Specific Activity Of Mutant Enzyme Research Articles

Improved thermostability of type I pullulanase from Bacillus thermoliquefaciens by error-prone PCR

Pullulanase (PulB) is a starch-debranching enzyme. In order to improve its catalytic performance, random mutagenesis was performed on the pullulanase gene derived from Bacillus thermoliquefaciens. Two rounds of error-prone PCR were carried out. Mutant T252S was screened in the first round of error-prone library, which had the highest catalytic activity. During the second round of mutations, mutant enzyme G250P/T252S/G253T/N255K was screened, which had further improved catalytic activity and the best thermostability. Compared with the parent enzyme, the specific activity of mutant enzyme G250P/T252S/G253T/N255K increased by 1.9 times, Km decreased by 22.7 %, kcat increased by 28.7 %, and kcat/Km increased by 68.4 %. The thermostability of the mutant enzyme improved significantly, showing that the half-life at 60 °C was extended to 7.5 h, which was 87.5 % higher than that of the parent enzyme. The mutation sites in these two rounds were concentrated in the 250–255 regions, indicating that this region was an important region affecting the catalytic activity and Thermostability. The reasons for the change of enzymtic properties was also preliminarily analyzed through three-dimensional simulation.

Conditions affecting the activity of glucocerebrosidase purified fom spleens of control subjects and patients with type 1 Gaucher disease

Glucocerebrosidase was purified to homogeneity from spleens of control subjects and Type 1 Gaucher disease patients by immunoaffinity chromatography. Activation of the enzyme by taurocholate, phosphatidylserine and sphingolipid activator protein (saposin C; SAP-2) was investigated by titration of combinations of various effectors in the absence and presence of Triton X-100. The specific activity of Type 1 Gaucher glucocerebrosidase was found to be less than 20% of the corresponding congrol value under most conditions. However, in the presence of optimal amounts of activator protein SAP-2 and phosphatidylserine (and in the absence of Triton X-100 and/or taurocholate), the specific activity of mutant enzyme towards artificial and natural lipid substrates was close to normal when measured at pH 5.0–5.5. At pH values below 5.0, the specific activity of mutant enzyme decreased more rapidly compared to that of control enzyme. The activity of Type 1 Gaucher glucocerebrosidase in the intact cell might, in a comparable manner, be highly dependent on the extent of activation by endogenous activators and on the intralysosomal pH. Values for residual glucocerebrosidase activity, as measured in vitro in extracts of cells and tissues from Type 1 Gaucher disease patients, are indeed highly dependent on the assay conditions employed. Consequently such measurements are of little value in the assessment of the actual capacity for glucosylceramide hydrolysis and for prediction of the clinical severity of the disease.