色褐链霉菌磷脂酶D基因pld的克隆与表达

利用表达载体pET-22b( ),实现了色褐链霉菌磷脂酶D基因在大肠杆菌BL21(DE3)中的高效表达.利用镍亲和柱对表达产物进行纯化,将纯化后的重组磷脂酶D作用于底物卵磷脂和丝氨酸定向合成磷脂酰丝氨酸,并用HPLC法检测酶活力.结果表明,目的蛋白可在短时间内进行大量表达.转酯反应6 h后卵磷脂的转化率达到31%,重组磷脂酶D活力达到39 U·(mg蛋白)-1.     

血浆特定蛋白检测在IgG层析法中试纯化工艺中的应用

目的通过血浆特定蛋白检测,对层析法纯化IgG的中试工艺进行质量控制。方法采用免疫散射比浊法及BNP特定蛋白分析仪及配套试剂,全面检测生产用和中试纯化工艺用原料血浆的组成,建立血浆中白蛋白(albumin,ALB)、IgG、IgA、IgM、补体3(compliment 3,C3)、C4、转铁蛋白(transferin,TRF)、α1-抗胰蛋白酶(α1-antitrypsin,AAT)、α2巨球蛋白(alpha2-macroglobin,A2M)、触珠蛋白(haptoglobin,HPT)、α1酸性糖蛋白(alpha1-acid glycoprotein,AAG)、铜蓝蛋白(ceruloplasmin,CER)、纤维蛋白原(fibrinogen,FIB)、抗凝血酶Ⅲ(antithrombinⅢ,ATⅢ)、IgG1、IgG2、IgG3、IgG4 18项特定蛋白分布的数据库;对3批中试纯化工艺各步骤中ALB、IgG、IgA、IgM、FIB、AAT的去除情况及IgG回收率进行监测,验证工艺的稳定性;将3批中试纯化工艺制备的原液及成品中IgG含量的检测结果与凯氏定氮法检测结果进行比较,验证免疫散射比浊法的准确度及不同人员重复检测的中间精密度;对中试纯化工艺及低温乙醇法制备的IgG原液的杂质含量及亚类进行比较分析。结果中试纯化工艺用原料混浆中仅TRF、A2M、ATⅢ含量与生产用原料混浆差异有统计学意义(P<0.05);中试纯化工艺用原料血浆经A2P亲和层析去除了大部分的ALB、AAT,辛酸盐沉淀去除了剩余的ALB、FIB和IgM,DEAE离子交换纯化去除了IgA和ALB,IgG总回收率高于70%,表明该工艺稳定性好,提高了目标蛋白的得率。中试纯化工艺制备的IgG原液及成品的检测CV值均<10%,回收率在90%¹10%之间,表明该方法准确度及精密度良好;与低温乙醇法制备的IgG原液相比,中试纯化工艺制备的原液杂质含量更低,IgG的亚类分布与所用的原料血浆相似。结论血浆特定蛋白检测在血浆蛋白纯化工艺的监控和优化中具有应用价值。     

产磷脂酶菌株的筛选鉴定及其应用

采用磷脂平板初筛和摇瓶复筛,从富油土样中筛选得到一株产磷脂酶菌株BIT-18。经菌株形态特征、生理生化特征及16S rRNA序列分析,鉴定其为荧光假单胞菌（Pseudomonas fluorescens）。以磷脂标准品（1-棕榈酰-2-油酰-Sn-甘油-3-磷脂酰胆碱）为底物,通过气相色谱分析反应产物的脂肪酸成分,定性鉴定P.fluorescens BIT-18表达的磷脂酶为B型磷脂酶。该酶为低温酶,最适温度和pH值分别为25℃和6.5,低浓度的金属离子有利于其酶促反应进行。以磷脂酶B为催化剂在自制间歇式反应器中对大豆油进行酶法脱胶,在加酶量500 U·kg^-1,加水量2%,温度40℃,pH 4.7的条件下反应6 h,脱胶油磷含量由90.1 mg·kg^-1降至4.6 mg·kg^-1,脱胶率高达94.9%,显示出良好的应用前景。     

