Entrapment of Rhizopus oryzae lipase displayed on Saccharomyces cerevisiae surface as whole cell biocatalyst for biodiesel synthesis

摘要

In this work, the whole cell biocatalyst of Rhizopus oryzae lipase (ROL), a versatile lipase for biodiesel production, was successfully developed through surface display technique using a-agglutinin 2 (Aga2) as an anchor protein and Saccharimyces cevevisiea as anchor yeast cell. The enzyme properties of displayed ROL were systematically investigated. Results showed that the maximal value of olive oil hydrolysis activity of displayed ROL reached 78.9 ± 2.1 U/g dried cells. It retained 93% original activity at 50 °C for 1 h incubation, and exhibited relatively high stability with remaining > 65% of original activity at pH 7.0 and 8.0. Interestingly, Ca~(2+) could significantly enhance the displayed ROL activity by more than 1.6-fold, while other metal ions (Na~+, K~+, NH_4~+, Mg~(2+), Ca~(2+), Zn~(2+), Cu~(2+), Mn~(2+), Al~(3+) and Fe~(3+)) showed no effects on its activity. The displayed ROL also showed higher tolerance abilities towards organic solvents and detergents in comparison with the native free ROL. Reaction kinetics showed that the parameter Vmax of the displayed ROL was calculated to be 1.62 mol/L/min, and K_m values were 14.65 mol/L for methanol substrate and 1.18 mol/L for soybean oil substrate, respectively. To further improve its operational stability for biodiesel production from waste cooking oil, the displayed ROL was respectively entrapped with sodium alginate (SA), sodium alginate-boric acid (SA-BA), polyvinyl alcohol-alginate-boric acid (PVA-SA-BA), and sodium alginate-glutaraldehyde (SA-GA). The highest biodiesel yield of 81.2% was obtained by the displayed ROL entrapped with SA-GA, and retained 85% of original activity after 7-batch recycles.