Statistical optimization of enzymatic degradation process for oil palm empty fruit bunch (OPEFB) in rotary drum bioreactor using crude cellulase produced from Aspergillus niger EFB1

Oil palm empty fruit bunch (OPEFB) was pretreated with 2% (v/v) HNO₃ and degraded by Aspergillus niger EFB1 crude cellulase. Through 2 Level Factorial Design (2LFD), it was found that OPEFB concentration, temperature, incubation time, concentration of Tween 80 and agitation speed have significant effect in reducing sugar production. A standard Response Surface Methodology (RSM) design known as Central Composite Design (CCD) was used to optimize the enzymatic degradation condition of OPEFB in rotary drum bioreactor. Reducing sugar level of 1.183 g/L was obtained with the following optimized degradation conditions: 1.95% (w/v) OPEFB, 0.5% (v/v) Tween 80, 55 °C, 87.5 rpm in the incubation period of 3 days and 16 h. The optimal degradation condition improved reducing sugar production by 1.07 fold compared to that before optimization in shake flasks culture. The optimization strategy of enzymatic degradation of OPEFB inside rotary drum bioreactor led to increase in glucose, xylose, arabinose, galactose and mannose production by 3, 2.5, 1.64, 19.37 and 22.52 fold, respectively. The improvement in reducing sugar and polyoses production were comparable with the reduction in OPEFB cellulose and hemicellulose content by 89.32% and 48.17% respectively after enzymatic degradation in optimized condition.     

Cellulase production and oil palm empty fruit bunch saccharification by a new isolate of Trichoderma koningii D-64

A new strain Trichoderma koningii D-64 was isolated from Singapore soil samples. It produced cellulase, xylanase, and laccase on a variety of carbon sources. Enzyme activities of 3.8 ± 0.3, 40.3 ± 5.1, 6.6 ± 0.3 and 98.8 ± 10.3 U/mL were respectively obtained for FPase, CMCase, β-glucosidase and xylanase in a mixture of 1% cellulose and 2% wheat bran. About 70–95% saccharification efficiency of oil palm empty fruit bunch was obtained using T. koningii D-64 enzymes alone without the supplement of any other commercial enzymes. Strain T. koningii D-64 is therefore a potential cellulase producer for the efficient lignocellulosic biomass conversion to fermentable sugars.     

Unwrapping the hydrolytic system of the phytopathogenic fungus Phoma exigua by secretome analysis

The present work describes the secretome profiling of a phytopathogenic fungus, Phoma exigua by liquid chromatography coupled tandem mass spectrometry (LC–MS/MS) based proteomics approach to highlight the suites of enzymes responsible for biomass hydrolysis. Mass spectrometry identified 33 proteins in the Phoma secretome when grown on α-cellulose as the sole carbon source. The functional classification revealed a unique extracellular enzyme system mainly belonging to the family of glycosyl hydrolase proteins (52%). This hydrolytic system consisted of cellulases (endo-1,4-β-glucanase, cellobiohydrolase I, exoglucanase, and β-glucosidase), hemicellulases (1,4-β-xylosidase and endo-1,4-β-xylanase) and other hypothetical proteins including GH3, GH5, GH6, GH7, GH11, GH20, GH32 and GH54. The synergistic action of this enzyme cocktail was assessed by the saccharification of alkali treated wheat straw. Since the Phoma secretome has limited β-glucosidase activity, it was supplemented with commercial β-glucosidase. After supplementation, this enzyme complex resulted in high yields of glucose (177.2 ± 1.0 mg/gds), xylose (209.2 ± 1.5 mg/gds) and arabinose (25.2 ± 0.3 mg/gds). The secretome analysis and biomass hydrolysis by P. exigua revealed its unique potential as a source of hydrolytic enzymes for lignocellulosic biomass hydrolysis.     

