Impact Of Peptide Linkers On T.Maritima Lipase Tm1350 Displayed On B.Subtilis Spore Surface Using CotB As Fusion Partner

Industrially used enzymes are the key sources for the development of foods,pharmaceuticals,chemical and other products,used during bio-catalytic reactions.They are enantio-,sterio-and region specific,and have the ability to recognize the substrate in a simple media such as water rather than specific media,such as organic solvents and other solutions.Lipases are ? or ? hydrolase folding protein,well known as fat splitter.Lipases from thermophilic organisms have astonishing features for industrial bioprocesses because of tolerating high temperatures.However the stability and expression of these enzymes can be improved by displaying it on the surface of microbial spores.Spore surface display is the most desirable with enhanced effects,low cost,less time consuming and the most promising technology for environment,medical and industrial development.Spores have various applications in industry,due to the ability of survival in harsh industrial processes including heat resistance,alkaline tolerance,chemical tolerance,easy recovery and reusability.Yeast and bacteria,including Gram positive and negative are the most frequently used organisms for the display of various proteins(eukaryotic and prokaryotic),but unlike spores,due to nutritive properties,susceptibility to heat,pH and chemicals they can rupture easily.Hence spores are the best choice to avoid these problems and has various applications over nonspore formers due to amenability for laboratory purposes.Various strains of Clostridium and Bacillus are spore formers,but the suitable choice for display is Bacillus subtilis because,according to WHO,it is safe to humans and considered as “GRAS”(generally recognized as safe).Spore surface display technology has various applications towards industries,vaccine development,environment and peptide libraries construction,with cell surface display for enhanced protein expression and high enzymatic activity.Different vectors,cote proteins and statistical analyses can be used for linker selection(fusion protein construction)to obtain greater expression and high activity of the displayed protein.Fusion protein constructs with suitable peptide linkers have the ability to provide prolonged conformation,extended stability and activity to the displayed protein.In this study a series of fusion between Tm1350 and CotB comprising of several peptide linkers,with different length,flexibility and orientations were constructed.The lipase Tm1350 with different peptide linkers and CotB genes were PCR amplified using T.maritima and Bacillus subtilis chromosomal genome as template respectively.The desired genes were inserted in E.coli-B.subtlis shuttle vector PHS.The recombinant plasmid PHS-CotB-Lx-Tm1350 was transferred to B.subtilis and sporulationwas inducted.The spores were purified and the expression of CotB-Lx-Tm1350 was confirmed through western blot analysis.Effects of various degree of temperature(ranging from 40-80 oC),different pH(ranging from 4.0-10)and different solvents were examined on the enzyme activity of spore surface displayed enzyme with various peptide linkers.The optimum temperature for all the fusion constructs were 75 oC whereas the optimum pH for L8 and L10 were(pH 8.5),for L0,L5,L6 and L9 have(pH 9.0)while for L1,L2,L3,L4 and L7 it was(pH 9.5).The fusion protein with longer flexible linkers L9(GGGGS-GGGGSGGGGS-GGGGS)and L7(GGGGS-GGGGS-EAAAK-EAAAK-GGGGS-GGGGS)possess1.29 and 1.16 fold higher activity than original,under optimum temperature and pH respectively.Moreover,L3(EAAAK-GGGGS)and L9(GGGGS-GGGGS-GGGGS-GGGGS)were the most thermo-stable linkers,maintaining 1.40 and 1.35 fold higher activity than original respectively,at 80 oC after 5 hours of incubation.The enzyme activity of linkers with different orientation and length was determined.L5,L6 and L7 contain 30 amino acid residues with different orientation where L7 maintained 1.05 and 1.27 fold higher activity than L6 and L5.Effect of 0.1% proteinase K,Bromelain,20% ethanol and 30% methanol was investigated.Linkers with appropriate Glycine residues(flexible)showed higher activity than Alanine residues(rigid).In summary,the activity of displayed enzyme can be improved by maintaining orientation and flexibility of peptide linkers up to some extent which would be the most promising technology for industrial applications.