Influence of the pH of glutaraldehyde and the use of dextran aldehyde on the preparation of cross-linked enzyme aggregates (CLEAs) of lipase from Burkholderia cepacia

The preparation of cross-linked enzyme aggregates (CLEAs) of lipase has been a challenge due the low amount of lysine residues that lipases have on their surface. The results show that CLEAs prepared using dextran aldehyde (100-200KDa) have a higher hydrolysis activity and particle size (activities between 3186 +/- 21 U/g of CLEA and 4800 +/- 30 U/g of CLEA and particle sizes between 52.6 +/- 18.7 um and 126.2 +/- 53.5 um) than CLEAs prepared with glutaraldehyde (0.1 KDa) (activities between 894 +/- 16 U/g of CLEA and 2874 +/- 20 U/g of CLEA and particle sizes between 21.2 +/- 5.1 um and 83.4 +/- 24.9 um); Thermal stability assays of bioctalysts at 60 ºC at pH 7.0 using phosphate buffer 25 mM showed that CLEAs prepared with dextran aldehyde have lower residual activity after 50 hrs (maximum residual activity of 46.8 percent in the CLEA) than CLEAs prepared with glutaraldehyde (maximum residual activity of 70.2 percent in CLEA). When considering hydrolysis activity, thermal stability and residual activity of CLEAs as a criteria for selecting the best preparation conditions, it has been found that the best condition for CLEAs preparation are to use glutaraldehyde as cross-linking reagent at pH 9.5, at a concentration of 3.5 g/l, and an enzyme/albumin ratio of 15.

Affinity and response of Burkholderia pseudomallei and Burkholderia cepacia to insulin.

The cells of Burkholderia pseudomallei, B. cepacia and Pseudomonas aeruginosa grown on agar plates were stained with fluorescently-labeled insulin. The former two species were stained positively indicating insulin binding but P. aeruginosa was not. Insulin exposure reduced phospholipase C and acid phosphatase activities of B. pseudomallei but did not affect those enzymatic activities of B. cepacia in the employed experimental conditions. It is suggested that B. pseudomallei have insulin receptors which may be associated with a signal transfer system involving phospholipase and protein tyrosine phosphatase.