Comparative features between recombinant lipases CALA-like from U. maydis and CALA from C. antarctica in thermal stability and selectivity
- 125 Downloads
Ustilago maydis lipase A (UMLA) expressed in Pichia pastoris was compared with Candida antarctica lipase A (CALA) to study its biochemical properties such as thermostability and selectivity.
UMLA had similar behavior to its homologue CALA regarding the effect of pH and temperature on enzymatic activity, substrate preference and selectivity. Both lipases were active on insoluble triglycerides as well as natural oils and hydrolyzed preferably esters with short and medium acyl and alkyl chains. Both enzymes were slightly selective for the (S)-glycidyl butyrate enantiomer and had a remarkable preference for the sn-2 position of triglycerides. The optimal activity was 40 and 50 °C for UMLA and CALA, respectively. However, temperature had a greater effect on the stability of UMLA compared to CALA, observing a half-life at 50 °C of 2.07 h and 12.83 h, respectively.
UMLA shares some biochemical properties with CALA such as the sn-2 preference on triglyceride hydrolysis and transesterification. However, the high thermostability attributed to CALA was not observed in UMLA; this can be due to the lack of stabilization via AXXXA motifs in helices and fewer proline residues at the surface.
KeywordsCandida antarctica Lipase Pichia pastoris sn-2-Selectivity Thermostability Ustilago maydis
Maria Marcela Robles-Machuca acknowledges the Doctoral fellowship received from The National Council of Science and Technology (CONACYT). This work was funded by SEP-CONACYT (242544-2014). The authors thank Ean Hundley from Peace Corps for revising the English manuscript.
Supplementary Table 1—Purification steps of the recombinant lipases. The lipolytic activity was monitored by PHIBLA method at 37 °C for 15 min using TG (8:0) for UMLA and TG (4:0) for CALA as substrates.
Supplementary Fig. 1—UMLA and CALA purity analysis by SDS-PAGE. a. Proteins in cell-free culture (line 1), ammonium sulfate precipitation (line 2), eluted UMLA fraction from Butyl-sepharose (line 3). b. Eluted CALA fraction from CM-Sepharose column (line 2). Molecular weight marker was Low range, Bio Rad (line 4a and 1b). SDS-PAGE (12%) gel under reducing conditions. Coomassie brilliant blue was used to stain the proteins.
Supplementary Fig. 2—Mass spectrum of recombinant UMLA (a) and CALA (b) proteins. Both enzymes after purification were analyzed by MALDI-TOF mass spectrometry. Intensity values were normalized.
Supplementary Fig. 3—Surface representation of UMLA (a) and CALA (b) structures. The structures are graphically depicted as the 180° rotated view, with the yellow surface corresponding to active site, the exposed prolines residues in red, and the sequence exposed AXXXA motifs in purple. Based on the crystallographic structure of UMLA (PDB ID: 3ZPX) and CALA (PDB ID: 2VEO).