Functional cello-oligosaccharides production from the corncob residues of xylo-oligosaccharides manufacture

An integrated process has been developed, consisting of the “adsorption–separation” of cellulase enzymes to selectively remove β-glucosidase, and multi-stage enzymatic hydrolysis of corncob residues from xylo-oligosaccharides manufacture with the β-glucosidase deficient cellulase, aiming to obtain a high yield of cello-oligosaccharides production. After the “adsorption–separation” process, 79.50% of the endo-glucanase was retained in substrate, whereas 90.67% of β-glucosidase was removed with the separated liquid fraction, utilizing the different adsorbability of these enzymes to the substrate. A three-stage enzymatic hydrolysis of corncob residues with the β-glucosidase deficient cellulase was proposed in which the first, the second and the third stage were conducted for 6, 6 h and 12 h, respectively. Analysis indicated that the removal of hydrolysis products (glucose and cello-oligosaccharides) at each stage improved cello-oligosaccharides productivity and enzymatic hydrolysis yield. The cello-oligosaccharides yield and enzymatic hydrolysis yield in three-stage enzymatic hydrolysis were significantly improved to 51.78% and 75.56%, respectively, which were 36.00% and 25.10% higher than single-stage hydrolysis with original cellulase enzymes.     

Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate

 Enzymatic hydrolysis of corncob and ethanol fermentation from cellulosic hydrolysate were investigated. After corncob was pretreated by 1% H₂SO₄ at 108 °C for 3 h, the cellulosic residue was hydrolyzed by cellulase from Trichoderma reesei ZU-02 and the hydrolysis yield was 67.5%. Poor cellobiase activity in T. reesei cellulase restricted the conversion of cellobiose to glucose, and the accumulation of cellobiose caused severe feedback inhibition to the activities of β-1,4-endoglucanase and β-1,4-exoglucanase in cellulase system. Supplementing cellobiase from Aspergillus niger ZU-07 greatly reduced the inhibitory effect caused by cellobiose, and the hydrolysis yield was improved to 83.9% with enhanced cellobiase activity of 6.5 CBU g⁻¹ substrate. Fed-batch hydrolysis process was started with a batch hydrolysis containing 100 g l⁻¹ substrate, with cellulosic residue added at 6 and 12 h twice to get a final substrate concentration of 200 g l⁻¹. After 60 h of reaction, the reducing sugar concentration reached 116.3 g l⁻¹ with a hydrolysis yield of 79.5%. Further fermentation of cellulosic hydrolysate containing 95.3 g l⁻¹ glucose was performed using Saccharomyces cerevisiae 316, and 45.7 g l⁻¹ ethanol was obtained within 18 h. The research results are meaningful in fuel ethanol production from agricultural residue instead of grain starch.     

Isatin based Schiff bases as inhibitors of α-glucosidase: Synthesis,characterization, in vitro evaluation and molecular docking studies

Isatin base Schiff bases (1–20) were synthesized, characterized by ¹H NMR and EI/MS and evaluated for α-glucosidase inhibitory potential. Out of these twenty (20) compounds only six analogs showed potent α-glucosidase inhibitory potential with IC₅₀ value ranging in between 2.2 ± 0.25 and 83.5 ± 1.0 μM when compared with the standard acarbose (IC₅₀ = 840 ± 1.73 μM). Among the series compound 2 having IC₅₀ value (18.3 ± 0.56 μM), 9 (83.5 ± 1.0 μM), 11 (3.3 ± 0.25 μM), 12 (2.2 ± 0.25 μM), 14 (11.8 ± 0.15 μM), and 20 (3.0 ± 0.15 μM) showed excellent inhibitory potential many fold better than the standard acarbose. The binding interactions of these active analogs were confirmed through molecular docking.     