磷脂酶A1高产菌株的筛选及其等离子体诱变

目的从富油土壤中筛选磷脂酶A1高产菌株,优化其发酵条件,并经等离子诱变提高其产酶能力。方法用筛选培养基和发酵培养基分别对富油土壤中磷脂酶A1产生菌进行初筛和复筛,经气相色谱鉴定产酶类型;对获得的高产菌株进行形态、生理生化特征和分子生物学鉴定;优化该菌株的发酵时间,温度,pH值及摇床转速;从该菌株出发进行等离子体诱变,绘制致死率曲线,进行平板初筛及摇瓶复筛,并检测获得菌株的遗传稳定性。结果经初筛和复筛,获得1株磷脂酶A1高产菌株W22,经气相色谱鉴定其产酶为磷脂酶A1;该菌株在普通琼脂培养基上呈圆形,质地软,表面光滑、扁平,乳白色,晕圈明显,为芽孢杆菌属蜡样芽孢杆菌;最佳发酵条件为:发酵时间12 h,初始pH 8.0,温度32℃,摇床转速200 r/min,优化后酶活为9.12 U/ml,较优化前(6.74 U/ml)提高了35.3%;等离子体诱变后,经平板及摇瓶筛选,获得的菌株W22-5酶活最高,为12.80 U/ml,较诱变前(9.12 U/ml)提高了89.91%,且具有良好的遗传稳定性。结论成功获得1株磷脂酶A1高产菌株W22,优化了该菌的发酵条件,且经等离子诱变进一步提高了菌株的产酶能力,为磷脂酶A1的生产和应用奠定了基础。     

米曲霉GX0011 β-果糖基转移酶的分离纯化

通过热变性、硫酸铵分级沉淀、DEAE-Sephadex A-50(柱1)、Octyl-Sepharose CL-4B、Sephacryl S-300、DEAE-Sephadex A-50(柱2)层析等分离纯化步骤对本实验室自行筛选诱变的米曲霉GX0011来源的β-果糖基转移酶进行了分离纯化,并通过SDS-PAGE检测了该酶的纯度和亚基分子量.结果表明电泳一条带,亚基表观分子量为5.1×104,小于多数文献报道的结果.纯化倍数为121.7,比活和活力回收分别为1975 U·mg-1和17%.酶活测定表明该酶只起到转果糖基的作用而无水解活性.     

戊唑醇农药降解菌的筛选及其降解效能初探

[目的]从自然环境中分离纯化得到戊唑醇农药降解菌株,为该农药生产及使用过程中带来的环境污染问题的解决提供菌种。[方法]通过富集培养和平板分离技术,利用高效液相色谱法和平板培养法测定菌株对戊唑醇的降解及耐受性能。[结果]得到4株(DZ-1、DZ-2、DZ-3、DZ-4)具有高效、高耐受性及遗传稳定性好的菌株,其降解戊唑醇农药的效率分别为91.5%、89.9%、92.2%、90.8%;菌株DZ-1、DZ-2、DZ-4和DZ-3对戊唑醇农药耐受限分别为3000、3000、2500、2500 mg/L;4株菌的传代菌株对戊唑醇农药降解效率分别为90.7%、89.5%、92.2%、90.4%,菌株DZ-1、DZ-2和DZ-3、DZ-4的传代菌株对戊唑醇农药耐受限分别为3 000、2 500 mg/L。[结论]试验采用的富集培养及平板分离技术能有效分离纯化到不同环境中的菌株;高效液相色谱法和平板培养法能够对降解和耐受性能进行准确检测。     

C.I.酸性橙7标准样品的研制

介绍了C.I.酸性橙7标准样品研制过程。该高纯度样品是用化学方法合成得到初纯,再经重结晶及硅胶柱层析纯化分离而得到。对纯化后的样品分装,装瓶后的标准样品再进行均匀性和稳定性评价,以上各项工作完成后确定标准样品的标准值。本文所研制的C.I.酸性橙7标准样品用于对食品中违禁添加的C.I.酸性橙7检测的质量保证与质量控制。     

