Preparation, Modification And Application Of Geotrichum Candidum Lipase

Nonaqueous biocatalysis with lipase has advantages over aqueous biocatalysis owing to its multiple reaction types. For a given lipase-catalyzed transformation process, it is very important to obtain suitable and effective enzyme preparations, since the catalytic activity and enzyme stability are key factors affecting biocatalysis efficiency. The major obstacles restricting lipase application in non-aqueous biocatalysis are low reaction rates and poor stability. Modifying or improving enzyme preparations with desirable catalytic properties through directed evolution or physical/chemical methodology has been a focus of recent research. In this study, constructions of recombinant strains for producing clear background Geotrichum candidum lipase were performed. Based on these, double imprint molecule bioimprinting coupled to other methods, as well as novel immobilization techniques including organic treatment, cross-linked enzyme aggregates (CLEAs), protein-coated micro-crystals (PCMCs) were employed to modify free lipase. These modified lipase preparations were successfully applied in esterification of oleic acid and methanol and hydrolysis of fish oil for enrichment of PUFAs. These approaches provided reference for the preparation of other enzyme biocatalysts by upgrading crude enzymes to refined biocatalysts with high activity and stability.1. Based on my previous work for master degree, which mainly focused on cloning of Geotrichum candidum lipase gene, constructions of recombinant strains of Pichia pastoris with pPIC9K, pPICZaA, pGAPZaA and GAP-pPIC9K were further performed here. The results showed that pPIC9K vector was favourable for expression of Geotrichum candidum lipase. Recombinant lipase was produced, purified and characterized. Characterization of the properties showed that the lipase exhibited maximum activity at 40-50?and pH 8.0, and was fairly stable between pH 6.0-10.0 and below the temperature 60?. The lipase was compatible with the presence of organic solvents such as n-heptane, hexane, cyclohexane, benzene and diethyl ether. The lipase showed a notable hydrolysis preference for vegetable oils and triacylglycerol substrates containing cis-9 unsaturated fatty acid. The lipase also exhibited a good hrdrolysis activity towards a wide range of natural oils. The above properties indicate that the lipase is a promising candidate for applications in biocatalysis.2. Geotrichum candidum lipase with enhanced activity and operational stability was prepared for use in esterification of oleic acid and methanol for the first time. A combined strategy comprising bioimprinting with dual imprint molecules and a co-solvent coupled to pH tuning, KCl salt activation, lecithin coating and immobilization on macroporous resin effectively enhanced the activity and operational stability of Geotrichum candidum lipase. The modified lipase enhanced 18.4-fold esterification activity towards methyl oleate synthesis, and retained 90% relative conversion by repeated usage of 10 times.3. Based on conventional immobilization, organic solvent pretreatment before immobilization and organic solvent treatment after immobilization, as well as cross-linking of enzyme aggregates and protein-coating were introduced. For reaction of esterification of oleic acid and methanol, compared to immobilized lipase in buffer, polar isopropanol and propanol treatment after immobilization gave higher esterification activity. Isopropanol-buffer pretreatment and octane-buffer pretreatment before immobilization gave higher esterification activity than immobilized lipase in buffer. Especially, the effect of octane-buffer pretreatment was more obvious. PEI-CLEAs and PCMCs also showed better biocatalysis efficiency (esterification activity, activity recovery and operational stability) than free lipase in esterification reaction of oleic acid and methanol.4. Based on non-aqueous modifications, adjustment of modification methods was performed. Namely, fish oil treatment substituted for oleic acid bioimprinting, and coupled to immobilization were carried out, and then applied these modified lipases into hydrolysis of fish oil successfully. For free lipase, being liable to agglomeration in reaction medium was overcome by immobilization. Interfacial activation was introduced by immobilization in octane-buffer mixture. Substrate-binding pockets were activated by treatment of fish oil. These modification procedures resulted in different enhancement in initial reaction rate and hydrolysis degree. For hydrolysis of fish oil, free lipase without any modification only gave 12% of hydrolysis degree. Lipase immobilized in buffer and immobilized in octane-buffer mixture gave 28% and 31% of hydrolysis degree, respectively. Lipase immobilized in buffer coupled to treatment by fish oil gave 36% of hydrolysis degree, while lipase immobilized in octane-buffer mixture coupled to treatment by fish oil gave 40% of hydrolysis degree. Initial reaction rate and hydrolysis degree by immobilized lipase coupled to treatment by fish oil were both higher than those of immobilized lipase coupled to bioimprinting using oleic acid as imprint molecule. However, modified by immobilization and treatment of fish oil stepwisely or simultaneously had no significant effect on initial reaction rate and hydrolysis degree. Strong polar and hydrophobic solvents had negative impact on immobilization-fish oil treatment lipase, low polar solvents were helpful to maintain the modification effect of immobilization-fish oil treatment lipases. After 5 batch of usage, the immobilization-fish oil treatment lipases still maintained more than 80% of relative hydrolysis degree.5. Cross-linking of enzyme aggregates was applied into hydrolysis of fish oil successfully. Based on acetone precipitation, PEI and glutaraldehyde and enzyme interaction, stable cross-linked enzyme aggregates PEI-CLEAs was prepared. PEI-CLEAs had more excellent temperature and organic solvent tolerance than free lipase and CLEAs, which could maintain more than 65% of hydrolysis degree in the temperature range of 50-55?, and maintain more than 85% of hydrolysis degree after being treated by acetone, tertiary butanol and octane. PEI-CLEAs increased hydrolysis degree to 42%. After 5 batch reactions, PEI-CLEAs still maintained more than 72% of relative hydrolysis degree. PEI-CLEAs had advantages over CLEAs and free lipase in initial reaction rate, hydrolysis degree and operational stability.6. Protein-coating was applied into hydrolysis of fish oil successfully. In the temperature range of 45-50?, PCMCs showed better thermostability than free lipase, and retained at least 86% of relative hydrolysis degree after treatment by acetone, octane, tertiary butanol and propanol. PCMCs increased hydrolysis degree to 48%. After continuous usage of 5 batches, PCMCs still maintained more than 52% of relative hydrolysis degree.7. For hydrolysis of fish oil, lipase preparations modified by immobilization-fish oil treatment, aggregation and cross-linking, and enzyme coating exhibited different modification effect. Different modification methods gave different degree of initial reaction rate, hydrolysis degree, required time of achieving the highest hydrolysis degree, thermostability, organic solvent tolerance and operational stability. Different lipase preparations showed advantage in different aspects, thus, when being applied, special lipase preparation matches specific target and condition.