High throughput screening atrazine chlorohydrolase mutants with enhanced activity through Haematococcus pluvialis expression system / 生物工程学报

Developing a high-throughput screening method is of great importance for directed evolution of atrazine chlorohydrolase. A mutagenesis library of atzA from Pseudomonas sp. ADP and Arthrobacter sp. AD1 was constructed using error-prone PCR and DNA shuffling. Candidate mutants were screened through Haematococcus pluvialis expression system, using atrazine as selection pressure. Sequence analysis showed that mutations in the obtained 12 mutants with enhanced activity were all point-substitutions and scattered throughout the gene. Enzymatic activity analysis showed that the mutants all had higher activities than that of the wild type. The activities were 1.8-3.6 fold of the wild-type enzyme when cultured in BBM medium with 1 mg/L atrazine, whereas 1.8-2.6 fold with 2 mg/L atrazine. These results indicated that Haematococcus pluvialis expression system is an ideal high throughput screening system for directed evolution of atrazine chlorohydrolase.

Obtainment and Characterization of The Evolved Enzymes From Arabidopsis thaliana Glutathione S-Transferase Zeta Class / 生物化学与生物物理进展

The Arabidopsis thaliana glutathione S-transferases zeta class (AtGSTZ) is a multi-functional enzyme, which plays important role in cellular metabolism and environmental purification. Error-prone PCR and cycles of DNA shuffling were used to construct a mutagenesis library of AtGSTZ. The screening of the resultant libraries was carried out by a pH indicator dye-based colorimetric assay. Nine mutants which enhanced the dichloroacetic acid dechlorination activity were obtained. Among them, NN23 contained 25 amino acid substitutions with the activity improving 120%, whereas NN20 contained 24 amino acid substitutions with the activity improving 102%. EC1 contained 2 amino acid substitutions with the activity improving 47%. The rest 6 mutants contained one amino acid substitution with their activity increasing from 9% to 60%. The enzymatic characterization showed that all the evolved enzymes increased their catalytic efficiencies towards dichloroacetic acid and binding affinity towards glutathione whereas some of them increased the renaturability. However there is no obvious change in their thermostability. Based on these data, functional residues related to catalysis and refolding of AtGSTZ were discussed.

Expression of human leucocyte antigen in ectopic endometrial tissue of endometriosis / 中华妇产科杂志

Objective To investigate the human leucocyte antigen (HLA) expression of ectopic endometrial epithelial cells in cases with endometriosis and its role in pathogenesis of endometriosis Methods By immunohistochemical analysis,HLA expression was observed in ectopic endometrial epithelial cells of 25 endometriosis patients Ectopic endometrial cells were cultured in vitro successfully and the expression of HLA were analyzed by flow cytometry in 10 out of the 25 patients Eutopic endometrium of 15 normal women were taken as control Results (1)Expression of HLA class Ⅰ molecule on ectopic endometrial epithelial cells of endometriosis measured by the immunohistochemical analysis was lower than those of controls, (4 0?0 5) score and (1 2?0 8) score respectively So was the expression rate by flow cytometry in vitro culture (63 38?11 88)% and (5 27? 2 88)% respectively ( P

Relationship between range of operative and postoperative ovary function in 55 endometriosis / 中国实用妇科与产科杂志

ObjectiveTo study the relationship between the range of operations and the incidence of climacteric syndrom,the time of episode, degree and estrogen level in endometriosis after operations. MethodsThesexual hormone level was measured and climacteric syndrome was followed up after operation in 55 endometriosis. ResultsThe incidence of climacteric syndrom in oophorectomy was more frequent and earlier than partial oophorectomy. Meanwhile, the syndrome was much severe and the level of E2 was lower in the former group than those in the later one. ConclusionSo we should retain the ovary tissue and blood supply as much as possible. 6 months postoperation is suggested to be suitable to begin hormone replacement therapy. The dosage should be adviced to remain the blood hormone level just like one ovary does.