植物乳杆菌凝集素的纯化及其体外抗HIV活性

目的纯化植物乳杆菌(L.plantarum PCL)凝集素,并检测其体外抗HIV活性。方法采用Sephadex G-25凝胶脱盐层析和HITrap Q Sepharose XL离子交换层析纯化L.plantarum PCL凝集素;测定纯化产物的兔红细胞凝集素活性、纯度及荧光光谱;采用MTT法检测其对C8166淋巴细胞的毒性作用,以光镜检查其对HIV-1ⅢB诱导C8166细胞形成合胞体的抑制作用,并计算TI值。结果纯化的L.plantarum PCL凝集素经尿素-SDS-PAGE,可见相对分子质量约85000的单一条带;荧光光谱可见在380nm附近有一个发射强度较大的凝集素特征荧光发射峰;L.plantarum PCL凝集素对C8166细胞的IC50为2.239μg/ml,对HIV-1ⅢB诱导C8166细胞合胞体形成的EC50为0.079μg/ml,TI值为28.34。结论已成功纯化了L.plantarum PCL凝集素,并具有抗HIV活性。     

肺炎链球菌补体结合蛋白C3-BP的纯化及其免疫效果

目的对9V型肺炎链球菌补体结合蛋白C3-BP进行纯化及免疫效果分析。方法通过三氧醋酸沉淀、离子交换层析、凝胶过滤层析和亲和层析等方法,从9V型肺炎链球菌培养上清中提取C3-BP,Lowry法检测其浓度,SDS-PAGE检测其纯度及大小,Western blot检测其补体C3结合活性。将纯化蛋白免疫NIH小鼠,ELISA法测定小鼠血清IgG抗体,MTT法测定小鼠脾淋巴细胞增殖情况,活菌攻击,检测其交叉保护效果。结果经纯化的表面蛋白C3-BP,其相对分子质量约为90 000,为单链蛋白,浓度为0.21 mg/ml,纯度为90.7%,具有与补体C3结合的特性。免疫小鼠后,抗体滴度达到1∶38 802。免疫组小鼠脾脏与胸腺淋巴细胞代谢活性明显增强,与对照组相比,二者刺激指数之间差异有显著意义。9V型C3-BP免疫小鼠后,不仅对9V型肺炎链球菌株有保护作用,对2型、6B型和14型肺炎链球菌菌株也有保护作用。结论摸索出了一种纯化肺炎链球菌表面蛋白C3-BP的方法,纯化的C3-BP具有不依赖血清型的交叉保护作用,而且体液免疫及细胞免疫效果良好。     

工艺研究

Na-9-芴甲氧羰基-氨基酸合成方法的研究；从微生物发酵液中纯化普伐他汀前体美伐他汀方法的研究；一类磷脂酶甲2的抑制剂合成方法的研究；含有磺酰基的二苯基苯并咪唑化合物的研究；用古龙酸制备粗维生素C新方法的研究；光学纯S-（-）-和R-（＋）-奥美拉唑制剂的研究……     

Protein kinase C-dependent stimulation of phospholipase D in phospholipase C-treated fibroblasts

Treatment of [¹⁴C]choline- or [¹⁴C]ethanolamine-labeled NIH 3T3 fibroblasts withBacillus cereus phosphatidylcholine-specific phospholipase C (PLC) enhanced phospholipase D (PLD)-mediated hydrolysis of the respective¹⁴C-labeled phospholipids. PLD activity was stimulated by 1.5 U/mL of POLC and by 100 nM of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) to similar extents. Treatment of¹⁴C]palmitic acid-labeled fibroblasts with PLC in the presence of ethanol also enhanced PLD-mediated formation of phosphatidylethanol; the effects of PLC and PMA were nonadditive. PLC had no effect on PLD activity in fibroblasts in which PKC was down-regulated by prolonged (24 h) treatment with 300 nM PMA. These data indicate that treatment of fibroblasts with exogenous PLC results in PKC-dependent activation of PLD.     

New Aqueous Injection Molding Process for Ceramic Powders

An aqueous injection molding process based on the gelling properties of certain naturally occurring polysaccharides, in particular agar, and its purified derivative, agarose, is described. The materials form nonviscous solutions at temperatures near 100°C which solidify to rigid gels upon cooling below the so-called gel point temperature, 37°C. Gels formed by these materials are strong, nominally 1500 g/cm² for agar and 2500 g/cm² for agarose at 3 wt% concentration. Molded parts can be dried and fired without use of absorbent powders or special debinding operations of any kind.     

