Overexpression And Characterization Of A Novel Cellulase (CTN1107) From Thermotoga Neapolitana:Application In Biomass Hydrolysis

Among different industrial applications of biomass,biofuel production is receiving increasing attention.Many factors influence the transformation of biomass into biofuels such as biomass composition,moisture content,and the desired final energy product.Among different methods,conversion of biomass to fermentable sugars by enzymatic hydrolysis offers the potential for higher yield,lower energy consumption,higher selectivity and eco-friendly process.In that regard,the stability of hyperthermophilic enzymes at extreme temperatures acquired them a unique potential for biomass conversion.In recent years,many trials were done to enhance the biological production and stability of thermostable enzymes.In addition to optimization of growth conditions and using chemical methods,many new genetic systems were developed.In the present study,hyperthermophilic cellulase?HTC?gene from Thermotoga neapolitana was isolated and expressed in E.coli using pHsh-T vector.The enzyme showed high activity at the maximum applied temperature and,therefore,95?was identified as the optimum temperature.The optimum pH was in the range of 6.0-6.5 with more stability in alkaline than acidic environments.In addition,the enzyme showed high thermostability when incubated at 95?,with maximum activity after 5 h of incubation.These superior properties of HTC strongly support the application of enzyme in cellulosic biomass hydrolysis for biofuel production or other industrial applications.The presence of lignin in lignocellulosic biomass and the need for de-polymerization of cellulose and hemicellulose into reducing sugars limit the efficiency of enzymatic hydrolysis for biomass conversion.Therefore,microalgae and cyanobacteria were discussed as a potential feedstock for biofuel(3^rd generation)due to the absence of lignin in their cell wall.Among different cyanobacteria,Spirulina platensis was discussed as a potential feedstock for different applications,including biofuel production.Therefore,the effect of static magnetic field?SMF?on Spirulina platensis biomass production,carbohydrates accumulation and its influence on cadmium ions(Cd²⁺)removal efficiency were studied.In addition,the effect of HTC on hydrolysis of lignin-free biomass?Spirulina platensis?grown under SMF and high concentrations of Cd²⁺was examined.Application of 6 h day^-1 SMF resulted in the highest significant biomass productivity of 0.198 g L^-1 day^-1.However,10 and 15 mg L^-1 of Cd²⁺resulted in significant reduction in biomass productivity by 8.8 and 12.5%,respectively,below the control.Combined SMF showed 30.1%significant increase in biomass productivity over the control.On the other hand,increase of initial Cd²⁺concentration resulted in significant reduction of Cd²⁺removal efficiency,representing 79.7%and 61.5%at 10 and 15 mg L^-1,respectively,after 16 days.Interestingly,application of SMF for 6 h day^-1 enhanced Cd²⁺removal efficiency counted by 91.4%and 82.3%after 20 days for cultures with initial Cd²⁺concentration of 10 and 15 mg L^-1,representing increase by 6.3 and 25.3%,respectively,over the SMF-untreated cultures.Moreover,application of SMF resulted in accumulation of carbohydrates with simultaneous enhanced growth,which led to pronounced increase in carbohydrate productivity over the untreated cultures.Fiber analysis showed 7.3%dw cellulose and 13.2%dw hemicellulose,while lignin was not detectable.Enzymatic hydrolysis of Spirulina biomass was compared with acid pretreatment as a widely used conventional method for biomass hydrolysis.Under the applied conditions,acid pretreatment showed the maximum amount of reducing sugars?1.37?g/mL?at the highest applied concentration?10%?.However,application of HTC showed increase of the hydrolysis efficiency by increasing the enzyme concentration and incubation time,reaching its maximum action?1.39?g/mL?using 10?L of enzyme and incubation with the substrate for 30 min.The structural changes and component decomposition of biomass were analyzed using FTIR to determine the extent of fibers degradation.In raw biomass,the band at 3422 cm^-1,corresponds O-H stretching,indicated the presence of cellulose and/or acids.Increasing of this peak by acid pretreatment was attributed to the saturation of sample with acid,while the significant reduction in the peak by HTC treatment confirms that cellulose structure became loose in the digested fiber due to destroying of hydrogen bonds in cellulose.The absorption band at 2945cm^-1 is related to the C–H stretching,confirming that methyl and methylene groups of cellulose were partly removed after both acid and enzyme pretreatment.In addition,the decrease in intensity of the peak at 1654 cm^-1?C=O stretching?in the enzyme-treated samples might be attributed to the partial degradation of hemicellulose due to hydrolysis of cellulose by the enzyme.Moreover,application of HTC on the pure crystalline cellulose showed the maximum relative activity after 12 h of incubation at 95?.However,increasing of incubation time up to48 h showed insignificant changes in the relative activity.Thus,the enzyme requires longer time to reach its maximum activity with crystalline cellulose?12 h?than cellulose?5 h?.Overall,the present results confirmed the significant action of HTC on cellulose and its crystalline form which will have wide industrial applications in the future.