Highly efficient, processive and multifunctional recombinant endoglucanase RfGH5_4 from Ruminococcus flavefaciens FD-1 v3 for recycling lignocellulosic plant biomasses.

Gene encoding endoglucanase, RfGH5_4 from R. flavefaciens FD-1 v3 was cloned, expressed in Escherichia coli BL21(DE3) cells and purified. RfGH5_4 showed molecular size 41 kDa and maximum activity at pH 5.5 and 55 °C. It was stable between pH 5.0-8.0, retaining 85% activity and between 5 °C-45 °C, retaining 75% activity, after 60 min. RfGH5_4 displayed maximum activity (U/mg) against barley ß-D-glucan (665) followed by carboxymethyl cellulose (450), xyloglucan (343), konjac glucomannan (285), phosphoric acid swollen cellulose (86), beechwood xylan (21.7) and carob galactomannan (16), thereby displaying the multi-functionality. Catalytic efficiency (mL.mg-1 s-1) of RfGH5_4 against carboxymethyl cellulose (146) and konjac glucomannan (529) was significantly high. TLC and MALDI-TOF-MS analyses of RfGH5_4 treated hydrolysates of cellulosic and hemicellulosic polysaccharides displayed oligosaccharides of degree of polymerization (DP) between DP2-DP11. TLC, HPLC and Processivity-Index analyses revealed RfGH5_4 to be a processive endoglucanase as initially, for 30 min it hydrolysed cellulose to cellotetraose followed by persistent release of cellotriose and cellobiose. RfGH5_4 yielded sufficiently high Total Reducing Sugar (TRS, mg/g) from saccharification of alkali pre-treated sorghum (72), finger millet (62), sugarcane bagasse (38) and cotton (27) in a 48 h saccharification reaction. Thus, RfGH5_4 can be considered as a potential endoglucanase for renewable energy applications.

Structure and dynamics analysis of multi-domain putative ß-1,4-glucosidase of family 3 glycoside hydrolase (PsGH3) from Pseudopedobacter saltans.

Structure and conformational behaviour of a putative ß-1,4-glucosidase of glycoside hydrolase family 3 (PsGH3) from Pseudopedobacter saltans was predicted by using in-silico tools. PsGH3 modeled structure constructed using Phyre2 displayed multidomain architecture comprising an N-terminal (ß/α)8-fold domain followed by (α/ß)6-sandwich domain, PA14 domain, and a C-terminal domain resembling an immunoglobulin fold. Ramachandran plot displayed 99.3% of amino acids in the allowed region and 0.7% residues in the disallowed region. Multiple sequence alignment (MSA) and structure superposition of PsGH3 with other homologues from GH3 family revealed the conserved residues, Asp274 and Glu624 present in loops LA and LB, respectively originating from N-terminal domain act as catalytic residues. The volume and area calculated for PsGH3 displayed a deep active-site conformation comparable with its homologues, ß-1,4-glucosidases (GH3) of Kluyveromyces marxianus and Streptomyces venezuelae. Molecular dynamic (MD) simulation of PsGH3 structure for 80 ns suggested stable and compact structure. Molecular docking studies revealed deeper active site conformation of PsGH3 that could house larger cellooligosaccharides up to 7° of polymerization (DP7). The amino acid residues, Ala86, Leu88, Cys275, Pro483, Phe493, Asn417, Asn491, Pro492, and Leu495 created a binding pocket near the catalytic cleft, crucial for ligand binding. MD simulation of PsGH3 in the presence of cellooligosaccharides, viz., cellobiose and celloheptaose showed stability in terms of RMSD, Rg, and SASA values till 80 ns. The calculation of average number of hydrogen bond (H-bond), interaction energy, and binding free energy confirmed the stronger binding affinity of the larger cellooligosaccharides such as celloheptaose in the binding cavity of PsGH3.

Sequential pretreatment of sugarcane bagasse by alkali and organosolv for improved delignification and cellulose saccharification by chimera and cellobiohydrolase for bioethanol production.

