Optimized protocol for the pilot-scale preparation of fungal cellulase

Biodegradation of microcrystalline cellulose in pH–pH recyclable aqueous two-phase systems with water-soluble immobilized cellulase

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was employed to study the sorption behaviors of cellulases on microcrystalline celluloses and hardwood pulp. The adsorption and recovery of cellulases from Aspergillus niger and Trichoderma reesei were investigated at 25 °C. Cellulase recovery was conducted by rinsing adsorbed enzymes with sodium acetate buffer, Milli-Q water, and sodium hydroxide solution. The initial, equilibrium, and recovered enzymes were analyzed using SDS-PAGE gels. Gels were scanned and analyzed using ImagePro software. The molecular weights of cellulase proteins were determined using a protein marker having seven known proteins. The cellulase system from Trichoderma reesei had a higher adsorption on all substrates studied than the cellulase system from Aspergillius niger, and higher pH favored desorption from the substrates studied. Experimental results also demonstrated that adsorption and desorption amounts determined by SDS-PAGE were proportional to protein concentrations in their crude mixtures.

Cellulases from Trichoderma reesei were recovered by adsorption in sodium acetate buffer at lower pH using cation exchange resins followed by desorption at higher pH. The weakly acidic ion exchange resin WK10 was found to be the best among the six resins tested in terms of the enzyme activity recovery. The optimal pH values for the adsorption and desorption were 4.0 and 8.0, respectively, and the optimal adsorption and desorption times were both 5 h. Almost 100% of the initial cellulase activity was recovered under the optimal conditions with the supplement of β-glucosidase, which was unable to be efficiently recovered due to its strong adsorption (95.7%) but poor desorption (1.9%).

Single cultures and co-cultures of Bacillus licheniformis and Bacillus paralicheniformis isolated from compost were evaluated for their carboxymethyl cellulase (CMCase) and filter paperase (FPase) production potential. Using a medium supplemented with microcrystalline cellulose (MCC), in the co-culture, CMCase and FPase activities increased 8.87- and 2.28-fold and 10.15- and 3.20-fold over B. licheniformis and B. paralicheniformis monocultures, respectively. The synergistic behavior of the two isolates might be due to the consumption of hydrolysis product (glucose, cellobiose) by one or both of the isolates, which improved their metabolic performance for cellulase secretion. Optimal conditions for cellulase production by this co-culture were a temperature of 45 °C, and pH 7 at 180 rpm in a medium containing rice bran at 1% (w/v) and chicken manure as nitrogen supplement at 2% (w/v). The maximum CMCase and FPase produced under the above conditions were 79.8 U/mL and 12.5 U/mL, respectively. This corresponds to 257.4- and 59.5-fold enhancement in CMCase and FPase activity, respectively, over B. licheniformis monoculture, and 306.9- and 83.3-fold increase with respect to the B. paralicheniformis monoculture. These results indicate that improved cellulase production can be achieved through co-culture and chicken manure nitrogen-supplement.

Synergistic β-glucosidases for improving cellulases recyclability and biomass enzymatic saccharification in wheat straw

Microcrystalline cellulose is still imported by the Pharmaceutical Industry in Indonesia even though Indonesia has large natural resources but they have not been utilized optimally. Microcrystalline cellulose can be obtained from residue (extraction dregs) in the lotus plant extraction process, namely in the form of simplicia powder which will not be used again (organic solid waste). This research aims to obtain microcrystalline cellulose from several parts of the white lotus plant (Nymphaea nouchali Burm. F.) using the enzymatic hydrolysis method using cellulase from the termite Coptotermes sp., then the quality of the powder will be tested and compared with Avicel PH 101. Each part The white lotus is extracted, then the residue is delignified to obtain α-cellulose, then hydrolyzed with crude extract from Coptotermes sp termite cellulase. so that microcrystalline cellulose is obtained which will then be characterized and compared with the commercial version, namely Avicel® PH 101. The highest yield of microcrystalline cellulose is found in leaves with a yield of 95.3%, followed by leaf stalks 89.3%, flower stalks 75.7%, and the yield lowest in interest with a percentage of 74%. The physical characteristics in the form of color reaction, organoleptics, solubility and pH of white lotus microcrystalline cellulose powder show similarities with the comparison standard. It can be concluded that microcrystalline cellulose powder from several parts of the white lotus plant can be an alternative in obtaining cellulose raw materials from natural sources.

