ABSTRACT
Background & objectives: Reusable biostrip consisting enzymes immobilized onto alkylamine glass beads affixed on plasticized PVC strip for determination of triglyceride (TG) suffers from high cost of beads and their detachments during washings for reuse, leading to loss of activity. The purpose of this study was to develop a cheaper and stable biostrip for investigation of TG levels in serum. Methods: A reusable enzyme-strip was prepared for TG determination by co-immobilizing lipase, glycerol kinase (GK), glycerol-3-phosphate oxidase (GPO) and peroxidase (HRP) directly onto plasticized polyvinyl chloride (PVC) strip through glutaraldehyde coupling. The method was evaluated by studying its recovery, precision and reusability. Results: The enzyme-strip showed optimum activity at pH 7.0, 35oC and a linear relationship between its activity and triolein concentration in the range 0.1 to 15 mM. The strip was used for determination of serum TG. The detection limit of the method was 0.1 mM. Analytical recovery of added triolein was 96 per cent. Within and between batch coefficients of variation (CV) were 2.2 and 3.7 per cent, respectively. A good correlation (r=0.99) was found between TG values by standard enzymic colrimetric method employing free enzymes and the present method. The strip lost 50 per cent of its initial activity after its 200 uses during the span of 100 days, when stored at 4oC. Interpretation & conclusions: The nitrating acidic treatment of plasticized PVC strip led to glutaraldehyde coupling of four enzymes used for enzymic colourimetric determination of serum TG. The strip provided 200 reuses of enzymes with only 50 per cent loss of its initial activity. The method could be used for preparation of other enzyme strips also.
ABSTRACT
Permeabilized cells containing trehalose synthase from Meiothermus sp.CBS-01 was immobilized by the integration of entrapment in alginate beads with cross-linking and electrostatic self-assembly coating technique.Coating on alginate beads with diazo-resin and poly(styrene sulfonate) can protect the beads from degradation in phosphate buffer,whereas cross-linking with carbodiimide improves the thermostability of trehalose synthase entrapped in alginate beads.By co-immobilization of permeabilized cells using entrapment-cross-linking- coating method,the enzyme recovery was 32%,and the optimum temperature of the enzyme was still 60℃,but the optimum pH was shifted from 6.5 to 7.In batch manner,a maximum conversion yield of 60% trehalose from maltose could be reached by the immobilized cells.Within 4 times repeated use of the immobilized cells,and each time was operated at 50℃ for 24h,more than 50% conversion yield of trehalose could be maintained with less than 20% loss of the enzyme activity.