Assessment of combination of pretreatment of Sorghum durra stalk and production of chimeric enzyme (ß-glucosidase and endo ß-1,4 glucanase, CtGH1-L1-CtGH5-F194A) and cellobiohydrolase (CtCBH5A) for saccharification to produce bioethanol.

Optimization of pretreatment and saccharification of Sorghum durra stalk (Sds) was carried out. The chimeric enzyme (CtGH1-L1-CtGH5-F194A) having ß-glucosidase (CtGH1) and endo ß-1,4 glucanase activity (CtGH5-F194A) and cellobiohydrolase (CtCBH5A) from Clostridium thermocellum were used for saccharification. Chimeric enzyme will save production cost of two enzymes, individually. Stage 2 pretreatment by 1% (w/v) NaOH assisted autoclaving + 1.5% (v/v) dilute H2SO4 assisted oven heating gave lower total sugar yield (366.6 mg/g of pretreated Sds) and total glucose yield (195 mg/g of pretreated Sds) in pretreated hydrolysate with highest crystallinity index 55.6% than the other stage 2 pretreatments. Optimized parameters for saccharification of above stage 2 pretreated biomass were 3% (w/v) biomass concentration, enzyme (chimera: cellobiohydrolase) ratio, 2:3 (U/g) of biomass, total enzyme loading (350 U/g of pretreated biomass), 24 h and 30 °C. Best stage 2 pretreated Sds under optimized enzyme saccharification conditions gave maximum total reducing sugar yield 417 mg/g and glucose yield 285 mg/g pretreated biomass in hydrolysate. Best stage 2 pretreated Sds showed significantly higher cellulose, 71.3% and lower lignin, 2.0% and hemicellulose, 12.2% (w/w) content suggesting the effectiveness of method. This hydrolysate upon SHF using Saccharomyces cerevisiae under unoptimized conditions produced ethanol yield, 0.12 g/g of glucose. Abbreviation: Ct-Clostridium thermocellum, Sds-Sorghum durra stalk, TRS-Total reducing sugar, HPLC-High performance liquid chromatography, RI-Refractive index, ADL-acid insoluble lignin, GYE-Glucose yeast extract, MGYP-Malt glucose yeast extract peptone, SHF-separate hydrolysis and fermentation, OD-Optical density, PVDF-Poly vinylidene fluoride, TS-total sugar, FESEM-Field emission scanning electron microscopy, XRD-X-ray diffraction, FTIR-Fourier transform infra-red spectroscopy and CrI-Crystallinity index.

Comparative analysis of pretreatment methods on sorghum (Sorghum durra) stalk agrowaste for holocellulose content.

This study compares different types of pretreatment methods, such as thermal pretreatment at 120 °C, autoclaving, microwaving and ultrasonication in the presence of water, dilute acid (1% H2SO4) or dilute alkali (1% NaOH) on Sorghum stalk with respect to the holocellulose and Acid Detergent/Insoluble Lignin content. Among all the methods, pretreatment with 1% NaOH along with autoclaving at 121 °C and 15 psi for 30 min was the most effective method for Sorghum stalk. Fourier Transform Infra-Red spectroscopy analysis of this pretreated biomass showed the removal of lignin and Field Emission Scanning Electron Microscope analysis displayed enhanced surface roughness. The enzymatic hydrolysis of raw and best pretreated Sorghum stalk using recombinant endo-ß-1,4-glucanase (CtCel8A) and ß-1,4-glucosidase (CtBgl1A) both from Clostridium thermocellum gave glucose yields, 22.4 mg/g raw biomass and 34 mg/g pretreated biomass, respectively, resulting in 1.5-fold increase of glucose yield after the pretreatment.

Molecular Characterization, Regioselective and Synergistic Action of First Recombinant Type III α-L-arabinofuranosidase of Family 43 Glycoside Hydrolase (PsGH43_12) from Pseudopedobacter saltans.

