Directed Evolution of E. coli Alkaline Phosphatase Towards Higher Catalytic Activity

Directed evolution was used to enhance the catalytic activity of E. coli alkaline phosphatase (EAP). Through two rounds of error-prone PCR and one round of DNA shuffling followed by a rapid, sensitive screening procedure, several improved variants were obtained. Their enzymatic kinetic properties, thermal stabilities and possible mechanism for the improvement were investigated. In 1.0 M Tris buffer, the specific activity of the most active EAP variant S2163 was 1500 units/mg protein, showing it to be 3.6 times more active than the D101S parent enzyme and ∼40 times more active than the wild-type EAP. At the same time, the K_m value of the S2163 variant decreased to 1491 μM from the 2384 μM of the D101S. As a result, the k_cat/K_m ratio of this variant showed a 5.8-fold enhancement over that of D101S parent enzyme. Three activating amino acid substitutions, K167R, G180S and S374C, which were located far away from the center of the catalytic pocket, were identified by sequencing the genes encoding evolved enzymes. Possible explanations for the improvement of activity were analyzed.     

In vitro activation of yeast pro-carboxypeptidase Y expressed as inclusion bodies in Escherichia coli

The genes encoding pro-carboxypeptidase Ys (pro-CPYs) from Saccharomyces cerevisiae and Hansenula polymorpha were cloned and expressed in Escherichia coli. Most of the expressed pro-CPY was present in the form of inclusion bodies, accounting for about 35% of the total cellular protein. The inclusion bodies solubilized in 6 M guanidinum chloride were renatured by dilution 1:60 into renaturation buffer. Further refolding and activation were followed by the addition of 60 g ml_–1 proteinase K into the diluted solution. The specific activities of in vitro activated S. cerevisiae and H. polymorpha CPYs were found to be similar, representing about 25% of that of native S. cerevisiae CPY.     

高比活力大肠杆菌磷酸转乙酰酶的纯化

从国内大肠杆菌菌株1.505提取磷酸转乙酰酶(PTA),经数步纯化荻得了高比活力酶制品。该酶制品比粗酶纯化了61倍,比活力达1040单位/mg,经聚丙烯酰胺凝胶电泳检查,仅有两条蛋白质区带。用经过纯化的酶测定辅酶A,其结果6.75单位/10微升酶液在光径1cm时,233nm的吸收值△E_(PTA)<0.009。     

双歧杆菌对实验性肠梗阻肠道G^—菌及血内毒素影响的研究

本实验采用大白鼠的急性、单纯性、机械性肠梗阻模型,将80只大鼠随机分成5组:正常对照组、梗阻未治疗组、新霉素治疗组、甲哨唑治疗组、双歧杆菌治疗组。分别将各制刑直接肠道给药。24h后,对梗阻近端回肠末段的肠杆菌、双歧杆菌进行定性及定量检测,并同时测定各组门静脉血内毒素值。结果表明,新霉素及甲哨唑治疗组双歧杆菌明显减少(P<0.01);门静脉血内毒素明显升高(P<0.01),达到未治疗组的水平(P<0.05)。新霉素治疗组肠杆菌明显减少(P<0.01),甲哨唑治疗组肠杆菌明显增加(P<0.01),达到未治疗组的水平(P>0.05)。未治疗组肠杆菌及门静脉血内毒素明显增加(P<0.01),双歧杆菌无明显减少。双歧杆菌治疗组双歧杆菌明显增加(P<0.01),肠杆菌保持在正常水平(P>0.05),门静脉血内毒素明显低于未治疗组和各抗生素治疗组(P<0.01)。本实验说明,急性肠梗阻时,肠道内肠杆菌数量及门静脉血内毒素可明显升高;外源性双歧杆菌在肠道内能抑制肠杆菌,降低门静脉血内毒素水平;新霉素及甲哨唑能引起肠道菌群紊乱,促使血内毒素过度升高。因此,利用双歧杆菌制剂作为急性肠梗阻治疗申的辅助用药,具有一定实际意义。     