Molecular docking studies and synthesis of novel bisbenzimidazole derivatives as inhibitors of α-glucosidase

A series of bisbenzimidazole derivatives starting from o-phenylenediamine and 4-nitro-o-phenylenediamine were prepared with oxalic acid. Most of the reactions were conducted using both the microwave and conventional methods to compare yields and reaction times. The operational simplicity, environmental friendly conditions and high yield in a significantly short reaction time were the major benefits. All substances’ inhibitory activities against α-glucosidase were evaluated. The results may suggest a significant role for the nature of bisbenzimidazole compounds in their inhibitory action against α-glucosidase. They showed different range of α-glucosidase inhibitory potential with IC₅₀ value ranging between 0.44 ± 0.04 and 6.69 ± 0.01 μM when compared to the standard acarbose (IC₅₀, 13.34 ± 1.26 μM). This has described a new class of α-glucosidase inhibitors. Molecular docking studies were done for all compounds to identify important binding modes responsible for inhibition activity of α-glucosidase.     

Expression of a bifunctional cellulase with exoglucanase and endoglucanase activities to enhance the hydrolysis ability of cellulase from a marine Aspergillus niger

Low exoglucanase and endoglucanase activities of marine Aspergillus niger cellulase decreased the hydrolyzing ability of cellulase. To increase the activity of halostable cellulase obtained from a marine A. niger, a cellulase with endoglucanase and exoglucanase activity was efficiently expressed by constructing a vector with promoter glaA. Exoglucanase and endoglucanase activities increased from 0.21 and 4.51 U/ml of the original strain to 0.89 U/ml and 15.12 U/ml of the transformant, respectively. Filter paper activity (FPA) increased by 7.1 folds from 0.63 to 4.47 U/ml. The release of glucose by hydrolysis of wheat straw with cellulase from the transformant was 1.37 folds higher than that with cellulase from the original strain under high salinity condition. Cellulase with endoglucanase and exoglucanase activities could be well expressed in marine A. niger. The cellulase from the transformant not only showed higher activity, but also retained halostability. An appreciate proportion of β-glucosidase, exoglucanase, endgolucanasein cellulase was important for hydrolyzing cellulose.     

Bioconversion of lignocellulosic palm byproducts into enzymes and lipid by newly isolated oleaginous fungi

Direct conversion of palm pressed fiber (PPF) and palm empty fruit bunches (EFB) into enzymes and lipid by oleaginous fungi were performed through solid-state fermentation (SSF). Among the strains tested, TSIP9 converted PPF and EFB into lipid with the highest yield of 31.1 ± 1.7 mg/gram dry substrate (gds) and 37.5 ± 2.2 mg/gds, respectively. It also produced high activity of cellulolytic enzymes. It was identified as Aspergillus tubingensis. The similar fatty acids of its lipid to those of plant oil indicate its suitable use as biodiesel feedstock. The cellulase and xylanase production by this strain was improved when EFB was pretreated with alkaline. When alkaline-pretreated EFB was added with palm kernel cake (PK) as an alternative nitrogen source and the culture conditions were optimized through response surface methodology (RSM), the production of lipid, cellulase and xylanase were increased up to 88.5 ± 4.9 mg/gds, 26.1 ± 0.1 U/gds and 59.3 ± 0.3 U/gds, respectively. This study reveals the potential use of in situ cellulolytic enzymes producing fungi and the optimal conditions for direct conversion of lignocellulosic biomass into lipid.     

Synthesis of thiobarbituric acid derivatives: In vitro α-glucosidase inhibition and molecular docking studies

Synthesis, structure, and evaluation of in vitro α-glucosidase enzyme inhibition of a new class of diethylammonium salts of aryl substituted thiobarbituric acid is described. This protocol is straight, environmentally benign and efficient, involving Aldol-Michael addition reaction in one pot fashion. The 3D chemical structures of the synthesized compounds were assigned based on spectroscopic methods and X-ray single crystal diffraction analyses. All synthesized compounds 3a-3n were evaluated for their in vitro α-glucosidase enzyme inhibitory activity, whereas acarbose was used as the standard drug (IC₅₀ = 840 ± 1.73 µM). All tested compounds were found to possess varying degree of α-glucosidase enzyme inhibition activity with (IC₅₀ = 19.46 ± 1.84–415.8 ± 4.0 µM). Compound 3i (IC₅₀ = 19.4 ± 1.84 µM) exhibited the highest activity. To the best of knowledge this is the first report of the in vitro α-glucosidase enzyme inhibition by the diethylamonium salts of aryl substituted thiobarbituric acid. Furthermore, molecular docking studies of selected compounds were also performed to see interactions between active compounds and binding sites.     

