基于表达元件和宿主优化促地衣芽胞杆菌高效表达L-天冬酰胺酶

L-天冬酰胺酶(L-asparaginase, L-ASN)广泛用于恶性肿瘤治疗及低丙烯酰胺食品生产,然而其较低的表达水平限制了应用推广。异源蛋白表达是提高目标酶表达水平的有效策略,芽胞杆菌广泛用于酶蛋白的高效生产,本研究拟通过表达元件及宿主优化提高芽胞杆菌(Bacillus)中L-天冬酰胺酶产量。首先,筛选了5种信号肽(SP_SacC、SP_AmyL、SP_AprE、SP_YwbN、SP_WapA)用于L-天冬酰胺酶的分泌表达,其中SP_SacC介导下L-天冬酰胺酶分泌效果最好,酶活达到157.61 U/mL。随后,选取了4种芽胞杆菌强启动子(P43、P_ykzA-P43、P_Ubay、P_{bac A}),其中串联启动子P_ykzA-P43介导的L-天冬酰胺酶表达量最高,较对照菌株提高了52.94%。最后,筛选了3种芽胞杆菌表达宿主：地衣芽胞杆菌(Bacillus licheniformi...     

大肠杆菌Nissle1917 L-天冬酰胺酶Ⅱ基因在大肠杆菌BL21中的表达与抗肿瘤活性

【目的】从大肠杆菌Nissle1917中获得L-天冬酰胺酶Ⅱ基因,并研究其抗肿瘤活性。【方法】以大肠杆菌Nissle1917基因组为模板PCR扩增L-天冬酰胺酶Ⅱ基因,克隆至可诱导表达载体pET28a上。将L-天冬酰胺酶Ⅱ表达载体pET28a-asp转化至大肠杆菌BL21(DE3)中并通过IPTG诱导表达,经聚丙烯酰胺凝胶电泳(SDS-PAGE)和液相色谱-质谱(LC-MS)对表达的L-天冬酰胺酶Ⅱ进行鉴定,并通过镍柱亲和层析纯化收集表达出的L-天冬酰胺酶Ⅱ。用纯化定量以后的L-天冬酰胺酶Ⅱ作用小鼠乳腺癌4T1细胞、人肝癌Hep-3B细胞和人脐静脉内皮细胞HUVEC。【结果】来自于大肠杆菌Nissle1917的L-天冬酰胺酶Ⅱ基因可在大肠杆菌BL21中高效表达并通过LC-MS得到鉴定,细胞毒性实验结果表明L-天冬酰胺酶Ⅱ对4T1细胞和Hep-3B细胞的生长具有较强的抑制作用,而对人脐静脉内皮细胞HUVEC的生长无明显抑制效果。【结论】来源于大肠杆菌Nissle1917的L-天冬酰胺酶Ⅱ能显著抑制4T1细胞和Hep-3肿瘤细胞的生长,而对人正常组织细胞的生长无明显抑制效果,为进一步研究L-天冬酰胺酶Ⅱ特异性抗肿瘤作用机制和对实体瘤的应用研究奠定了重要基础。     

米黑根毛霉来源l-天冬酰胺酶的分子改造及高效表达

l-天冬酰胺酶能够水解l-天冬酰胺生成l-天冬氨酸和氨,广泛存在于微生物、植物和部分啮齿类动物的血清中,在医药和食品行业中都具有重要应用。然而无论是在医药还是在食品行业中,l-天冬酰胺酶依然存在一些问题,如催化效率低、热稳定性差、产量低等。文中通过理性设计及5′非翻译区 (5′ untranslated region, 5′ UTR)改造提高米黑根毛霉Rhizomucor miehei来源的l-天冬酰胺酶 (RmAsnase) 的酶活及蛋白表达量。结果显示,通过同源建模结合序列比对分析构建的6个突变菌株中,突变酶A344E比酶活较野生酶提高了1.5倍。继而构建食品安全菌株枯草芽孢杆菌Bacillus subtilis 168/pMA5-A344E,对其进行UTR改造,获得重组菌株B. subtilis 168/pMA5 UTR-A344E,其酶活较原始菌提高了7.2倍,对重组菌B. subtilis 168/pMA5 UTR-A344E进行5 L罐研究,最终产量为489.1 U/mL。该酶活提高的重组菌株对l-天冬酰胺酶的工业化应用具有重要价值。     