Nasal glucagon delivery using microcrystalline cellulose in healthy volunteers

The powder flow plays an important role in the manufacture of dosage form such as direct compression tablets. The objective of this research was to investigate the individual and interaction effects of magnesium stearate (MgSt) (Merck Ltd., Germany) as lubricant and sodium starch glycolate (SSG) (Primojel?) as superdesintegrator on powder flowability. Powder formulations were prepared by mixing different concentration of MgSt (0.25% and 5%) and SSG (0.5% and 4%) with microcrystalline cellulose (MCC) (Vivapur?101) or spray-dried lactose (LAC) (Super Tab 21AN) as diluents. Eight powder formulations were prepared using as filler LAC and MCC, as superdesintegrator SSG and as lubricant MgSt in ratios as follows: P1 (LAC : 0.5% SSG : 0.25% MgSt); P2 (LAC : 4% SSG : 5% MgSt); P3 (LAC : 0.5% SSG : 5% MgSt); P4 (LAC : 4% SSG : 5% MgSt); P5 (MCC : 0.5% SSG : 0.25% MgSt); P6 (MCC : 4% SSG : 5% MgSt); P7 (MCC : 0.5% SSG : 5% MgSt); P8 (MCC : 4% SSG : 0.25% MgSt). The powder formulations were evaluated using indirect methods of flowability evaluation according to 9th European Pharmacopoeia: measurement of tapped and bulk density (Ph.Eur.9, 2.9.34) and angle of repose (Ph.Eur. 2.9.36). Also, compressibility index, Hausner?s ratio were calculated. A full factorial design with three factors at two levels and response surface methodology were applied to evaluate the influence of various concentrations of MgSt and SSG on powder flowability. The results indicate that flow properties improved with decrease of SSG content and with increase of MgSt content. Magnesium stearate in powder formulations with microcrystalline cellulose has a greater influence on the improvement of the flow properties than powder formulations with spray-dried lactose.

Production of fungal cellulase was performed by the isolate of Penicillium expansum MDFS2 on rice straw, rice bran, and wheat straw under solid-state fermentation. The greatest potential growth was detected using rice bran as the carbon source substrate. On the fifth day of fermentation, filter paperase, carboxymethyl cellulase, and β-glucosidase obtained their maximal activities of 4.91 U/g substrate, 36.51 U/g substrate, and 12.21 U/g substrate, respectively. The optimum temperature for filter paperase was reported at 40 °C, whereas carboxymethyl cellulase and β-glucosidase were optimally active at 50 °C. Filter paperase and carboxymethyl cellulase showed maximum activity at pH 5.0. However, β-glucosidase proved to be maximally active at pH 6.0. According to the thermal stability results, all the three components of the cellulolytic enzyme complex proved to be less thermally resistant at 60 °C, as compared to 50 °C. β-glucosidase and carboxymethyl cellulase depicted the highest and the lowest thermal resistance, respectively. β-glucosidase and filter paperase stored for one week at -20 °C proved to be the most and least stable enzymes, respectively. It is hoped that current research findings will help in the cost-effective production of industrially important cellulases using agro-industrial by-products as fermentation substrates.

A novel form of cellulose powder was evaluated as a filler-binder in tablets. The particle, powder, flow and binding properties of this experimental cellulose material were compared with those of two commercial microcrystalline celluloses, Avicel PH 101 and Emcocel. The effect of various storage conditions on the physical stability of tablets compressed from celluloses was also evaluated. The particle size and shape of experimental cellulose powder differed markedly from those of microcrystalline celluloses. Experimental cellulose contained mainly large and roughly spherical agglomerates of particles, among which were few smaller regularly shaped particles. Because of spherical particle shape, the experimental cellulose powder flowed better than microcrystalline celluloses, which consisted of much more irregularly shaped particles. Experimental cellulose formed stronger tablets than microcrystalline celluloses. It also acted more effectively than microcrystalline celluloses as a binding material in tablets containing poorly compressible ascorbic acid and acetaminophenone. This may be due to the extensive surface area of the particles of experimental cellulose powder. The specific surface area of this material was over 50 times as great as that of microcrystalline celluloses. This indicates an extremely porous structure of cellulose agglomerates. Tablets containing experimental cellulose powder were able to resist a permanent loss in tablet strength at different storage conditions better than tablets containing microcrystalline celluloses. According to the results of this study an experimental agglomerated form of cellulose powder is a very advantageous material as a filler-binder for direct compression of tablets.

