[Heterologous expression, purification and characterization of phospholipase C from Bacillus cereus in Kluyveromyces lactis].

Authors :: Xiao C
Zhang L
Li Y
Xin Y
Chen G
Yang S
Source :: Wei sheng wu xue bao = Acta microbiologica Sinica [Wei Sheng Wu Xue Bao] 2017 Jan 04; Vol. 57 (1), pp. 87-96.
Publication Year :: 2017
Abstract: Objective: In this study, we constructed recombinant Kluyveromyces lactis strains to produce phospholipase C (PLC) of Bacillus cereus. The recombinant enzymes were purified and characterized.<br />Methods: We cloned the PLC encoding gene bcplc of Bacillus cereus. And the amplified fragments were inserted into pKLAC1 to obtain expression plasmids. K. lactis harboring the above plasmids was cultivated to express PLC that was purified by HisTrapTM affinity chromatography and characterized.<br />Results: PLC of B. cereus was cloned and expressed in K. lactis. The recombinant enzyme had shown activity of 19251 U/mg when using p-nitrophenyl phosphorycholine as substrate. Purified PLC exhibited optimum temperature at 80 °C and optimal pH at 9.0. The recombinant enzyme was stable below 40 °C and pH between 7.0 and 8.0. Cu2+ and Co2+ inhibited its activity whereas Zn2+, Mn2+, Ca2+ and Mg2+ stimulated its activity.<br />Conclusion: It is the first time to express and characterize the PLC gene in K. lactis. These research results provide reference for the study of recombinant PLC.

Subjects :: Bacterial Proteins genetics
Bacterial Proteins metabolism
Cloning, Molecular
Enzyme Stability
Hydrogen-Ion Concentration
Kluyveromyces metabolism
Plasmids genetics
Plasmids metabolism
Temperature
Type C Phospholipases genetics
Type C Phospholipases metabolism
Bacillus cereus enzymology
Bacterial Proteins chemistry
Bacterial Proteins isolation & purification
Gene Expression
Kluyveromyces genetics
Type C Phospholipases chemistry
Type C Phospholipases isolation & purification

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