α-L-Arabinofuranosidase (PsGH43_12) of family 43 glycoside hydrolase and subfamily 12 from Pseudopedobacter saltans was cloned, over-expressed and biochemically characterized. PsGH43_12 displayed molecular mass, ~ 65 kDa. It exhibited activity in pH (5-9) and temperature range (35-55 °C) with maxima at pH 6.5 and 50 °C. PsGH43_12 gave 88.7 U/mg specific activity against rye arabinoxylan and 78.9 U/mg against wheat arabinoxylan. PsGH43_12 displayed Km and Vmax, 3.02 mg/ml and 103 µmole/min/mg, respectively, against rye arabinoxylan and 2.17 mM and 100.7 µmole/min/mg, respectively, against pNP-α-L-arabinofuranoside. 10 mM Mg2+ or Ca2+ ions enhanced PsGH43_12 activity by 54% or 33%, respectively. PsGH43_12 hydrolyzed rye arabinoxylan and released only L-arabinosyl moiety as main product, confirming its specificity towards α-L-arabinofuranoside. The regioselective analysis by NMR showed that PsGH43_12 belongs to type III α-L-arabinofuranoside. The synergistic behavior of PsGH43_12 in saccharification of mild alkali pretreated finger miller stalk (FMS) along with xylanase (CtXyn11A) from Clostridium thermocellum and xylosidase (BoGH43) from Bacteroides ovatus gave twofold higher total reducing sugar (TRS) yield. TLC analysis of pretreated FMS hydrolysed by CtXyn11A and BoGH43 showed xylooligosaccharides and xylose. Addition of PsGH43_12 to above combination gave mostly xylose and arabinose confirming their synergistic behavior and displaying their applicability in hydrolysis of hemicellulosic biomass.

Enzymatic hydrolysis of hemicellulose from pretreated Finger millet (Eleusine coracana) straw by recombinant endo-1,4-ß-xylanase and exo-1,4-ß-xylosidase.

This study focuses on enzymatic saccharification of hemicellulose part of the pretreated Finger millet straw (FMS) for production of xylose. The variation in the carbohydrate composition of FMS was analysed when subjected to different pretreatments. The recombinant endo-1,4-ß-xylanase (CtXyn11A) was most active on the FMS pretreated with 1% (w/v) NaOH combined with oven heating at 120 °C for 20 min, resulting in a total reducing sugar yield (TRS) of 32 mg/g pretreated biomass. The pretreatment aided in concentrating the holocellulose content from 69.3% of raw powdered FMS to 76.4%. The post-treatment solid biomass yield was 0.36 g/g raw biomass. The two-step optimization of hemicellulose saccharification from the above pretreated FMS with i) endo-1,4-ß-xylanase (CtXyn11A) at 55 °C and ii) exo-1,4-ß-xylosidase (BoGH43A) at 37 °C, both at pH 7.5 by Box-Behnken design yielded the TRS of 70 mg/g pretreated biomass. The percentage conversion of xylan to xylose by CtXyn11A and BoGH43A was 24.7%.

Development of bi-functional chimeric enzyme (CtGH1-L1-CtGH5-F194A) from endoglucanase (CtGH5) mutant F194A and ß-1,4-glucosidase (CtGH1) from Clostridium thermocellum with enhanced activity and structural integrity.

Site-directed mutagenesis of ß-1,4-endoglucanase from family 5 glycoside hydrolase (CtGH5) from Clostridium thermocellum was performed to develop a mutant CtGH5-F194A that gave 40 U/mg specific activity against carboxymethyl cellulose, resulting 2-fold higher activity than wild-type CtGH5. CtGH5-F194A was fused with a ß-1,4-glucosidase, CtGH1 from Clostridium thermocellum to develop a chimeric enzyme. The chimera (CtGH1-L1-CtGH5-F194A) expressed as a soluble protein using E. coli BL-21cells displaying 3- to 5-fold higher catalytic efficiency for endoglucanase and ß-glucosidase activities. TLC analysis of hydrolysed product of CMC by chimera 1 revealed glucose as final product confirming both ß-1,4-endoglucanase and ß-1,4-glucosidase activities, while the products of CtGH5-F194A were cellobiose and cello-oligosaccharides. Protein melting studies of CtGH5-F194A showed melting temperature (Tm), 68 °C and of CtGH1, 79 °C, whereas, chimera showed 78 °C. The improved structural integrity, thermostability and enhanced bi-functional enzyme activities of chimera makes it potentially useful for industrial application in converting biomass to glucose and thus bioethanol.