Tissue preparation for laser capture microdissection and RNA extraction from fresh frozen breast tissue

The amino acids involved in the coordination of two Zn2+ ions and one Mg2+ ion in the active site are well conserved from EAP (Escherichia coli alkaline phosphatase) to BIAP (bovine intestinal alkaline phosphatase), whereas most of their surrounding residues are different. To verify the consequences of this heterology on their specific activities, we compared the activity and structure recoveries of the metal-free forms (apo) of EAP and of BIAP. In the present study, we found that although the sensitivities of EAP and BIAP to ions remained similar, significant differences in dimeric structure stability of apo-enzymes were observed between EAP and BIAP, as well as in the kinetics of their activity and secondary structure recoveries. After mild chelation inactive apo-EAP was monomeric under mild denaturing conditions, whereas inactive apo-BIAP remained dimeric, indicating that the monomer-monomer contact was stronger in the mammalian enzyme. Dimeric apo-EAP (0.45 microM, corresponding to 4 units/ml) recovered approx. 80% of its initial activity after 3 min incubation in an optimal recovery medium containing 5 microM Zn2+ and 5 mM Mg2+, whereas dimeric apo-BIAP (0.016 microM, corresponding to 4 units/ml) recovered 80% of its native activity after 6 h incubation in an optimal recovery medium containing 0.5 microM Zn2+ and 5 mM Mg2+. Small and different secondary structure changes were also observed during activity recoveries of apo-BIAP and apo-EAP, which were not in parallel with the activity recoveries, suggesting that distinct and subtle structural changes are required for their optimal activity recoveries.

Powder formulations of fluorescein were prepared with carriers, including microcrystalline cellulose (MCC), hydroxypropylcellulose (HPC), and lactose or polystyrene particles (PP), and administered to rabbits intranasally. The order of fluorescein absorption in terms of the AUC was MCC > PP > HPC > lactose. To clarify the differences between the carriers, absorption and elimination of fluorescein from the nasal cavity were simultaneously determined using a newly developed in vivo method. Elimination of carriers was also determined by the method. To quantify insoluble MCC and PP, a new image-analysis method was developed. These results, with in vitro fluorescein release studies, suggest that the schematic role of carriers in nasal delivery is as follows. Both MCC and PP are insoluble and showed rapid fluorescein release. PP was eliminated rapidly, while MCC was retained in the cavity. This suggests that bioadhesive MCC probably forms a local environment with a higher fluorescein concentration between the particles and membrane, thus resulting in enhanced absorption. In the HPC formulation, fluorescein or HPC eliminations and fluorescein release were sustained due to its gel-forming property. In the lactose formulation, lactose dissolves rapidly, and there may be no means for fluorescein to stay near the mucous membrane. As a result, fluorescein was eliminated rapidly by mucus flow, thus resulting in poor absorption.

The purpose of this study was to investigate the effect of granulating water level on the physical-mechanical properties of microcrystalline cellulose (MCC) and silicified microcrystalline cellulose (SMCC). Granulations containing either MCC or SMCC were manufactured at different water levels using a high-shear mixer and were then tray-dried. The water level ranged from 0 to 100%. The granules were evaluated for size, granular and true density, porosity, flow, compactibility, compressibility, and strain-rate sensitivity index (SRS). Increasing the water level affected the size, increased the granular density and flow properties of the granules, and decreased the porosity and compactibility. The compactibilities for both materials were similar and acceptable at each granulating water level up to 40%. They both showed poor compactibility at higher water levels. Yield values and SRSs revealed that MCC and SMCC have similar compressibility, and that both exhibit a plastic component to the deformation process. The granulating water level had no statistically significant effect on the compressibility or the SRS for MCC or SMCC. SMCC did not offer practical advantages over MCC, other than better flow in the powder form, which could be attributed to slightly larger particle size and the presence of silicon dioxide in its structure.

A variety of methods to detect cellulase secretion by microorganisms has been developed over the years, none of which enables the real-time visualization of cellulase activity on a surface. This visualization is critical to study the interaction between soil-borne cellulase-secreting microorganisms and the surface of plant roots and specifically, the effect of surface features on this interaction. Here, we modified the known carboxymethyl cellulase (CMC) hydrolysis visualization method to enable the real-time tracking of cellulase activity of microorganisms on a surface. A surface was formed using pure CMC with acridine orange dye incorporated in it. The dye disassociated from the film when hydrolysis occurred, forming a halo surrounding the point of hydrolysis. This enabled real-time visualization, since the common need for post hydrolysis dyeing was negated. Using root-knot nematode (RKN) as a model organism that penetrates plant roots, we showed that it was possible to follow microorganism cellulase secretion on the surface. Furthermore, the addition of natural additives was also shown to be an option and resulted in an increased RKN response. This method will be implemented in the future, investigating different microorganisms on a root surface microstructure replica, which can open a new avenue of research in the field of plant root–microorganism interactions.