Nutrient control for stationary phase cellulase production in Trichoderma reesei Rut C-30

This work describes the use of nutrient limitations with Trichoderma reesei Rut C-30 to obtain a prolonged stationary phase cellulase production. This period of non-growth may allow for dependable cellulase production, extended fermentation periods, and the possibility to use pellet morphology for easy product separation. Phosphorus limitation was successful in halting growth and had a corresponding specific cellulase production of 5 ± 2 FPU/g-h. Combined with the addition of Triton X-100 for fungal pellet formation and low shear conditions, a stationary phase cellulase production period in excess of 300 h was achieved, with a constant enzyme production rate of 7 ± 1 FPU/g-h. While nitrogen limitation was also effective as a growth limiter, it, however, also prevented cellulase production.     

Kinetics of cellulose hydrolysis by halostable cellulase from a marine Aspergillus niger at different salinities

Kinetics of cellulose hydrolysis with halostable cellulase from a marine Aspergillus niger was analyzed at different salinities. Cellulase activity in 8% NaCl solution was 1.43 folds higher than that in NaCl free solution. Half saturation constant, K_m (15.6260 g/L) and the rate constant of deactivation, K_de (0.3369 g/L h) in 8% NaCl solution was lower than that (18.6364 g/L), 0.3754 (g/L h) in NaCl free solution. The maximum initial hydrolysis velocity, V_max (25.5295 g/L h), in 8% NaCl solution was higher than that in NaCl free solution (25.0153 g/L h). High salinity increased affinity to the cellulase to the substrate and thermostability. Halostable cellulase from a marine Aspergillus niger was valuable for cellulose hydrolysis under high salinity conditions.     

Discovery of 3,3-di(indolyl)indolin-2-one as a novel scaffold for α-glucosidase inhibitors: In silico studies and SAR predictions

3,3-Di(indolyl)indolin-2-ones 4a-4n were synthesized and evaluated for their in vitro α-glucosidase inhibitory activity. These newly synthesized compounds showed moderate to potent α-glucosidase inhibitory activity with IC₅₀ range from 5.98 ± 0.11 to 145.95 ± 0.46 μM, when compared to the standard drug acarbose. Among this series of 3,3-di(indolyl)indolin-2-ones, compound 4j (5.98 ± 0.11 μM) having a 2-fluorobenzyl group on the indole ring was found to be the most active compound. Molecular docking studies showed that compound 4j have high binding affinities with the active site of α-glucosidase enzyme through hydrogen bonds, arene-cation, π-π stacking and hydrophobic interactions. This study showed these 3,3-di(indolyl)indolin-2-ones as a new class of α-glucosidase inhibitors.     

Dihydropyrano [2,3-c] pyrazole: Novel in vitro inhibitors of yeast α-glucosidase

Inhibition of α-glucosidase enzyme activity is a reliable approach towards controlling post-prandial hyperglycemia associated risk factors. During the current study, a series of dihydropyrano[2,3-c] pyrazoles (1–35) were synthesized and evaluated for their α-glucosidase inhibitory activity. Compounds 1, 4, 22, 30, and 33 were found to be the potent inhibitors of the yeast α-glucosidase enzyme. Mechanistic studies on most potent compounds reveled that 1, 4, and 30 were non-competitive inhibitors (K_i = 9.75 ± 0.07, 46 ± 0.0001, and 69.16 ± 0.01 μM, respectively), compound 22 is a competitive inhibitor (K_i = 190 ± 0.016 μM), while 33 was an uncompetitive inhibitor (K_i = 45 ± 0.0014 μM) of the enzyme. Finally, the cytotoxicity of potent compounds (i.e. compounds 1, 4, 22, 30, and 33) was also evaluated against mouse fibroblast 3T3 cell line assay, and no toxicity was observed. This study identifies non-cytotoxic novel inhibitors of α-glucosidase enzyme for further investigation as anti-diabetic agents.     

