In silico and experimental characterization of chimeric Bacillus thermocatenulatus lipase with the complete conserved pentapeptide of Candida rugosa lipase.

Mostafa Hosseini, Ali Asghar Karkhane, Bagher Yakhchali, Mehdi Shamsara, Saeed Aminzadeh, Dena Morshedi, Kamahldin Haghbeen, Ibrahim Torktaz, Esmat Karimi, Zahra Safari

Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology-NIGEB, P.O. Box 14965/161, Tehran, Iran.

Applied biochemistry and biotechnology 2013 Feb

filter terms:

Lipases are one of the highest value commercial enzymes as they have broad applications in detergent, food, pharmaceutical, and dairy industries. To provide chimeric Bacillus thermocatenulatus lipase (BTL2), the completely conserved pentapeptide (¹¹²Ala-His-Ser-Gln-Gly¹¹⁶) was replaced with similar sequences (²⁰⁷Gly-Glu-Ser-Ala-Gly²¹¹) of Candida rugosa lipase (CLR) at the nucleophilic elbow region. For this purpose, three mutations including A112G, H113E, and Q115A were inserted in the conserved pentapeptide sequence of btl2 gene. Based on the crystal structures of 2W22, the best structure of opened form of the chimeric lipases were garnered using the MODELLER v9.10 software. The native and chimeric lipases were docked to a set of ligands, and a trial version of Molegro Virtual Docker (MVD) software was used to obtain the energy values. Docking results confirmed chimeric lipase to be better than the native lipase. Following the in silico study, cloning experiments were conducted and expression of native and chimeric btl2 gene in Pichia pastoris was performed. The native and chimeric lipases were purified, and the effect of these mutations on characteristics of chimeric lipase studied and then compared with those of native lipase. Chimeric lipase exhibited 1.6-fold higher activity than the native lipase at 55 °C. The highest percentage of both lipases activity was observed at 60 °C and pH of 8.0. The ion Ca²⁺ slightly inhibited the activity of both lipases, whereas the organic solvent enhanced the lipase stability of chimeric lipase as compared with the native lipase. According to the results, the presence of two glycine residues at the conserved pentapeptide region of this chimeric lipase (¹¹²Gly-Glu-Ser-Ala-Gly¹¹⁶) may increase the flexibility of the nucleophilic elbow region and affect the enzyme activity level.

Citation

Mostafa Hosseini, Ali Asghar Karkhane, Bagher Yakhchali, Mehdi Shamsara, Saeed Aminzadeh, Dena Morshedi, Kamahldin Haghbeen, Ibrahim Torktaz, Esmat Karimi, Zahra Safari. In silico and experimental characterization of chimeric Bacillus thermocatenulatus lipase with the complete conserved pentapeptide of Candida rugosa lipase. Applied biochemistry and biotechnology. 2013 Feb;169(3):773-85