| Nitrilase, as one kind of valuable industrial enzyme, was utilized to enzymatic conversion of toxic nitrile compounds directly to corresponding non-toxic and highvalue acids with liberating ammonia, which has been obtained wide application. That nitrilase-mediated biocatalytic reactions for preparation of some carboxylic acids has replaced some conventional chemical approachs because it provides an ecofriendly alternative allowing clean and mild synthesis combined with high yield and selectivity. In recent years, the application researches of nitrilase are mainly subject to the factors: the bad anti-resistance, few species, narrow substrate spectrum and others. So screening for more excellent nitrilases still remains huge significance and focused on by researchers. B.subtilis spore surface display technology has emerged as a novel immobilization method for protein and aroused wide attention. However, few literatures were reported using spore surface display technology for immobilized anti-resistance chemical enzymes. The B.subtilis spores possess high stability and robust resistance, which made B.subtilis spore an excellent vehicle for displaying anti-resistance chemical enzyme on its surface. This thesis firstly expressed and characterized the hyperthermophiles Thermotoga maritima MSB nitrilase and displayed it on the spore surface of B.subtilis, and then preliminarily optimized the linker between the coat protein and the nitrilase.In the present study, the nitrilase gene from Thermotoga maritima MSB nitrilase was firstly amplified, and then was inserted into the prokaryotic expression vector PET-28 a, the constructed recombinant plasmid PET-28a-nit was transformed into E.coli BL21, followed by SDS-PAGE analysis after IPTG induction. The molecular mass was about 30 k Da, which was consistent with the predicted molecular mass of the protein.The properties of Thermotoga maritima MSB nitrilase was investigated. The optimal temperature and p H of the purified enzyme were 45°C and 7.5, respectively when 3-cyanopyridine was used as the substrate. The thermal stability experiments indicated that this nitrilase could retain nearly 50% residual activity after incubation at 75 °C for 0.5h. The p H stability experiments indicated that this nitrilase was sensitive for alkaline condition. Mn2+, Zn2+, and Cu2+ could obviously inhibit the nitrilase activity. Mg2+ and Fe2+ could slightly enhance the nitrilase activity. Other metal ions and chelating reagent EDTA had no significant inhibitory effects on enzyme activity. The reductant DTT could somewhat increase the nitrilase activity. Some other reductants, surfactant, protease inhibitor and organic solvents all inhibited the nitrilase activity to some degree. The kinetic constants Vmax and Km of this nitrilase were 3.12 μmol/min/mg and 7.63 m M, respectively. The catalytic efficiency kcat/Km was 0.44 /m M/s toward succinonitrile. Substrate spectrum experiments indicated this nitrilase was specific to aliphatic dinitriles.Bacillus spores were used as a matrix for displaying nitrilase. An E. coli– B. subtilis shuttle vector(p HS-Cot G-nit) was constructed. The successful display of Cot Gnit fusion protein on the spore surface of B. subtilis was verified by western blot analysis and activity measurement. Namely, the nitrilase immobilization. The optimal temperature and p H of dispalyed nitrilase were observed to be 50°C and p H 8.0, respectively, which were higher than the free nitrilase. The displayed nitrilase retained 79% and 97% at 75°C and p H 8.0 after 1 h of incubation, whereas it were 32% and 52%, respectively, for free nitrilase. Furthermore, the reusability experiments indicated that the activity of displayed nitrilase could retain 83% of the initial activity at the fifth cycle.This study preliminarily investigated the effects of various linkers on the spore surface-displayed nitrilase. The results showed that the optimal temperature and p H of displayed nitrilase containing linkers L0, L3 and L4, L4, L5, L6, L9 were 45 ℃ and 7.5, respectively, L2, L10, L11 were 50 ℃ and 8.0, L8(GGGGSEAAAKGGGGS) were 55 ℃ and 8.5. After 3 h of incubation at 85 ℃, the residual activity of L8 was the highest and reached to 19.5%, and L0 without linker was 7.3%, the former is almost 3 times of the latter, so the excellent linker for thermal stability was L8. After 3h of incubation in p H9.0, the residual activity of L8 was the highest and reached to 21.5%, and L0 without linker was 11.3%, the former is almost the latter 2 times, so the excellent linker for alkali stability was L8. In the presence of 1 m M DTT, 1% v/v SDS and 20% v/v ethanol, the excellent linkers for stability were MGSSN, GGGGSEAAAKGGGGS and(GGGGS)3, respectively.This study provides a novel method for the anti-resistance nitrilase and some other chemical enzymes about the immobilization on the spore surface and optimization on the linkers so as to improve the immobilization effect, which would meet the demand for industrial biocatalyst under harsh environment. |
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