Subject(s)
Abnormalities, Multiple/diagnosis , Gigantism/diagnosis , Abnormalities, Multiple/drug therapy , Abnormalities, Multiple/genetics , Blood Glucose/analysis , Brain Damage, Chronic/prevention & control , Congenital Hypothyroidism , Diagnosis, Differential , Female , Gigantism/drug therapy , Gigantism/genetics , Hernia, Umbilical/congenital , Hernia, Umbilical/diagnosis , Hernia, Umbilical/drug therapy , Hernia, Umbilical/genetics , Humans , Hypoglycemia/drug therapy , Hypothyroidism/diagnosis , Infant , Male , Phenytoin/therapeutic use , Precancerous Conditions , Sucrose/therapeutic use , Syndrome , Tongue/abnormalitiesABSTRACT
Substitution of the N-terminus of Streptomyces olivaceoviridis xylanase XYNB to generate mutant TB has been previously shown to increase the thermostability of the enzyme. To further improve the stability of this mutant, we introduced a disulfide bridge (C109-C153) into the TB mutant, generating TS. To assess the effect of the disulfide bridge in the wild-type enzyme, the S109C-N153C mutation was also introduced into XYNB, resulting in XS. The mutants were expressed in Pichia pastoris, the recombinant enzymes were purified, and the effect of temperature and pH on enzymatic activity was characterized. Introduction of the disulfide bridge (C109-C153) into XYNB (XS variant) and TB (TS variant) increased the thermostability up to 2.8-fold and 12.4-fold, respectively, relative to XYNB, after incubation at 70 degrees C, pH 6.0, for 20 min. In addition, a synergistic effect of the disulfide bridge and the N-terminus replacement was observed, which extended the half-life of XYNB from 3 to 150 min. Moreover, XS and TS displayed better resistance to acidic conditions compared with the respective enzymes that did not contain a disulfide bridge.
Subject(s)
Disulfides/chemistry , Endo-1,4-beta Xylanases/chemistry , Streptomyces/enzymology , Dithiothreitol/pharmacology , Hydrogen-Ion Concentration , Mutation , TemperatureABSTRACT
Insect-specific scorpion neurotoxin AaIT gene inserted into a binary vector was transferred into a hybrid poplar clone N-106(P. deltoides x P. simonii) growing in the Southern of China. We obtained sixty-two regenerated plants by Agrobacterium tumefaciens transferring system. PCR and PCR-Southern analysis showed that AaIT gene was incorporated into the genome of some recovered poplar plants. One of the transformed plants named A5 was significantly resistant to feeding by first instar larvae of Lymantria dispar, compared with the untransformed control plant. It caused a decrease in leaf consumption by larvae, a lower larval weight gain and a higher larval motality rate of Lymantria dispar. ELISA analysis proved that AaIT gene was expressed in this transfomed poplar plant.