Co-immobilization of Carbohydrases and Proteases by Nanomagnetic Combi-CLEAs Method for Oil and Protein Hydrolysates Extraction from Oil Seeds in Aqueous Phase at Single Step Process

BACKGROUND AND OBJECTIVESFunctionalized magnetic nanoparticles are effective enzyme carriers since they have several advantages such as easily separation under external magnetic fields as well as enhancing mass transfer and reusability of enzymes. In this article, three types of enzymes included Viscozyme L (carbohydrases), Alcalase ۲.۴ L (endo-peptidase), and Flavourzyme (Exo-peptidase) Simultaneously were immobilized by nanomagnetic cross-linked enzyme aggregates (CLEAs) method. Then assessment of the results obtained was performed. At first, Fe۳O۴ nanoparticles were synthesized by chemical co-precipitation then, surface coating procedure was used for functionalization of Fe۳O۴ by lysine amino acid. Thereafter, enzyme aggregation and cross-linking was achieved by using of enzymes with functionalized Fe۳O۴ and glutaraldehyde in saturated ammonium sulfate, and/or solvents such as, acetone, acetonitrile, tert-butanol, isopropanol, and ethanol at ۳-۴° C separately. Afterthat, for size reduction of formed NM-Combi-CLEAs, high speed homogenizer and then ultrasonic waves were applied. Then, produced NM-Combi-CLEAs was hold at ۳-۴° C for ۳-۲۴ hours and cross-linked enzymes were separated from liquid phase by centrifuge at ۱۵۰۰۰ rpm accurately. Finaly, activity assay, FE-SEM images and EDX, DLS and zeta potential analysis, FTIR, degree of hydrolysis (DH%), kinetic parameters (Km, Vmax, kcat, kcat/Km, t۱/۲, kd) and thermodynamic parameters (ΔG, ΔH, ΔS, Ea(in)) of immobilized enzymes compared to native enzymes mixtures was evaluated. The NM-Combi-CLEAs kept ۷۵-۸۰% of its original activity after ۱۰ cycles, which proposes strong operational stability. In conclusion, the NM-Combi-CLEAs are thermo-stable, reusable, and efficient nanobiocatalyst for enzymatic oil and protein hydrolysates extraction in aqueous phase.In this present work, a new nanobiocatalyst is fabricated by using of co-immobilization of various enzymes (Viscozyme, Alcalase, Flavourzyme) in nano scale. This method consists of the simultaneous nano aggregation of the free enzymes in an appropriate solvent and cross-linking of aggregates containing functionalized Fe۳O۴ by glutaraldehyde via ε-NH۲ groups of accessible surface lysine residues. ۲۸۰The produced efficient nanobiocatalysts displayed improvement in thermal stability and reusability. Moreover, mass transfer limitation, missed fine CLEAs during recovery processes are eliminated.MATERIALS AND METHODSViscozyme L produced from a selected strain of Aspergillus aculeatus, Alcalase ۲.۴ L, (from Bacillus licheniformis) and Flavourzyme ۱۰۰۰ L (from Aspergillus oryzae) were supplied by Novozymes (Bagsvaerd, Denmark). starch, pectin, casein, galacturonic acid, glutaraldehyde (۲۵% v/v in water), sodium potassium tartrate, ۳,۵ dinitrosalicylic acid, FeCl۲, FeCl۳, D(+) glucose, L-lysine monohydrochloride (>۹۹%), and ascorbic acid were purchased from Merck. Bovine serum albumin (BSA) was obtained from Fluka company. Coomassie brilliant blue (G-۲۵۰) was purchased from GE Healthcare (Uppsala, Sweden). All other chemicals were supplied by Merck (Darmstadt, Germany).RESULTS AND DISCUSSIONIn the present research, multi-enzyme immobilization in aqueous phase process in a single step process was used for oil and protein hydrolysates extraction from sesame seeds and rice bran. At first, the enzyme activity, protein content, and optimum process conditions for Viscozyme, Flavourzyme, and Alcalase, were evaluated. Then, the effect of coinciding use of proteases on the activity of Viscozyme was assessed. The results revealed that, ۲:۱ ratio of water/oil seeds, Viscozyme L amount ۱.۵ kg per ton of oil seeds, ۰.۱% Alcalase, ۰.۵% Flavourzyme per oil seeds protein at ۵۵ ° C, pH ۵.۵, ۶ h reaction time, and inactivation of enzymes at ۸۵-۸۰ ° C for ۱۵ minutes were the optimal process conditions. The oil extraction efficiency was ۹۳% with ۸۳-۸۴% oleic and linoleic essential fatty acids. The protein hydrolysates efficiency was about ۳ times that of the original protein with molecular mass of ۱۰-۲۰ kDa and its methionine was raised about ۲ times. The essential amino acid and nutritional indices were enhanced ۵۱.۳۴%, and ۵۱.۴۱% respectively compared to raw protein.CONCLUSIONThe removal of toxic hexane solvent, using the simultaneously three types of enzymes in the aqueous phase process for oil and protein hydrolysates extraction with high efficiency is the novelty of this research. Thus, this process introduces as an efficient, eco-friendly and safe method.

کلیدواژه ها:

Enzyme immobilization ، Nanomagnetite ، Cross-linked enzyme aggregates ، nanobiocatalyst ، Kinetics and Thermodynamics.