RESUMO
In this study lipolytic biocatalysts GD-95RM, GDEst-95 and GDEst-lip were immobilized by encapsulation in calcium alginate beads. All three immobilized biocatalysts demonstrated significantly increased thermal stability at 60-70 °C temperatures and the activity of GD-95RM lipase increased by 50% at 70-80 °C following the immobilization. Moreover, encapsulated GDEst-95 esterase retained higher than 50% lipolytic activity after 3 months of incubation with butanol (25%) and ethanol (50%); GDEst-lip enzyme possessed 50% activity after 2 months of treatment with ethanol (25%) and methanol (25%); and GD-95RM lipase displayed higher that 50% activity after two-week incubation with methanol (50%). All three immobilized enzymes displayed long-term storage capability (>50% activity) at least until 3 months at 4 °C. It was also detected that immobilized GD-95RM and GDEst-lip can perform flow hydrolysis of both avocado oil and p-NP dodecanoate in prototype packed-bed column reactor. The analysis of continuous transesterification of avocado or sunflower oil with ethanol or methanol as substrates confirmed that encapsulated GD-95RM and GDEst-lip enzymes is a useful approach to produce fatty acid alkyl esters.
Assuntos
Geobacillus/enzimologia , Lipase/química , Lipase/metabolismo , Óleos de Plantas/química , Alginatos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Butanóis/farmacologia , Cápsulas , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Esterificação , Etanol/farmacologia , Meia-Vida , Temperatura Alta , Hidrólise , Ácidos Láuricos/química , Metanol/farmacologia , Persea/química , Óleo de Girassol/químicaRESUMO
The prospects of industrial uses of microbial enzymes have increased greatly during the 21st century. Fused lipolytic enzymes (where one or both fused domains possess lipolytic activity) is a rapidly growing group of industrial biocatalysts. However, the most effective fusion strategy, catalytic behavior of each domain and influence of added linkers on physicochemical and kinetic characteristics of such biocatalysts has not been yet explored. In this study the functionality of individual domains in fused lipolytic enzymes, while using GDEst-lip, GDLip-lip and GDEst-est enzymes as a model system, is analyzed for the first time. Analysis of mutant GDEst-lip, GDLip-lip and GDEst-est variants, where one domain is inactive, showed that both domains retained their activity, although the reduction in specific activity of individual domains has been detected. Moreover, experimental data proposed that the N-terminal domain mostly influenced the thermostability, while the C-terminal domain was responsible for thermal activity. GDEst-lip variants fused by using rigid (EAAELAAE) and flexible (GGSELSGG) linkers indicated that a unique restriction site or a rigid linker is the most preferable fusion strategy to develop new chimeric biocatalysts with domains of Geobacillus lipolytic enzymes.
Assuntos
Esterases/química , Geobacillus/enzimologia , Lipase/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Catálise , Estabilidade Enzimática , Esterases/metabolismo , Geobacillus/metabolismo , Cinética , Lipase/metabolismo , Lipólise , Especificidade por SubstratoRESUMO
This study presents a new microbial lipolytic enzyme GD-95RM designed via random mutagenesis using previously characterized GD-95 lipase as a template. The improvement in activity of GD-95 lipase was caused by E100K, F154V and V174I mutations. Compared with GD-95 lipase, the GD-95RM lipase had 1.3-fold increased specific activity (2000 U/mg), demonstrated resistance to higher temperatures (75-85 °C), had fourfold increased Vmax towards p-NP dodecanoate and showed 2.5-fold lower KM for p-NP butyrate. It retained > 50% of its lipolytic activity when hydrolyzing short, medium and long acyl chain substrates at 30 °C and 55 °C reaction temperatures after 20 days' incubation with 25% of ethanol. GD-95RM also displayed long-term tolerance (40 d) to 5% NaCl, trisodium citrate, sodium perborate, urea, 0.1% boric acid, citric acid and Triton X-100. Moreover, oil hydrolysis and transesterification results revealed the capability of GD-95RM lipase to produce fatty acids or fatty acid esters through eco-friendly hydrolysis and transesterification reactions using a broad range of vegetable and fish oils, animal fat and different alcohols as substrates. GD-95RM lipase was successfully applied in synthesis reactions for ethyl oleate, octyl oleate and isoamyl oleate without giving to use additional reaction compounds or special reaction conditions.