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Abstract

Objective: To obtain industrial standard lipase with high activity and stability, Butelase 1 ligase was used to connect the N- and C-termini of Thermomyces lanuginosus lipase (TLL), and then the protein characteristics, including heat stability, protease resistance, and enzymatic kinetics were analyzed. Methods: His6 affinity column was used to purify recombinant Butelase 1 (rButelase 1) and TLL (rTLL) from mammalian cells and E. co- li; rButelase 1 to was utilized connect the N- and C-termini of rTLL to generate circular rTLL (cTLL); the enzymatic activity, the heat stability, the protease resistance, the resistance to heat-induced precipitation, and the enzymatic kinetics were analyzed to determine the difference of rTLL and cTLL. Results: Recombinant rButelase 1 and rTLL expressed in mammalian cells and E. co-li were purified; cTLL was obtained, and the comparable enzymatic activity was found in both cTLL and rTLL. After heat treatment at 70 ℃ for 180 min, most of cTLL remained soluble and 95% of its activity, while rTLL was almost completely precipitated and lost most of its activity. cTLL has characteristics of stable proteins, such as uniform particle size and molecular weight distribution. Conclusion: Butelase 1 can effectively circularize lipase from T. lanuginosus. cTLL maintained its enzymatic activity. Meanwhile, its protein characteristics, including thermal stability and protease resistance, has also been improved.

Publication Date

10-16-2022

First Page

40

Last Page

43,54

DOI

10.13652/j.spjx.1003.5788.2022.60068

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