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 pPICZalphaA vector and integrated further into the genome of Pichia pastoris GS115. One of the best transformants harboring the linearized plasmid pPalpha-BTL2 integrating into the P. pastoris genomic DNA was cultivated in a 5-L bioreactor filled with 4L of the culture medium BMMY. The BTL2 lipase was produced as an extracellular protein in large quantities of 309,000U/L supernatant. The lipase was purified using butyl-Sepharose with a specific activity of 23,000U/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 lambdaP(L), yielding 600U/g or 54,000U/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.
Quyen
Dinh Thi
DT
National Center of Natural Science and Technology, Institute of Biotechnology, 18 Hoang Quoc Viet Road, Nghiado, Distr. Caugiay, 10600 Hanoi, Viet Nam. quyen@ibt.ac.vn
Schmidt-Dannert
Claudia
C
Schmid
Rolf D
RD
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D003902:Detergents / Q000494:pharmacology; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012997:Solvents / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
D003902:Detergents; D011994:Recombinant Proteins; D012997:Solvents; D008049:Lipase