聚乙二醇修饰重组人天冬酰胺酶的研究

天冬酰胺酶对治疗急性淋巴细胞白血病和某些肿瘤疾病有很好的疗效,但由于其在人体内所产生的副作用———过敏反应限制了它的应用。目的:运用化学修饰剂聚乙二醇对天冬酰胺酶进行化学修饰,以降低其免疫原性,提高其在体内的半衰期;并使修饰后的天冬酰胺酶的活性保持较高的水平。结果:修饰后的天冬酰胺酶的免疫原性降低为原来的20-30%;用胰酶水解24h没有变化;而其比活力为未修饰酶的23%。该结果已经具备了很大的临床应用价值。     

生物转化法制备L-天冬酰胺

探索生物转化法制备L-天冬酰胺的技术与工艺。通过分子生物学方法,克隆来源于大肠杆菌(Escherichia coli, E.coli)JM109的天冬酰胺合成酶A基因asnA,并于E. coli BL21(DE3)中表达,利用构建的E.coli基因工程菌E.coli BL21(DE3)/pET28a(+)-asnA全细胞高密度催化L-天冬氨酸生产L-天冬酰胺,以PITC柱前衍生-高效液相检测底物和产物。表达的蛋白质分子质量约为37kDa,与预期大小相符,比酶活力为1786.6U/g。L-天冬氨酸转化率为95.8%,L-天冬酰胺产量可达126.5g/L,生产速率为15.81g/(L·h)。结果表明,已成功构建高效表达天冬酰胺合成酶A基因工程菌株,并用于催化L-天冬氨酸转化生产L-天冬酰胺,解决了L-天冬酰胺生物转化生产工艺中ATP成本过高的难题,为L-天冬酰胺制备提供新的绿色途径。     

天冬酰胺酶及PEG_2-天冬酰胺酶对L-天冬酰胺、谷氨酰胺亲和性的研究

本文报导了天冬酰胺酶及PEG_2-天冬酰胺酶对废物L-天冬酰胺、谷氨酰胺亲和性的研究,结果表明:PEG_2-天冬酰胺酶对谷氨酰胺的亲和性明显强于天冬酰胺酶(Km值分别为7.35×10~(-3)mol/L和7.14×10~(-2)mol/L),对天冬酰胺的亲和性略强于天冬酰胺酶(Km值分别为2.9×10~(-5)mol/L和4.0×10~(-5)mol/L)。天冬酰胺酶和PEG_2-天冬酰胺酶的CD光谱表明:天冬酰胺和谷氨酰胺对天冬酰胺酶和PEG_2-天冬酰胺酶的构象影响较大,但天冬酰胺酶和PEG_2-天冬酰胺酶的构象变化趋势有明显的不同。     

蓖麻蚕丝腺L-天冬酰胺酶的纯化及其性质

本文总结从蓖麻蚕5龄幼虫的丝腺经过匀浆、离心、氯化锰沉淀、硫酸铵盐析、加热处理、离子交换柱层析、吸附层析、凝胶过滤等步骤纯化L-天冬酰胺酶的过程,并对其生化性质进行了研究。纯化后L-天冬酰胺酶的比活力为原始上清液的246倍。该酶的分子量为112,000,由4个亚基组成;在溶液中以及冻于过程中,酶分子会相互聚合成多聚体。该酶在pH8.5,0.1M硼酸缓冲液中的K_m值为1.1×10^-4M,最适pH为8.5,在pH7.5-10.0的范围内,都具有较高的活力。PO₄^≡、SO₄⁼、Cl^-等离子对该酶有激活作用。蓖麻蚕丝腺L-天冬酰胺酶很不稳定,特别在偏碱性溶液中极易失去活力;L-天冬氨酸、二硫苏糖醇、乙二胺四乙酸对其有保护作用,其中以L-天冬氨酸的作用最为明显。另外,还分析了该酶的氨基酸组成等,并与不同来源的L-天冬酰胺酶进行了比较研究。     