Sequential pretreatments for sugarcane bagasse (scb) by NaOH followed by organosolv under mild conditions were evaluated for cellulose recovery and dilignification. The best-optimized sequential pretreatment of scb was obtained at 10% (w/v) of raw scb loading at 1% (w/v) NaOH (50 °C, 2 h) followed by treatment with organosolv (85%, v/v phosphoric acid, 50 °C, 1 h) with chilled acetone. This sequentially pretreated scb showed cellulose recovery, 66.1% (w/w) and delignification, 83.2% (w/w). NaOH or organosolv pretreated scb showed lower cellulose recovery 47.4% (w/w) or 54.5% (w/w) with lower delignification, 61% (w/w) or 56% (w/w), respectively. Pretreated solid residue of sequentially pretreated scb was enzymatically saccharified by chimera (ß-glucosidase and endoglucanase, CtGH1-L1-CtGH5-F194A) and cellobiohydrolase (CtCBH5A) cloned from Clostridium thermocellum. Enzymatic hydrolysate of best sequentially pretreated scb gave total reducing sugar (TRS) yield, 230 mg/g and glucose yield, 137 mg/g pretreated scb. Only organosolv pretreated scb gave TRS yield, 112.5 mg/g and glucose yield, 72 mg/g of pretreated scb. Thus, sequentially pretreated scb resulted in 37% higher enzymatic digestibility than only orgnaosolv pretreated scb. Higher enzymatic digestibility was supported by higher crystallinity index CrI (45%) than those obtained with only organosolv pretreated (38%) or raw scb (25%). Field Emission Scanning Electron Microscope (FESEM) and Fourier-transform infrared (FT-IR) analyses showed enhanced cellulose exposure in sequentially pretreated scb. Preliminary investigation of bioethanol production at small scale by separate hydrolysis and fermentation (SHF) of enzymatic hydrolysate from best sequentially pretreated scb by Saccharomyces cerevisiae gave maximum ethanol yield of 0.42 g/g of glucose. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02600-y.

Role of glycine 256 residue in improving the catalytic efficiency of mutant endoglucanase of family 5 glycoside hydrolase from Bacillus amyloliquefaciens SS35.

Wild-type, BaGH5-WT and mutant, BaGH5-UV2 (aspartate residue mutated to glycine), endoglucanases belonging to glycoside hydrolase family 5 (GH5), from wild-type, and UV2 mutant strain of Bacillus amyloliquefaciens SS35, respectively, were earlier cloned in pHTP0 cloning vector. In this study, genes encoding BaGH5-WT or BaGH5-UV2 were cloned into pET28a(+) expression-vector and expressed in Escherichia coli BL-21(DE3)pLysS cells. BaGH5-UV2 showed 10-fold (43.6 U/mg) higher specific activity against carboxymethylcellulose sodium salt (CMC-Na), higher optimal temperature by 10°C at 65°C, and 22-fold higher catalytic efficiency against CMC-Na, than BaGH5-WT. BaGH5-UV2 showed stability in wider acidic pH range (5.0-7.0) unlike BaGH5-WT in narrow basic pH range (7.0-7.5). BaGH5-UV2 displayed a mutation, Asp256Gly in L11 loop, connecting ß6 -sheet with α6 -helix, near active site toward the domain surface of (α/ß)8 -TIM barrel fold. Molecular dynamics simulation studies showed more stable structure, accessibility of substrate for a catalytic site, and increased flexibility of loop L11 of BaGH5-UV2 than the wild type, suggesting enhanced catalysis by BaGH5-UV2. Molecular docking analysis displayed enhanced hydrogen bond interactions of cello-oligosaccharides with BaGH5-UV2, unlike BaGH5-WT. Thus, Gly256 residue of loop L11 plays an important role in enhancing catalytic efficiency, and pH stability of GH5 endoglucanase. Therefore, these results help in protein engineering of GH5 endoglucanase for improved biochemical properties.

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.

Combined SAXS and computational approaches for structure determination and binding characteristics of Chimera (CtGH1-L1-CtGH5-F194A) generated by assembling ß-glucosidase (CtGH1) and a mutant endoglucanase (CtGH5-F194A) from Clostridium thermocellum.

