Enzymatic Properties And Immobilization Of Cold-adapted Lipase From Marine Bacteria Exiguobacterium Sp.

Lipase is a versatile enzyme that catalyzes various biological reactions,including hydrolysis,alcoholysis,acidolysis,and esterification.However,free lipase has poor activity,stability,and recyclability,which have limited its wide spread use in industrial production.This study aimed to analyze the existing issues associated with lipase and provide a theoretical basis for overcoming these limitations by applying different strategies.The main contents and results of this study are as follows:（1）In this study,a lipase was screened from Exiguobacterium sp.and expressed it through recombination.The recombinant lipase was purified and named EaLIP27,with a molecular weight of 27 k Da.The enzymatic properties of the lipase were studied,and the results showed that it was a cold-resistant lipase with an optimum temperature of 35℃and an optimal pH of 8.0,and it had a wide pH stability range.Bioinformatics was used to predict the structure of the lipase,and low-temperature activity was further confirmed.The optimal substrate for the lipase was p-nitrophenyl butyrate.The adaptation of the recombinant lipase to different metal ions was also determined,showing that Fe³⁺and Fe²⁺strongly inhibited its activity,while Ca²⁺,K⁺,Na⁺,and Mg²⁺increased its activity by 1.1-fold.The effect of organic solvents on the stability of the lipase was studied,and the results showed that n-hexane and iso-octane increased its activity,while methanol and n-butanol decreased its activity by 50%.In the presence of surfactants,the hydrolytic activity of the lipase was positively promoted,making it a potential application in detergents.（2）In this study,hydrophobic modified magnetic polydopamine nanoparticles were prepared and used in lipase-Pickering emulsifier to make olive oil enzyme interpret fatty acids.Experimental results showed that the nanoparticles had a core-shell structure with a Zeta potential of-39.2667 m V,an average size of 605.8±21.54 nm,and a contact angle of55.9°.By adding solid particles to a lipase solution and immobilizing them,a Pickering emulsion was constructed.The optimal stability conditions were found to be a magnetic polydopamine nanoparticle concentration of 0.05 g/100 m L,maintaining an oil-to-water ratio of 1:9,and applying ultrasonic power at 500W for a duration of 10 min.Microscopic results showed that the emulsion droplet size was smaller and more evenly distributed after10 min of ultrasound treatment.Lipase was adsorbed at the oil-water interface to form enzyme-Pickering emulsions for olive oil hydrolysis.Compared with non-emulsions,the release of fatty acids increased by 1.2 times.This study provides a new immobilization method for lipase interface catalysis and a theoretical basis for the application of Pickering emulsions.（3）This study is based on magnetic polydopamine nanoparticles,which are covalently assembled with oxidized polysaccharides to synthesize a fixed material with flexibility,biocompatibility,and biomimetic adhesion.Various techniques were used to characterize the nanocomposite materials,and magnetic polydopamine nanoparticles modified with dialdehyde chitosan were used as carriers to immobilize lipase and improve its performance.The results showed that the optimal immobilization conditions for lipase at a concentration of 10 mg/m L were 50℃,pH 7.0,and 2 h of immobilization,with a maximum immobilization efficiency of 66.8%.Compared with free enzymes,immobilized enzymes have higher acid and alkali resistance and thermal stability,making them more adaptable to reaction environments.After incubating at 50°C for 180 minutes,the relative enzyme activity recovery of immobilized lipase was 75.3%.Immobilized enzymes were reused 8times,with a recovery rate still reaching 79%.This magnetic biocomposite material composed of natural compounds provides a new strategy for immobilizing enzymes.