L-天冬酰胺酶与产物的相互作用

本文从几方面探讨了产物L-天冬氨酸和L-天冬酰胺酶之间的相互作用。产物与珀琥酸对酶荧光影响的比较研究以及产物存在下对碘离子淬灭酶荧光性质的观察表明产物和酶之间存在直接的相互作用。产物保护天冬酰胺酶抗胰蛋白酶水解及抗热失活作用暗示它结合在酶的活性部位附近。     

L-天冬酰胺酶工程菌株培养条件及稳定性

L-天冬酰胺酶工程菌株的酶活和表达水平受菌体生物量和诱导时间的影响。在生物量Ａ６００03×１０左右,热诱导4h酶活力和表达水平可达到较高水平。葡萄糖对酶的生成有阻遏作用,当葡萄糖浓度大于025%时,对工程菌酶的合成造成阻遏。确定了工程菌培养的培养基、pH值、接种量等因素。重组质粒pASN在\%E.coli\% JM105,TG1和AS1357等宿主菌中具有很好的稳定性,工程菌培养50代以上重组质粒保留90%以上,在LB和M\|3培养基中也较稳定。     

白蚁与动物及微生物的共生类型及其机制

综述了白蚁螱客的主要种类、共生关系及相关机制的研究进展。白蚁螱客中,已报道的动物种类达170种。在与动物的共生关系中存在偏利共生（宾主共栖和异种共栖）、互利共生和无关共生三种;在与微生物的共生关系中,存在与内生菌（原生动物、细菌、真菌和放线菌）和外生菌（蚁巢伞菌等）间的互利关系。指出了白蚁与螱客研究中存在的问题,给出了解决方案,并提出了今后可能的研究热点或方向,为白蚁的综合利用（如纤维素酶）及今后研究物种间的协同进化提供了基础资料。     

New amino-dithiaphospholanes and phosphoramidodithioites and their rhodium and iridium complexes

New sulfur derivatives of phosphoramidite ligands were synthesized and the impact of the sulfur unit on the spectroscopic properties of their rhodium and iridium complexes was investigated. The new ligands Bn₂NPSCH₂CH₂S^a(P-S^a) (Bn = benzyl, 4), Bn₂NPSCHCHS^a(CH₂)₃C^aH₂(P-S^a)(C^a-S^a) (6) and Bn₂NP(4-XC₆H₄OMe)₂ (X = S, 7a; X = O, 7b) were converted to the rhodium and iridium complexes trans-[Rh(CO)Cl(L)₂] (L = 4, 6, 7), [RhCl(COD)(L)] (L = 4, 6, 7), [IrCl(COD)(7a)] and [IrCl₂Cp∗(6)]. For comparison, some phosphoramidite complexes of these formulations also were synthesized. The new metal complexes were spectroscopically analyzed. For the carbonyl complexes, the ν_CO IR stretching frequencies were lower than for the corresponding phosphite and phosphoramidite ligands. The ¹J_PRh coupling constants for the rhodium complexes with the new ligands were also smaller than for the respective phosphoramidite and phosphite complexes. Finally, the ¹J_PSe coupling constants of the selenides of the new ligands were lower than those of the phosphoramidite ligands but higher than for PPh₃. The spectroscopic data reveal that the new thio ligands 4, 6 and 7a are more electron donating than phosphites and phosphoramidites but less electron donating than PPh₃.     

Astrocytes and Synaptosomes Transport and Metabolize Lactate and Acetate Differently

Astrocytes transport the monocarboxylate acetate, but synaptosomes do not. The reason for this is unknown, because both preparations express monocarboxylate transporters (MCT). The transport and metabolism of lactate, another monocarboxylate, was examined in these two preparations, and the results were compared to those for acetate. Lactate transport is more rapid in astrocytes than in synaptosomes, but of lower affinity (Kms of 17 and 4 mM, respectively). Lactate (0.2 mM) is metabolized to CO2 more rapidly in synaptosomes than in astrocytes (rates of 0.37 and 0.07 nmol x mg protein(-1) x min(-1), respectively). The reason for this is unclear, but cellular differences in lactate dehydrogenase isotype expression may be involved. Acetate is metabolized to CO2 more rapidly in astrocytes than in synaptosomes (rates of 0.43 and 0.02 nmol x mg protein(-1) x min(-1), respectively). This is likely due to cellular differences in the expression of monocarboxylate transporter subtypes.     

