protein in large quantities of 309,000 U/L supernatant. The lipase was purified using butyl-Sepharose with a specific activity of 23,000 U/mg protein towards tributyrin. The pure enzyme was characterized and its physicochemical properties were compared to those of the BTL2 lipase, which had previously been expressed in Escherichia coli under the control of its native promoter on pUC18 or under the control of the strong temperature inducible promoter λP L, yielding 600 U/g or 54,000 U/g wet cells, respectively. The three proteins showed the same N-terminal sequence and had very similar pH optimum, pH stability, temperature optimum, thermostability, and substrate specificity profiles. Three enzymes were extremely stable in the presence of several organic solvents and detergents.
ISSN:1046-5928
1096-0279
DOI:10.1016/S1046-5928(02)00679-4