On the Enhanced Antibacterial Activity of Antibiotics Mixed with Gold Nanoparticles

The bacterial action of gentamicin and that of a mixture of gentamicin and 15-nm colloidal-gold particles on Escherichia coli K12 was examined by the agar-well-diffusion method, enumeration of colony-forming units, and turbidimetry. Addition of gentamicin to colloidal gold changed the gold color and extinction spectrum. Within the experimental errors, there were no significant differences in antibacterial activity between pure gentamicin and its mixture with gold nanoparticles (NPs). Atomic absorption spectroscopy showed that upon application of the gentamicin-particle mixture, there were no gold NPs in the zone of bacterial-growth suppression in agar. Yet, free NPs diffused into the agar. These facts are in conflict with the earlier findings indicating an enhancement of the bacterial activity of similar gentamicin–gold nanoparticle mixtures. The possible causes for these discrepancies are discussed, and the suggestion is made that a necessary condition for enhancement of antibacterial activity is the preparation of stable conjugates of NPs coated with the antibiotic molecules.     

An improved assay for the determination of phospholipase C activity

Phospholipases C (PLC, EC 3.1.4.3) are enzymes that specifically hydrolyze the C‐O‐P bond in phospholipids, yielding sn‐1,2(2,3)‐diacylglycerides and the phosphate residue bearing the corresponding headgroup. The biochemical characterization of PLC requires methods for the reliable determination of their activity. Here, an assay is described in which the phosphate residue released by the PLC is cleaved with an alkaline phosphatase. The phosphate formed is then extracted with n‐butanol and quantified as phosphomolybate complex. The applicability of this method is demonstrated for a concentration range from 10 nM to 10 mM for a range of phospholipids bearing different headgroups in an aqueous and a two‐phase system. The method has the additional advantage that the crude enzyme can be used without the need for purification.     

Critical surfactant concentration in the interaction between nonionic surfactants and polymeric acids. effects of temperature,pH and salts

Critical surfactant concentrations at which the binding of nonionic surfactants to polyacrylic acid, or complex formation, abruptly occurred in aqueous solution were lower than the CMC and were temperature-independent. The complex was precipitated by pH lowering or salt addition. At low pH, precipitation limit surfactant concentration (PLC) existed, below which no precipitation of the complex took place, and the PLC coincided with the critical concentration mentioned above. In this case the PLC did not change with temperature either. In the precipitation caused by A1C1```3`` addition, the PLC was a little higher than that at low pH, because Al ions induced not only shrinking and agglomeration of the complex but at the same time blocked the sites on the polymeric acid for hydrogen bonding with the surfactant. By NaCl or CaCl```2`` addition no PLC was found, because in both cases the salting-out effect dominated. Martially presented at the 77th Annual AOCS-JOCS Joint Meeting in May 1986 in Honolulu.     

Oxaliplatin Causes Transient Changes in TRPM8 Channel Activity

Oxaliplatin is a third-generation platinum-based anticancer drug that is widely used as first-line treatment for colorectal carcinoma. Patients treated with oxaliplatin develop an acute peripheral pain several hours after treatment, mostly characterized by cold allodynia as well as a long-term chronic neuropathy. These two phenomena seem to be causally connected. However, the underlying mechanisms that trigger the acute peripheral pain are still poorly understood. Here we show that the activity of the transient receptor potential melastatin 8 (TRPM8) channel but not the activity of any other member of the TRP channel family is transiently increased 1 h after oxaliplatin treatment and decreased 24 h after oxaliplatin treatment. Mechanistically, this is connected with activation of the phospholipase C (PLC) pathway and depletion of phosphatidylinositol 4,5-bisphosphate (PIP₂) after oxaliplatin treatment. Inhibition of the PLC pathway can reverse the decreased TRPM8 activity as well as the decreased PIP₂-concentrations after oxaliplatin treatment. In summary, these results point out transient changes in TRPM8 activity early after oxaliplatin treatment and a later occurring TRPM8 channel desensitization in primary sensory neurons. These mechanisms may explain the transient cold allodynia after oxaliplatin treatment and highlight an important role of TRPM8 in oxaliplatin-induced acute and neuropathic pain.     

