Assessment of molecular recognition element for the quantification of human epidermal growth factor using surface plasmon resonance

Background: A method for the selection of suitable molecular recognition element (MRE) for the quantification of human epidermal growth factor (hEGF) using surface plasmon resonance (SPR) is presented. Two types of hEGF antibody, monoclonal and polyclonal, were immobilized on the surface of chip and validated for its characteristics and performance in the quantification of hEGF. Validation of this analytical procedure was to demonstrate the stability and suitability of antibody for the quantification of target protein. Results: Specificity, accuracy and precision for all samples were within acceptable limit for both antibodies. The affinity and kinetic constant of antibodies-hEGF binding were evaluated using a 1:1 Langmuir interaction model. The model fitted well to all binding responses simultaneously. Polyclonal antibody (pAb) has better affinity (K D = 7.39e-10 M) than monoclonal antibody (mAb) (K D = 9.54e-9 M). Further evaluation of kinetic constant demonstrated that pAb has faster reaction rate during sample injection, slower dissociation rate during buffer injection and higher level of saturation state than mAb. Besides, pAb has longer shelf life and greater number of cycle run. Conclusions: Thus, pAb was more suitable to be used as a stable MRE for further quantification works from the consideration of kinetic, binding rate and shelf life assessment.

Nutrients and culture conditions requirements for the degradation of phenol by Rhodococcus UKMP-5M.

The capability of Rhodococcus UKMP-5M, isolated from petroleum contaminated soil, in the degradation of phenol was studied using shake flask culture. The effect of nutrients and cultivation conditions on growth of this bacterium and phenol degradation was investigated. Among the different types of medium tested (M1, M2, M3 and M4), M1 was found to be the preferred medium for growth of this bacterium and phenol degradation. The optimized cultivation conditions for growth of Rhodococcus UKMP-5M and phenol degradation were; 30oC, initial pH 7.5 and buffer concentration ranged from 5 to 50 mM. Improvement of growth and phenol degradation was achieved in medium supplemented with 2 g l-1 glucose. In addition, NaCl at a concentration of 0.1 g l-1 was required to enhance growth and phenol degradation. The addition of 0.4 g l-1 (NH4)2SO4 into the culture medium greatly enhanced phenol degradation. At optimal medium composition and cultivation condition, Rhodococcus UKMP-5M was able to utilize phenol at concentration up to 900 mg l-1. Results of this study showed that Rhodococcus UKMP-5M has potential to be used in the degradation of phenol.

Effect of extrinsic and intrinsic parameters on inulinase production by Aspergillus niger ATCC 20611

Background: Inulinase is a versatile enzyme from glycoside hydrolase family which targets the beta-2, 1 linkage of fructopolymers. In the present study, the effect of medium composition and culture conditions on inulinase production by Aspergillus niger ATCC 20611 was investigated in shake-flasks. Results: The highest extracellular inulinase (3199 U/ ml) was obtained in the presence of 25 percent (w/v) sucrose, 0.5 percent (w/v) meat extract, 1.5 percent (w/v) NaNO3 and 2.5 mM (v/v) Zn2+, at initial pH of 6.5, temperature 35ºC and 6 percent (v/v) of spores suspension in the agitation speed of 100 rpm. Surfactants showed an inhibitory effect on enzyme production. The optimum temperature for inulinase activity was found to be 50ºC. TLC analysis showed the presence of both exo- and endo-inulinase. Conclusion: Sucrose, Zn2+, and aeration were found to be the most effective elements in inulinase production by A. niger ATCC 20611. TLC analysis also showed that the crude enzyme contained both endo and exo-inulinases. The strain is suggested as a potential candidate for industrial enzymatic production of fructose from inulin.

High performance enzymatic synthesis of oleyl oleate using immobilised lipase from Candida antartica

High performance enzymatic synthesis of oleyl oleate, a liquid wax ester was carried out by lipase-catalysed esterification of oleic acid and oleyl alcohol. Various reaction parameters were optimised to obtain high yield of oleyl oleate. The optimum condition to produce oleyl oleate was reaction time; 5 min, organic solvents of log P is greater than or equal to 3.5, temperature; 40-50 ºC, amount of enzyme; 0.2-0.4 g and molar ratio of oleyl alcohol to oleic acid; 2:1. The operational stability of enzyme was maintained at >90 percent yield up to 9 cycles. Analysis of the yield of the product showed that at optimum conditions, >95 percent liquid wax esters were produced.