ABSTRACT
Immobilization of enzymes has many advantages for their application in biotechnological processes. In particular, the cross-linked enzyme aggregates (CLEAs) allow the production of solid biocatalysts with a high enzymatic loading and the advantage of obtaining derivatives with high stability at low cost. The purpose of this study was to produce cross-linked enzymatic aggregates (CLEAs) of LipMatCCR11, a 43 kDa recombinant solvent-tolerant thermoalkaliphilic lipase from Geobacillus thermoleovorans CCR11. LipMatCCR11-CLEAs were prepared using (NH4)2SO4 (40% w/v) as precipitant agent and glutaraldehyde (40 mM) as cross-linker, at pH 9, 20 °C. A U10(56) uniform design was used to optimize CLEA production, varying protein concentration, ammonium sulfate %, pH, glutaraldehyde concentration, temperature, and incubation time. The synthesized CLEAs were also analyzed using scanning electron microscopy (SEM) that showed individual particles of <1 µm grouped to form a superstructure. The cross-linked aggregates showed a maximum mass activity of 7750 U/g at 40 °C and pH 8 and retained more than 20% activity at 100 °C. Greater thermostability, resistance to alkaline conditions and the presence of organic solvents, and better durability during storage were observed for LipMatCCR11-CLEAs in comparison with the soluble enzyme. LipMatCCR11-CLEAs presented good reusability by conserving 40% of their initial activity after 9 cycles of reuse.
Subject(s)
Bacterial Proteins/chemistry , Geobacillus/enzymology , Lipase/chemistry , Protein Aggregates , Bacterial Proteins/genetics , Cross-Linking Reagents/chemistry , Enzyme Stability , Geobacillus/genetics , Lipase/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
Production of recombinant thermo-alkali-stable lipase LipMatCCR11, expressed in Escherichia coli BL21 (DE3), was investigated via response surface methodology by using a face-centered design with three levels of each factor. Additionally, improvement of the catalytic performance of expressed lipase was assessed by immobilization on microporous polypropylene. Results showed that inducer (isopropyl ß-d-1-thiogalactopyranoside [IPTG]) concentration and temperature were found to be the significant factors (P < 0.05). The maximum lipase expression was obtained at IPTG 0.6 mM, 16 °C, and 18 H, with a specific lipase activity of 7.29 × 106 U/mg, which was 36.4 times higher (over 1,300-fold increase) than lipase activity measured under nonoptimized conditions. On the other hand, immobilized lipase showed a high biocatalytic activity, particularly in the synthesis of aroma esters.