Random mutagenesis on the Pseudomonas lipase activator protein, LipB: exploring amino acid residues required for its function

LipB, lipase activator protein from Pseudomonas aeruginosa TE3285, specifically recovers the enzymatic activity of denatured inactive lipase. To find important amino acid residues of LipB in this reactivation, random mutagenesis using error-prone PCR was performed on a gene encoding the functional region of LipB. The resultant DNA library was introduced into the lipase expression system using Escherichia coli, and LipB mutants lacking lipase activity were selected by two screening procedures. First, on agar plates containing tributyrin as a substrate for lipase, single colonies lacking active lipase secretion were selected as clones missing the active LipB. Second, to exclude nonsense and frameshift mutants, the molecular size of LipB in the given clones was confirmed by Western blotting. From the selected mutants, of which multiple residues are replaced, five single- residue substituted mutants were newly prepared. Consequently, Y99C, Y99H, S102R and R115C mutants formed no detectable complex with the lipase and lost the in vitro reactivation activity. In the case of Y99C and R115C, their single cysteine residue formed the intermolecular disulfide bridge. Thus, Tyr99 and Arg115 are likely to exist on the molecular surface of LipB, and are candidates for residues that make direct interaction with the denatured lipase in the reactivation process.      

乌鸦果根甲醇提取物的抑真菌作用研究

乌鸦果根经甲醇回流提取、石油醚脱脂得到鸟鸦果根甲醇提取物。采用双层培养基法测定其抑真菌活性、琼脂梯度稀释法测定最小抑菌浓度和最小杀菌浓度。结果表明,乌鸦果根甲醇提取物（1000mg·mL^-1）对供试的21种真菌均有不同程度的抑菌活性。最小抑菌浓度和最小杀菌浓度均为31．2-250mg·mL^-1;且提取物抑菌能力与其浓度呈正相关关系。表明乌鸦果根甲醇提取物具有广谱抑制真菌作用,可以作为植物源农药进一步深入研究,具有潜在的开发利用价值。     

Semicrystalline thermoplastic polyimide + polymer liquid crystal blends: Nonisothermal calorimetry and thermogravimetry

Blends of a thermoplastic polyimide (TPI) and a polymer liquid crystal (PLC) were studied using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The presence of PLC enhances orientation in the system and lowers the crystallization temperature-—a manifestation of the channeling effect predicted theoretically (38). The degradation studies show high temperature stability of all blends with the degradation onset consistently above 520°C. The onset decreases with PLC concentration but reaches a plateau above 30 wt% PLC when the PLC-rich islands are formed. The amount of moisture absorbed decreases with the PLC concentration while the moisture does not affect the degradation of the samples significantly. A phase diagram is constructed for the PLC + TPI blends from the DSC and TGA data. A comparison of amorphous and semicrystalline TPI and the characterization of amorphous TPI + PLC blends will be reported later.     

Hybrid Hydrogels Based on Poly(vinyl alcohol) (PVA)/Agar/Poly(ethylene glycol) (PEG) Prepared by High Energy Electron Beam Irradiation: Investigation of Physico‐Mechanical and Rheological Properties

A series of hybrid hydrogels based on poly(vinyl alcohol) (PVA)/agar/poly(ethylene glycol) (PEG) prepared by a solution casting method using e‐beam irradiation are investigated to determine the effect of agar and PEG content (1, 2, and 4 wt%) on their physicomechanical and rheological properties. The gel content of the hydrogels decreases with increasing agar and PEG contents. The equilibrium swelling of PVA hydrogel decreases on blending with agar while adding PEG to PVA/agar increases the swelling by about 400%. No obvious change in the dehydration behavior of the hybrid hydrogels is observed on changing agar and PEG contents. The solid‐like rheological behavior of the hydrogels is not significantly affected by agar content, while it approaches a liquid‐like behavior at high PEG loading. The tensile strength of the hybrid hydrogels is improved by increasing agar content, while its elongation‐at‐break is decreased. On the other hand, the opposite results are found regarding the influence of PEG and its content on the mechanical properties of the hybrid hydrogels.