Production, characterization, and immobilization of protease from the yeast Rhodotorula oryzicola.

The protease was produced extracellularly in submerged fermentation by the yeast Rhodotorula oryzicola using different sources of nitrogen and maximum activity (6.54 × 10-3 U/mg) was obtained in medium containing 2% casein (w/v). Purification of the protease by gel filtration chromatography resulted in a 3.07-fold increase of specific protease activity. The optimal pH and temperature for enzyme activity were 6.51 and 63.04 °C, respectively. Incubation in the presence of some salts enhanced enzyme activity, which peaked under 0.01 M BaCl2 . The enzyme retained about 90% of enzymatic activity at temperatures 50-60 °C. The commercially available enzyme carriers evaluated, silica gel, Celite 545, and chitosan effectively immobilized the protease. The enzyme immobilized in Celite 545 retained 73.53% of the initial activity after 15 reuse cycles. These results are quite promising for large-scale production and immobilization of protease from R. oryzicola, as the high operational stability of the immobilized enzyme lowers production costs in biotechnological applications that require high enzymatic activity and stability under high temperatures.

Synthesis of Feruloyl Ester Using Bacillus subtilis AKL 13 Lipase Immobilized on Celite® 545.

The lipophilic antioxidants, glyceryl ferulate and feruloyl glyceryl linoleate, were synthesized using lipase from Bacillus subtilis AKL 13. The extracellular lipase was produced by cultivation of the strain in modified minimal medium and the enzyme was recovered by fractionation at 80% ammonium salt saturation. The concentrated enzyme with the specific activity of (4647±66) U/mg was immobilized on Celite® 545 and crosslinked using glutaraldehyde. The prepared enzyme catalyst was used for esterification of ferulic and linoleic acids with glycerol separately in hexane butane solvent system at 50 °C and 3.144×g agitation. The maximum ester conversion of 94% of feruloyl glyceryl linoleate was achieved at 48 h, whereas only 35% of glyceryl ferulate was synthesized. The reaction products were characterized using RP-HPLC, FTIR, 1H NMR, 13C NMR and fluorescence spectrophotometry. The kinetic parameters of esterification reaction were determined according to ping-pong bi-bi model. The Km and υmax were found to be 69.37 and 3.46 mmol, and 0.387 and 1.02 mmol/(min·g) for glyceryl ferulate and feruloyl glyceryl linoleate, respectively. The kinetic parameters were simulated in MATLAB and the experimental data were in good agreement. Furthermore, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity of the blend of feruloyl ester and palm oil was higher than of the plain palm oil and was closer to α-tocopherol.

An insight into methylene blue adsorption characteristics onto functionalized alginate bio-polymer gel beads with λ-carrageenan-calcium phosphate, carboxymethyl cellulose, and celite 545.

Herein, λ-carrageenan-calcium phosphate (λ-Carr-Cp), carboxymethylcellulose (CMC) and celite 545 (Ce545) were used to functionalize sodium alginate bio-polymer (SAB). The water content in the wet beads was about 98%. Fourier Transform Infra-Red (FT-IR) showed that SAB@λ-Carr-Cp, SAB@CMC, and SAB@Ce545 could interact through intermolecular hydrogen and electrostatic interactions. Scanning Electron Microscopy (SEM) revealed that the morphology of the functionalized beads had a porous sheet structure. Energy Dispersive X-Ray (EDX) depicted that the peak intensity of the carbon element became more intense in the case of organic modification. The appearance of mineral elements characteristics of Ce545 (Si, Al) and Cp (P) proved the incorporation of these inorganic reagents into the alginate matrix. The data from batch adsorption experiments indicated that the adsorption of methylene blue (MB) depended on pH, time, temperature, and dye concentration. At equilibrium, the adsorption capacities followed the order: SAB@Ce545 (7.5 mg g-1 wet) > SAB@λ-carr-Cp (6.8 mg g-1 wet) > SAB@CMC (6.5 mg g-1 wet) > SAB (5.77 mg g-1 wet). Kinetic data agreed with pseudo second-order suggesting chemi-sorption mechanism. The equilibrium data fitted for Freundlich. In summary, this provided assembly strategy could offer a new research opportunity for developing functionalized biopolymers with new functions and potential applications.

Whole cell immobilization of refractory glucose isomerase using tris(hydroxymethyl)phosphine as crosslinker for preparation of high fructose corn syrup at elevated temperature.

Glucose isomerase (GI) responsible for catalyzing the isomerization from d-glucose to d-fructose, was an important enzyme for producing high fructose corn syrup (HFCS). In a quest to prepare HFCS at elevated temperature and facilitate enzymatic recovery, an effective procedure for whole cell immobilization of refractory Thermus oshimai glucose isomerase (ToGI) onto Celite 545 using tris(hydroxymethyl)phosphine (THP) as crosslinker was established. The immobilized biocatalyst showed an activity of approximate 127.3 U/(g·immobilized product) via optimization in terms of cells loading, crosslinker concentration and crosslinking time. The pH optimum of the immobilized biocatalyst was displaced from pH 8.0 of native enzyme to neutral pH 7.0. Compared with conventional glutaraldehyde (GLU)-immobilized cells, it possessed the enhanced thermostability with 70.1% residual activity retaining after incubation at 90°C for 72 h. Moreover, the THP-immobilized biocatalyst exhibited superior operational stability, in which it retained 85.8% of initial activity after 15 batches of bioconversion at 85°C. This study paved a way for reducing catalysis cost for upscale preparation of HFCS with higher d-fructose concentration.