Ionic liquids (ILs) have been used as an alternative green solvent for lignocelluloses pretreatment. However, being a salt, ILs exhibit an inhibitory effect on cellulases activity, thus making the subsequent saccharification inefficient. The aim of the present study is to produce salt-tolerant cellulases, with the rationale that the enzyme also tolerant to the presence of ILs. The enzyme was produced from a locally isolated halophilic strain and was characterized and assessed for its tolerance to different types of ionic liquids. The results showed that halophilic cellulases produced from Aspergillus terreus UniMAP AA-6 exhibited higher tolerance to ILs and enhanced thermo stability in the presence of high saline conditions.
The protocol for the enzymatic deinking of laser printed waste papers on a laboratory scale using cellulase (C) and hemicellulase (H) of Aspergillus niger (Amano) was developed as an effective method for paper recycling. A maximum deinking efficiency of almost 73% by the enzyme combination of C:H was obtained using the deinking conditions of pulping consistency of 1.0% (w/v) with the pulping time of 1.0min, temperature of 50 degrees C, pH=3.5, agitation rate of 60rpm, pulp concentration of 4% (w/v), concentration of each enzyme of 2.5U/g air dried pulp and the enzyme ratio of 1:1. The deinking efficiency was further enhanced to 95% using the optimized flotation system consisting of pH=6.0, Tween 80 of concentration 0.5% (w/w), working air flow rate of 10.0L/min and temperature of 45 degrees C. The deinked papers were found to exhibit properties comparable to the commercial papers suggesting the effectiveness of the enzymatic process developed.
Aminoacylase I (EC. 3.5.1.14) was immobilized by covalent crosslinking to alginate molecules with 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide HCl followed by calcium alginate bead formation for the production of L-phenylalanine from the racemic mixtures of N-acetyl-DL-phenylalanine. Different concentrations of the coupling reagent were tested and the coupling process was optimized. The immobilized and the partially purified aminoacylase were characterized in terms of the activity, operational stability, thermal stability, pH and temperature optima and kinetic constants, Km and Vmax. The activity of the enzyme covalently immobilized in calcium alginate beads was enhanced by about 75% compared to that of free enzyme. The beads showed stable activity under operational conditions, they lost about 40% of their activity after four reaction cycles. The immobilized aminoacylase was more stable over a broader pH range. Thus this simple method provides irreversible immobilization of aminoacylase to give a biocatalyst with good operational stability and enhanced activity.
Aminoacylase I (E.C.3.5.1.14) was immobilized by entrapment in calcium alginate beads coated with polyethyleneimine for the production of L-phenylalanine by the hydrolysis of a racemic mixture of N-acetyl-DL-phenylalanine. The operational stability in terms of batch operation and continuous reaction in packed-bed bioreactor were studied. Kinetic constants, Km and Vmax values of free and immobilized enzymes were studied. Polyethyleneimine treatment was found to enhance the operational stability of the enzyme though its activity was substantially reduced. When polyethyleneimine-coated calcium alginate beads were packed into packed bed bioreactor, it was stable for at least 25 days under continuous operation without appreciable loss of activity.
Immobilization of cross-linked tannase on pristine multiwalled carbon nanotubes (MWCNT) was successfully performed. Cross-linking of tannase molecules was made through glutaraldehyde. The immobilized tannase exhibited significantly improved pH, thermal, and recycling stability. The optimal pH for both free and immobilized tannase was observed at pH 5.0 with optimal operating temperature at 30°C. Moreover, immobilized enzyme retained greater biocatalytic activities upon 10 repeated uses compared to free enzyme in solution. Immobilization of tannase was accomplished by strong hydrophobic interaction most likely between hydrophobic amino acid moieties of the glutaraldehyde-cross-linked tannase to the MWCNT.
Deep Eutectic Solvents (DESs) have recently emerged as a new generation of ionic liquids for lignocellulose pretreatment. However, DESs contain salt components which tend to inactivate cellulase in the subsequent saccharification process. To alleviate this problem, it is necessary to evaluate the applicability of the DESs-Cellulase system. This was accomplished in the present study by first studying the stability of cellulase in the presence of selected DESs followed by applicability evaluation based on glucose production, energy consumption and kinetic performance. Results showed that the cellulase was able to retain more than 90% of its original activity in the presence of 10% (v/v) for glycerol based DES (GLY) and ethylene glycol based DES (EG). Furthermore, both DESs system exhibited higher glucose percentage enhancement and lower energy consumption as compared to diluted alkali system. Among the two DESs studied, EG showed comparatively better kinetic performance.