人白细胞介素1受体拮抗剂cDNA克隆及其表达影响因素

采用ＰＣＲ技术成功地从人胎盘ｃＤＮＡ文库中扩增出人白细胞介素１受体拮抗剂（ｈＩＬ １ｒａ）成熟蛋白编码区基因,并以此为模板突变其ｃＤＮＡ５;其克隆产物经物理图谱证实与文献报道完全一致,利用含不同ＳＤ的同一表达质粒分别表达含不同５的ｃＤＮＡ,结合文献分析其表达影响因素,同时对ｈＩＬ １ｒａＥ．ｃｏｌｉ表达产物的可能存在形式进行了推论。     

手机辐射对大肠杆菌生长影响作用的研究

目的：探索了解手机辐射对大肠杆菌生长与繁殖的影响。方法：用不同型号的手机以通话方式对大肠杆菌进行辐射,检测大肠杆菌培养后的茵落的大小和数量,比较实验组与对照组的差异。结果：对照组与手机辐射组茵落数量相近,经统计学分析,两组菌落数差别无显著性,但是经手机辐射后,菌落的直径大于对照组,不同型号的手机之间也有差别,且差别有显著性（P〈0．05）。结论：手机辐射对大肠杆菌生长可能有影响,但其具体机制还有待进一步的研究进行阐明。     

Insight into the remarkable affinity and selectivity of the aminobenzosuberone scaffold for the M1 aminopeptidases family based on structure analysis

Aminopeptidases are ubiquitous hydrolases that cleave the N‐terminal residues of proteins and oligopeptides. They are broadly distributed throughout all kingdoms of life and have been implicated in a wide variety of physiological processes, including viral infection, parasite metabolism, protein processing, regulation of peptide hormones, and cancer cell proliferation. Members of the M1 family, also termed gluzincins, are defined by two highly conserved motifs in the catalytic domain: a zinc‐binding motif, HEXXH‐(X18)‐E; and an exopeptidase motif, GXMEN. We report the high‐resolution X‐ray structures of E. coli aminopeptidase N (PepN) in complex with three aminobenzosuberone scaffolds that display various Ki values (50, 0.33, and 0.034 µM) and provide a compelling view of the outstanding selectivity of these chemical entities for the M1 aminopeptidases. This series of inhibitors interacts as transition state mimics with highly conserved residues of the catalytic machinery and substrate recognition sites. Structural comparisons and model‐building studies allowed a deep interpretation of the SAR observed for bacterial, as well as mammalian enzymes. Proteins 2017; 85:1413–1421. © 2017 Wiley Periodicals, Inc.     

Epithelial Membrane Protein 2 and β1 integrin signaling regulate APC-mediated processes

Adenomatous Polyposis Coli (APC) plays a critical role in cell motility, maintenance of apical-basal polarity, and epithelial morphogenesis. We previously demonstrated that APC loss in Madin Darby Canine Kidney (MDCK) cells increases cyst size and inverts polarity independent of Wnt signaling, and upregulates the tetraspan protein, Epithelial Membrane Protein 2 (EMP2). Herein, we show that APC loss increases β1 integrin expression and migration of MDCK cells. Through 3D in vitro model systems and 2D migration analysis, we have depicted the molecular mechanism(s) by which APC influences polarity and cell motility. EMP2 knockdown in APC shRNA cells revealed that APC regulates apical-basal polarity and cyst size through EMP2. Chemical inhibition of β1 integrin and its signaling components, FAK and Src, indicated that APC controls cyst size and migration, but not polarity, through β1 integrin and its downstream targets. Combined, the current studies have identified two distinct and novel mechanisms required for APC to regulate polarity, cyst size, and cell migration independent of Wnt signaling.     

T4-like coliphage ΦKAZ14 virulent to pathogenic and extended spectrum β-lactamase-producing Escherichia coli of poultry origin

<正>Dear Editor,Bacteriophages(otherwise called phages)are a type of virus that infect bacteria.This viral type has found useful applications in the control of bacterial pathogens in foods and food processing environments.In addition,phages may be useful to prevent colonization and shedding of bacteria into the surrounding environment.