Chimera (CtGH1-L1-CtGH5-F194A) developed by fusing ß-glucosidase (CtGH1) at N-terminal and endoglucanase (CtGH5-F194A) at C-terminal was structurally characterized. Its secondary structure analysis by CD showed 38% α-helix, 9.3% ß-sheets and 52.7% random coils corroborating with prediction. In-silico modeled structure of Chimera comprised two modules, CtGH1 and CtGH5-F194A displaying (α/ß)8 fold. Ramachandran plot of Chimera showed 99.9% residues in allowed region. Binding interaction of Chimera with cello-oligosaccharides suggested active forms of CtGH1 and CtGH5-F194A and their involvement in catalysis. MD simulation of cellohexaose bound endoglucanase module of Chimera showed favourable flexibility in loops, LA with H-bond formation with Asn510 and in loop LC relocation of Tyr687 away from active site efficiently releasing the product after catalysis. Higher short range interaction energy of Chimera, -383 kJ/mol than the individual endoglucanase, 254 kJ/mol against cellohexaose suggested higher efficient catalysis by Chimera. ß-Glucosidase module of Chimera showed fluctuations in outer loops suggesting conformational changes that might be contributing to improved hydrolysis. SAXS analysis of Chimera displayed monodispersed state. Guinier analysis of Chimera showed globular shape (Rg= 3.15 ± 0.10 nm). Kratky plot confirmed fully folded and flexible behaviour in solution. Gasbor modeled structure of Chimera displayed an elongated structure with two modules having shape similar to bean-bag contour.

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.

The predictivity of mandibular third molar position as a risk indicator for pericoronitis: A prospective study.

AIM: The aim of this study was to describe the characteristics of the mandibular third molar at highest risk for acute pericoronitis using clinical and radiographic analysis. MATERIALS AND METHODS: A total of 120 patients ranging in age from 18 to 55 years suffering from pericoronitis were examined. Subjective and objective observations were recorded that included the age, gender, angulation of partially impacted mandibular third molar, the frequency of pericoronitis in a year, the presence of impinging maxillary third molar, the extent of soft tissue coverage over the impacted mandibular third molar, the clinical signs evaluated in the patient, the class and position of the impacted mandibular third molar, and the presence of distal radiolucency with respect to the impacted mandibular third molar. RESULTS: The results obtained in the study indicate that pericoronitis is associated more in the age group of 26-35 years and is more commonly reported in the female gender. Distoangular partially impacted mandibular third molars impacted at Class II and position B seem to be at the highest risk of developing pericoronitis. CONCLUSION: The results obtained in the study indicate that pericoronitis is associated more in the age group of 26-35 years and is more commonly reported in the female gender. Distoangular partially impacted mandibular third molars impacted at class II and position B seem to be at the highest risk of developing pericoronitis.

Is screening in dental office an effective method of detecting undiagnosed hypertension?

BACKGROUND: Hypertension is an important worldwide public health challenge because of its high frequency and risk of cardiovascular and renal disease. OBJECTIVE: The objective of this study was to investigate the prevalence of undiagnosed hypertension as well as inadequately controlled hypertension among general population who sought tooth extraction at Army College of Dental Sciences, Secunderabad. MATERIALS AND METHODS: Only 1200 patients in the age group of 20-60 years who sought tooth extraction were included in the study. Blood pressure (BP) was measured for three times in all patients. The readings were quantized into four categories which included normal, prehypertensive stage, and Stage 1 and Stage 2 of hypertension. The BP was assessed for the following variables - gender, habits of gutkha chewing, smoking and alcohol, regular exercise, age, and effect of local anesthesia. RESULTS: Nearly 24.4% of new cases of hypertension were diagnosed among all participants reported to the dental clinic. After giving local anesthesia, 16.71% increase in BP was observed in Stage 1 and 2.35% increase in Stage 2 hypertension. CONCLUSION: This study reveals that dentists play an important role in the early diagnosis of hypertension of many dental patients who are unaware of being hypertensive. This role should be emphasized in our specialty as a standard of care to prevent life-threatening complications.