The effect of enzymatic pretreatment on the degree of corn and mung bean starch derivatization by propylene oxide was investigated. The starch was enzymatically treated in the granular state with a mixture of fungal alpha-amylase and glucoamylase at 35 degrees C for 16 h and then chemically modified to produce enzyme-hydrolyzed-hydroxypropyl (HP) starch. Partial enzyme hydrolysis of starch in the granular state appeared to enhance the subsequent hydroxypropylation, as judged from the significant increase in the molar substitution. A variable degree of granule modification was obtained after enzyme hydrolysis, and one of the determinants of the modification degree appeared to be the presence of natural pores in the granules. Enzyme-hydrolyzed-HP starch exhibited significantly different functional properties compared to hydroxypropyl starch prepared from untreated (native) starch. It is evident that the dual modification of starch using this approach provides a range of functional properties that can be customized for specific applications.
Aminoacylase I (EC 3.5.1.14) encapsulated in calcium alginate beads stabilized with poly-L-lysine was used for the production of L-phenylalanine by the hydrolysis of a racemic mixture of N-acetyl-DL-phenylalanine. The immobilized aminoacylase was studied with respect to operational stability, thermal stability, effects of pH and temperature and kinetic constants. The leakage of enzyme from the stabilized beads was eliminated. The immobilized enzyme retained high biological activity. The Km and Vmax values for the stabilized beads were 11.11 mmol dm-3 and 0.076 mumol min-1 respectively. The optimum pH and temperature for the hydrolysis were 6.5 and 55 degrees C respectively. Scanning electron micrographs revealed crosslinked structures on the surface of the beads. The operational performances of the beads in a batch reaction and a packed-bed bioreactor for continuous reaction were investigated. With batch reaction, only about 5% of enzyme activity was lost within ten reaction cycles and there was no significant loss of activity over 600 h of continuous operation after equilibrium was reached, and a conversion yield of about 80% was obtained.
Although laccase has been recognized as a wonder molecule and green enzyme, the use of low yielding fungal strains, poor production, purification, and low enzyme kinetics have hampered its large-scale application. Thus,this study aims to select high yielding fungal strains and optimize the production, purification, and kinetics of laccase of Aspergillus sp. HB_RZ4. The results obtained indicated that Aspergillus sp. HB_RZ4 produced a significantly large amount of laccase under meso-acidophilic shaking conditions in a medium containing glucose and yeast extract. A 25 μM CuSO4 was observed to enhance the enzyme yield. The enzyme was best purified on a Sephadex G-100 column. The purified enzyme resembled laccase of A. flavus. The kinetics of the purified enzyme revealed high substrate specificity and good velocity of reaction,using ABTS as a substrate. The enzyme was observed to be stable over various pH values and temperatures. The peptide structure of the purified enzyme was found to resemble laccase of A. kawachii IFO 4308. The fungus was observed to decolorize various dyes independent of the requirement of a laccase mediator system.Aspergillus sp. HB_RZ4 was observed to be a potent natural producer of laccase, and it decolorized the dyes even in the absence of a laccase mediator system. Thus, it can be used for bioremediation of effluent that contains non-textile dyes.
Production cost of enzyme is largely determined by the type of the strain and raw material used to propagate the strain. Hence, selection of the strain and raw materials is crucial in enzyme production. For Glucose oxidase (GOx), previous studies showed Aspergillus terreus UniMAP AA-1 offers a better alternative to the existing sources. Thus, a lower production cost could be logically anticipated by growing the strain in a cheaper complex media such as molasses. In this work, sugar cane molasses, supplemented with urea and carbonate salt and a locally isolated strain Aspergillus terreus UniMAP AA-1 were used to produce a crude GOx enzyme in a small scale. A statistical optimization approach namely Response Surface Methodology (RSM) was used to optimize the media components for highest GOx activity. It was found that the highest GOx activity was achieved using a combination of molasses, carbonate salt and urea at concentration 32.51, 4.58 and 0.93% (w/v), respectively. This study provides an alternative optimized media conditions for GOx production using locally available raw materials.
Microbial mannanases have become biotechnologically important in industry but their application is limited due to high production cost. In presents study, the extraction of mannanase from fermented Palm Kernel Cake (PKC) in the Solid State Fermentation (SSF) was optimized. Local isolate of Aspergillus terreus SUK-1 was grown on PKC in (SSF) using column bioreactor. The optimum condition were achieved after two washes of fermented PKC by adding of 10% glycerol (v/v) soaked for 10 h at the room temperature with solvent to ratio, 1:5 (w/v).