Rapporteur report: Basics and technology and metrology and standards

The first and second sessions of the Workshop focussed on the basics of ultrasound and infrasound, their applications in both industry and medicine, and metrology and protection standards for ultrasound applications.     

Relative and combined effects of ethanol and protein deficiency on strontium and barium bone content and fecal and urinary excretion

To elucidate accumulation of minerals in human iliac arteries with aging, the content of minerals was analyzed by inductively coupled plasma atomic emission spectrometry. Bilateral common, internal, and external iliac arteries of 16 men and 8 women, ranging ages from 65 to 93 yr, were examined. It was found that an extremely high accumulation of calcium and phosphorus occurred in the common iliac artery at old age, being higher than that of the internal and external iliac arteries. It should be noted that the accumulation of calcium and phosphorus is the highest in the common iliac artery among the human arteries examined to date. Regarding sexual differences, the content of calcium and phosphorus in the common and internal iliac arteries was higher in women than in men, whereas their content in the external iliac artery was lower in women than in men.     

Prostaglandin and thromboxane production by human and guinea-pig macrophages and leucocytes

The ability of partially purified human and guinea-pig haematogenous cell populations, when cultured in vitro, to metabolise arachidonic acid (AA) has been studied. Supernatants from 24 hour cell culture have been subjected to analysis for products of AA metabolism by gas chromatography with electron-capture detection.The cell types studied were human peripheral blood monocytes (both glass adherent and non-adherent), neutrophils, eosinophils and leukemic leucocytes; thoracic duct lymphocytes and lung alveolar macrophages. From the guinea-pig, induced and non-induced macrophage or neutrophil enriched peritoneal exudate populations, lymph node cells, peritoneal eosinophils and peripheral blood platelets were examined. Supernatants were assayed for the presence of PGE₂, PGD₂, PGF_2α, TXB₂ and 6-keto-PGF_1α. In all types studied PGE₂ and TXB₂ were the major products formed. The identification of PGE₂ and TXB₂ was confirmed by GC/MS with multiple ion monitoring.The results have been compared with other reports and their possible significance discussed in relation to the proposed role of prostaglandins as mediators and modulators in immunopathology.     

Chemokines and cytokines: axis and allies in asthma and allergy

Allergic asthma can be precipitated by many factors. For the atopic person, fungus, pollen, dust mites, cockroach antigens, and diesel exhaust are all agents that may trigger an allergic attack. Cytokines and chemokines are integral mediators of fungal asthma. From the earliest time points, they recruit and activate the cells required for the clearance of fungus as well as being critical factors involved in the immunopathology of this disease. In the final analysis, it is clear that these mediators can act to the benefit or the detriment of the host.     

Oxidation and nitration of ribonuclease and lysozyme by peroxynitrite and myeloperoxidase

In spite of the many studies on protein modifications by reactive species, knowledge about the products resulting from the oxidation of protein-aromatic residues, including protein-derived radicals and their stable products, remains limited. Here, we compared the oxidative modifications promoted by peroxynitrite and myeloperoxidase/hydrogen peroxide/nitrite in two model proteins, ribonuclease (6Tyr) and lysozyme (3Tyr/6Trp). The formation of protein-derived radicals and products was higher at pH 5.4 and 7.4 for myeloperoxidase and peroxynitrite, respectively. The main product was 3-nitro-Tyr for both proteins and oxidants. Lysozyme rendered similar yields of nitro-Trp, particularly when oxidized by peroxynitrite. Hydroxylated and dimerized products of Trp and Tyr were also produced, but in lower yields. Localization of the main modified residues indicates that peroxynitrite decomposes to radicals within the proteins behaving less specifically than myeloperoxidase. Nitrogen dioxide is emphasized as an important protein modifier.