BIOCATALYTIC ACTIVITY OF RECOMBINANT HUMAN Β-Mannosidase IMMOBILIZED ONTO MAGNETIC NANOPARTICLES FOR BIOPROCESS

Abstract

Recombinant human β-mannosidase (rhMANB) is an important glycosidase enzyme that degrades mannose-linked glycoproteins and mannan polysaccharides. rhMANB was purified and covalently immobilized onto magnetic nanoparticles. The immobilization of the enzyme was confirmed by Fourier-transform infrared spectroscopy (FTIR) and magnetic nanoparticles linked immunosorbent assay (MagLISA). Antibodies against rhMANB were raised, purified and characterized for MagLISA. The binding of rhMANB onto magnetic nanoparticles was found to be 65%. The V( max ) and K( m ) of immobilized rhMANB was observed 3.0-fold higher and 2.024-fold lower, respectively, as compared to unbound rhMANB. The stability and activity of immobilized enzyme was observed at different pH, temperature, and after storage at 4°C. Metal chelators (oxalic acid, citric acid, and ascorbic acid) did not affect the enzyme activity of immobilized enzyme, whereas ethylenediamine tetraacetic acid reduced the activity. The results obtained from thin-layer chromatography indicate that immobilized rhMANB is more efficient than the unbound form to hydrolyze mannobiose, mannotriose, mannotetraose, mannopentose, galactoglucomannan, and locust bean gum. Magnetic nanoparticles suspended gel-permeation chromatography showed that 29% locust bean gum hydrolyzed efficiently during flow in the column. The immobilization of rhMANB will be a good process for gelling and saccharification of mannan polymers at industrial scale.

Immobilization of indigenous holocellulase on iron oxide (Fe 2 O 3 ) nanoparticles enhanced hydrolysis of alkali pretreated paddy straw

“…In one such study, Khoshnevisan et al [12] reported cellulase loading to magnetic NP ratio of 0.033 which was very low. However, some other researchers have obtained a higher protein loading of 117 mg/g silica NP and 130 mg/g magnetic NP for cellulase and mannosidase respectively [36,37]. The enzyme loading depends on properties of enzyme, shape and size of NPs and ligand properties [42].…”

“…Out of total 93 GHs identified, GH3 (22), GH 18 (14), GH 5 (7), GH10 (7) and GH 31 (6) Table 2. Various researchers have reported immobilization efficiencies ranging from 50-145% for different enzymes immobilized on various solid supports [12,[36][37][38]. Physical adsorption method is very simple and does not require any cross linker which makes this method less expensive.…”

Anticancer Medicines (Doxorubicin and Methotrexate) Conjugated With Magnetic Nanoparticles for Targeting Drug Delivery Through Iron

“…The number of amino groups attached onto MNP (less than 0.2 mm) was determined by using the molar extinction coefficient 6623 M À1 =cm. [27,28] The particles were stored at 4 C in water.…”

“…The dried pellets were processed for Fourier-transform infrared (FTIR) analysis as described. [27] Development of Anti-DOX and Anti-MTX Antibodies DOX was conjugated with bovine serum albumin (BSA) and oval albumin (OVA) by a two-step glutaraldehyde method. Similarly, MTX was also conjugated with bovine serum albumin (BSA) and oval albumin (OVA) by a two-step glutaraldehyde method.…”

“…[27] The MNP were suspended in double-distilled water and sonicated in a sonicator (soniprep 150) at 30 kHz five times in 2-min intervals on ice. The sonicated and dispersed magnetic nanoparticles were separated and passed through a 0.2-mm filter.…”