Improved expression and immobilization of Geobacillus thermoleovorans CCR11 thermostable recombinant lipase

Abstract

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 β-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 × 10 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.

Lipase catalysis in organic solvents: advantages and applications

“…In the last decade some other organic solvent-tolerant lipases and esterases have been isolated from various microorganisms such as Pseudomonas spp., Burkholderia cepacia strains, Bacillus spp., thermophilic archaea, fungi and yeast [ 56 ]. For example P. aeruginosa LST-03, B. cepacia ST-200 [ 25 ], Bacillus sphaericus 205y [ 57 ], Staphylococcus saprophyticus M36 [ 24 ] and Geobacillus thermoleovorans CCR11 [ 58 ] were also reported to be organic solvent-tolerant bacteria.…”

“…Many enzymes are easily denatured and inactivated in the presence of organic solvents. Therefore, protein engineering and several physical and chemical methods, such as immobilization, modification and entrapment for stabilizing enzymes in the presence of organic solvents have been developed [ 58 – 60 ]. Literature survey revealed that extracellular enzymes secreted by organic solvent-tolerant microorganisms for their growth are stable in the presence of organic solvents.…”

Advances in Recombinant Lipases: Production, Engineering, Immobilization and Application in the Pharmaceutical Industry

“…In the latter method, the formation of covalent bonds can be achieved through amide, ether, thio-ether or carbamate bonds between the support and the enzyme [124][125][126] The immobilization of an enzyme by adsorption is a simple technique with high commercial value due to its simplicity, regular use in large-scale processes, low cost, retention of high enzyme activity, and relatively chemical-free enzyme binding [127]. Supports with different degrees of hydrophobicity were previously used such as butyl Sepabeads and octadecyl Sepabeads [126], decaoctil-Sepabeads [128], macroporous resin HPD826 [129], polypropylene powder [124,130,131] and pore-expanded mesoporous silica (SBA-15) [132]. The use of hydrophobic supports is of particular interest, because these supports mimic the enzymes' natural media and can often promote hyperactivation, highly selective adsorption, purification, increased enantioselectivity, and strong but reversible immobilization, making support reuse possible after the enzyme has been deactivated [133,134].…”

Production and Characterization of Cross-Linked Aggregates of Geobacillus thermoleovorans CCR11 Thermoalkaliphilic Recombinant Lipase

“…In a previous study, LipMatCCR11 was used in lyophilized form for the synthesis of flavors and fragrance esters with hexane as a reaction medium [3]. Later, using response surface methodology, conditions for maximum expression were investigated and a 1300-fold increase in lipolytic activity was achieved; this enzyme was also immobilized in Accurel EP-100 and used to synthetize aromatic esters [34]. The aims of the present work were to establish the best conditions to produce cross-linked aggregates of the recombinant thermoalkaliphilic lipase LipMatCCR1 using ammonium sulfate as precipitation agent and glutaraldehyde as cross-linking agent, and to characterize the immobilized product.…”