ABSTRACT
One pair of primers were designed and synthesized on the base of the cDNA sequence encoding Schizosaccharomyces pombe N-glycanase reported on the GenBank. The cDNA sequence encoding Peptide N-glycanase was cloned from the Schizosaccharomyces pombe by RT-PCR. And then the RT-PCR product was cloned into the expression vector pET-15b. The expression vector pET-15b(+)/Png1p was transformed into E. coli BL21(DE3). The results showed that the relative molecular weight of the enzyme was determined to be approximately 39 kD using SDS-PAGE. The expression products after induction and purification can catalyze the cleavage of N-linked oligosaccharides from glycoprotein coped with heat, but have no action on the native glycoprotein with the help of DTT. The percentage of deglycosylated RNase B treated with equate Png1p in different reaction temperature, pH, concentration of DTT and denatured temperature showed that the optimum temperature, the optimum pH is 30 degrees C; the optimum concentration of DTT is 10 mmol/L and the optimum denatured temperature is 100 degrees C.
Subject(s)
Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Glycosylation , Hydrogen-Ion Concentration , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase , Genetics , Metabolism , Recombinant Proteins , Genetics , Schizosaccharomyces , Genetics , TemperatureABSTRACT
To study the effect of the osmotic stress in the microenvironment on the growth and metabolism of the encapsulated cells under aerobic condition, Osmo-sensitive yeast Y02724 and high-osmotic resistant yeast Hansel were used as models to explore the growth and metabolism state of the cells cultivated inalginate-chitosan-alginate (ACA) microcapsules. The changes of the yeast cells' specific growth rate, maximum product quantity and the secretion of ethanol and glycerol were analyzed. For Y02724, the yield of ethanol was increased in the ACA microenvironment compared to suspension cultivation. For Hansel, the maximum growth speed of microencapsulated cultivation had no obvious difference compared to the suspension cultivation. Moreover, after encapsulation, the production of glycerol was decreased for both Y02724 and Hansel compared to suspension cultivation. In conclusion, osmotic stress existed in the ACA microcapsules and affected the growth and metabolism of the cells.