Use of Saccharum spontaneum (wild sugarcane) as biomaterial for cell immobilization and modulated ethanol production by thermotolerant Saccharomyces cerevisiae VS3

Saccharum spontaneum is a wasteland weed consists of 45.10 ± 0.35% cellulose and 22.75 ± 0.28% of hemicellulose on dry solid (DS) basis. Aqueous ammonia delignified S. spontaneum yielded total reducing sugars, 53.91 ± 0.44 g/L (539.10 ± 0.55 mg/g of substrate) with a hydrolytic efficiency of 77.85 ± 0.45%. The enzymes required for hydrolysis were prepared from culture supernatants of Aspergillus oryzae MTCC 1846. A maximum of 0.85 ± 0.07 IU/mL of filter paperase (FPase), 1.25 ± 0.04 IU/mL of carboxy methyl cellulase (CMCase) and 55.56 ± 0.52 IU/mL of xylanase activity was obtained after 7 days of incubation at 28 ± 0.5 °C using delignified S. spontaneum as carbon source under submerged fermentation conditions. Enzymatic hydrolysate of S. spontaneum was then tested for ethanol production under batch and repeated batch production system using “in-situ” entrapped Saccharomyces cerevisiae VS₃ cells in S. spontaneum stalks (1 cm × 1 cm) size. Immobilization was confirmed by the scanning electron microscopy (SEM). Batch fermentation of VS₃ free cells and immobilized cells showed ethanol production, 19.45 ± 0.55 g/L (yield, 0.410 ± 0.010 g/g) and 21.66 ± 0.62 g/L (yield, 0.434 ± 0.021 g/g), respectively. Immobilized VS₃ cells showed maximum ethanol production (22.85 ± 0.44 g/L, yield, 0.45 ± 0.04 g/g) up to 8th cycle during repeated batch fermentation followed by a gradual reduction in subsequent cycles of fermentation.     

Synthesis, α-glucosidase inhibitory,cytotoxicity and docking studies of 2-aryl-7-methylbenzimidazoles

Benzimidazole analogs 1–27 were synthesized, characterized by EI-MS and ¹H NMR and their α-glucosidase inhibitory activities were found out experimentally. Compound 25, 19, 10 and 20 have best inhibitory activities with IC₅₀ values 5.30 ± 0.10, 16.10 ± 0.10, 25.36 ± 0.14 and 29.75 ± 0.19 respectively against α-glucosidase. Compound 6 and 12 has no inhibitory activity against α-glucosidase enzyme among the series. Further studies showed that the compounds are not showing any cytotoxicity effect. The docking studies of the compounds as well as the experimental activities of the compounds correlated well. From the molecular docking studies, it was observed that the top ranked conformation of all the compounds fit well in the active site of the homology model of α-glucosidase.     

Synthesis,molecular docking studies of hybrid benzimidazole as α-glucosidase inhibitor

Thiourea derivatives having benzimidazole 1–17 have been synthesized, characterized by ¹H NMR, ¹³C NMR and EI-MS and evaluated for α-glucosidase inhibition. Identification of potential α-glucosidase inhibitors were done by in vitro screening of 17 thiourea bearing benzimidazole derivatives using Baker’s yeast α-glucosidase enzyme. Compounds 1–17 exhibited a varying degree of α-glucosidase inhibitory activity with IC₅₀ values between 35.83 ± 0.66 and 297.99 ± 1.20 μM which are more better than the standard acarbose (IC₅₀ = 774.5 ± 1.94 μM). Compound 10 and 14 showed significant inhibitory effects with IC₅₀ value 50.57 ± 0.81 and 35.83 ± 0.66 μM, respectively better than the rest of the series. Structure activity relationships were established. Molecular docking studies were performed to understand the binding interaction of the compounds.     

