Research About The Immobilization Of Thermomicrobium Roseum Lipase Recombinant Expressed In Pichia Pastoris

Lipase is a highly efficient biocatalyst with a wide range of applications in different industrial fields such as pharmaceuticals,food and fine chemicals.However,requirements for industrial manufacturing,such as high activity,stability,and recyclability,are generally could not be fully fulfilled by the available lipase.Hence,it is desirable to obtain lipase with good properties and to investigate a simple,fast and effective immobilization method so as to improve the application range of lipase.In this study,the heterogeneous expression of lipase gene derived from the archaea Thermomicrobium roseum in Pichia pastoris was completed via genetic engineering technology.A series of immobilization method was investigated for the purpose to improve the enzymatic activity and stability of the synthesized lipase.The main research contents and results are as follows:（1）Heterogeneous expression of lipase derived from the archaea Thermomicrobium roseum in Pichia pastorisThe recombinant expression vector p Pic9k was constructed to integrate the lipase gene into the Pichia pastoris GS115 chromosomal genome by electroporation.Positive transformants containing different lipase gene copy numbers were screened by geneticin plates with concentrations ranging from low to high(0.5 mg·m L^-1～4 mg·m L^-1),and a total of 8 strains were obtained.To confirm the number of copies in the bacterial genome,the standard plasmids PT-GAPDH and PT-lip of the internal reference gene and lipase gene were constructed respectively.After establishing a standard curve of copy number and Ct value by RT-q PCR,the number of copies of the lipase gene was found to vary from 2 to 26.The lipase activity was then measured by having the eight strains fermented and induced.By comparing the tested enzymatic activity,it was observed that when the number of copies of the lipase gene in the genome was 13,the activity reached a maximum of 0.0284 U·m L^-1.Based on the above result,the identified strain was further cultured for fermentation and induction.Showing on SDS-PAGE,a single band with a molecular weight of 36.4 KDa,which was in consistent with the theoretical value,was obtained after purification by nickel column affinity chromatography.The specific enzyme activity of lipase was then measured as 1.05 U·mg^-1.The fermentation condition was optimized by determining the optimized fermentation duration,initial medium p H,induction temperature and methanol concentration.The results indicated that the enzyme activity of the supernatant(0.226 U·m L^-1)could be improved by 3.3 times when having the strain fermented and induced for 108 h at an induction temperature of 30°C by controlling the initial medium p H to be 8.0 and providing 2%methanol addition every 24 h.（2）Research about the preparation and characterization of inorganic thermophilic lipase hybrid nanometerIn this study,sonochemistry was introduced to have the free lipase hybridized with copper phosphate precipitation within 10 min.Scanning electron microscope（SEM）,X-Ray diffraction（XRD）and Fourier transform infrared spectroscopy（FT-IR）analysis were performed for the characterization of the Cu-Lip hybrid nanoflowers.Subsequently,the effect of metal ions,types of copper salt and reaction conditions on the enzymatic activity of immobilized lipase was investigated.The results indicated that Cu²⁺showed the best immobilization efficiency and the enzyme activity of Cu-Lip would be affected by the type of copper salts.The encapsulation ratio was 100%when using Cu SO₄ for immobilization,while the introduce of Cu Cl₂ was found to improve the relative enzyme activity to 145.7%.The catalytic efficiency(k_cat/km)of Cu SO₄-Lip and Cu Cl₂-Lip was measured as 106%and 134%referring to that of free lipase.In addition,the current immobilization strategy was verified with the potential to improve the storage stability by maintaining 80%relative activity after a storage of 30 days at room temperature.Moreover,the hybrid nanoflowers was found to exhibit 40%relative activity after five consecutive reactions.Overall,with providing promising method for the improvement of the applicability of lipase in industry,the current result also lays the foundation for its further application in the field of biodiesel synthesis,detergent and industrial production requiring high temperature.（3）Research about the preparation and characterization of lipase immobilized by hydrophobic materialsHerein,three hydrophobic materials,octyl agarose,phenyl agarose,and butyl agarose,were used for lipase immobilization.The measured loading capacity of phenyl agarose was found to reach 4.69 mg·g^-1（enzyme/Vector）with a relative enzyme activity of 110%compared with the free enzyme.After the characterization of the phenyl agarose immobilized enzyme by performing Scanning electron microscope（SEM）,Energy Dispersive Spectrometer（EDS）and X-ray photoelectron spectroscopy（XPS）analysis,the enzymatic properties were determined as well.It turns out that the optimal reaction temperature was improved to 95℃,which was 10℃higher than that of the free enzyme,while the optimal reaction p H was 9 but without a significant change of the p H tolerance capability.The storage stability was investigated by having the phenyl agarose immobilized enzyme stored at room temperature for 30 days.The remaining enzyme activity was measured to be 60%of original activity,which is significantly higher than that of the free enzyme（44%）.In addition,the phenyl agarose immobilized enzyme was found to maintain 50%of the enzyme activity after 9 consecutive uses.According to the above results,with obtaining thermophilic lipase with relative high enzyme activity via genetic engineering technology,the current research also identify possible immobilization method for improving the activity and stability of the obtained free enzyme,thereafter to provide meaningful theoretical foundation for enhancing the applicability of lipase in the field of industrial biocatalysts.