Improvement of catalytic capability of Paecilomyces thermophila J18 thermostable beta-1,3-1,4-glucanase under acidic condition by directed evolution / 生物工程学报

Directed evolution was used to improve the performance of beta-1,3-1,4-glucanase (designated as PtLicl6A) from Paecilomyces thermophila J18 under acidic condition. A mutant library was constructed by error-prone PCR and DNA shuffling, and positive clones were screened by Congo red staining. More than 1 500 mutants were selected. One mutant (PtLic16AM1) exhibited an optimal activity at pH 5.5, while the optimal pH of the wild-type enzyme was 7.0. The mutant PtLic16AM1 kept the high specific activity and thermotolerence of the wild-type enzyme. Sequence analysis revealed that the mutant enzyme has four sense substitutions which caused four amino acid substitutions - namely T58S, Y110N, G195E and D221G.. Homology modeling showed that among the four amino acid substitutions, Y110N was near the active site of the enzyme, while the other three was distant. T58S and G195E may play key roles in the change of optimal pH. This study provided a new perspective of obtaining applicable 3-1,3-1,4-glucanase for industrial use.

Antiproliferation and apoptosis of K562 cells by Astragalus Mongholicus Lectin / 中国药理学通报

Aim To investigate the effects of Astragalus Mongholicus Bunge Lectin (AMML) on tumor cells proliferation,cell cycle and apoptosis by using human leukemia cell line (K562 cells).Methods The antiproliferation effect of AMML on K562 cells was detected by the colorimetric MTT assay.The apoptosis induced by AMML on K562 cells was explored by means of cell morphological and flow cytometry.Results AMML showed strong inhibiton of the growth of K562 cells in a time-and concentration-dependence. After incubation of K562 cells with AMML at a concentration of 60 mg?L-1 for 72 h,the inhibition ratio was 89%.Morphological observation showed that AMML-treated K562 cells displayed outstanding apoptosis characteristics,such as nuclear fragmentation,chromatin condensation. AMML induced significant cell cycle arrest at S phase in K562 cells,and the apoptosis of K562 cells was confirmed by flow cytometry.Conclusion AMML can inhibit the growth of K562 cells through S arrest and induce the apoptosis of K562 cells. Thus,AMML may be valuable for the treatment of cancer.

Isolation and purification of a lectin from roots of Astragalus membranaceus / 中草药

Objective To isolate and purify a lectin from the roots of Astragalus membranaceus.Methods The protein was purified using a combination of 20%—60% ammonium sulfate fraction and ConA-Sepharose 4B affinity chromatography.Results The purified protein appeared as a single band with molecular mass of 3.15?104 on SDS-PAGE and the relative molecular mass was estimated by gel filtration on a calibrated Superdex 75 column with apparent molecular weight of 3.35?104.This lectin was a glycoprotein with a neutral carbohydrate content of 10.7%.Conclusion A lectin is isolated and purified from the roots of A.membranaceus for the first time.It is a monomer glycoprotein and its specific activity is 391.9 U/mg.