Synthesis,biological evaluation and molecular docking study of N-arylbenzo[d]oxazol-2-amines as potential α-glucosidase inhibitors

A novel series of N-arylbenzo[d]oxazol-2-amines (4a–4m) were synthesized and evaluated for their α-glucosidase inhibitory activity. Compounds 4f–4i, 4k and 4m displayed potent inhibitory activity against α-glucosidase with IC₅₀ values in the range of 32.49 ± 0.17–120.24 ± 0.51 μM as compared to the standard drug acarbose. Among all tested compounds, compound 4g having 4-phenoxy substitution at the phenyl ring was found to be the most active inhibitor of α-glucosidase with an IC₅₀ value of 32.49 ± 0.17 μM. Analysis of the kinetics of enzyme inhibition indicated that compound 4g is a noncompetitive inhibitor of α-glucosidase with a K_i value of 31.33 μM. Binding interaction of compound 4g with α-glucosidase was explored by molecular docking simulation.     

Pyridine sulfonamide as a small key organic molecule for the potential treatment of type-II diabetes mellitus and Alzheimer’s disease: In vitro studies against yeast α-glucosidase,acetylcholinesterase and butyrylcholinesterase

This paper presents the efficient high yield synthesis of novel pyridine 2,4,6-tricarbohydrazide derivatives (4a–4i) along with their α-glucosidase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition activities. The enzymes inhibition results showed the potential of synthesized compounds in controlling both type-II diabetes mellitus and Alzheimer’s disease. In vitro biological investigations revealed that most of compounds were more active against yeast α-glucosidase than the reference compound acarbose (IC₅₀ 38.25 ± 0.12 μM). Among the tested series the compound 4c bearing 4-flouro benzyl group was noted to be the most active (IC₅₀ 25.6 ± 0.2 μM) against α-glucosidase, and it displayed weak inhibition activities against AChE and BChE. Compound 4a exhibited the most desired results against all three enzymes, as it was significantly active against all the three enzymes; α-glucosidase (IC₅₀ 32.2 ± 0.3 μM), AChE (IC₅₀ 50.2 ± 0.8 μM) and BChE (IC₅₀ 43.8 ± 0.8 μM). Due to the most favorable activity of 4a against the tested enzymes, for molecular modeling studies this compound was selected to investigate its pattern of interaction with α-glucosidase and AChE targets.     

Application of an integrated statistical design to optimize the cold enzyme hydrolysis conditions for ethanol production

Cold enzyme hydrolysis was investigated on the ethanol production by Saccharomyces cerevisiae during simultaneous saccharification and fermentation (SSF) processing. An integrated statistical design, which incorporated single factor design, response surface methodology (RSM) and weighting coefficient method, was used to determine the optimum hydrolysis conditions leading to maximum biomass, ethanol concentration and starch utilization ratio. After the studied ranges of α-amylase, glucoamylase and liquefaction time were identified by single factor design, RSM was used to further optimize the hydrolysis conditions for each objective. The results showed that, under hydrolysis condition optimized with RSM, biomass, ethanol concentration and starch utilization ratio reached 4.401 ± 0.042 × 10⁸ cells/ml, 14.81 ± 0.23% (wt.%) and 94.52 ± 0.53%, respectively. Finally, multi-objective optimization (MOO) was applied to obtain a compromised result of three desirable responses by weighting coefficient methodology. Biomass of 4.331 ± 0.038 × 10⁸ cells/ml, ethanol concentration of 14.12 ± 0.21% (wt.%) and starch utilization ratio of 92.88 ± 0.21% were simultaneous obtained when hydrolysis at pH 5.9 for 114 min with 233 IU/g_starch α-amylase and 778 IU/g_starch glucoamylase. The optimized conditions were shown to be feasible and reliable through verification tests.