Production of 3-hydroxypropionate homopolymer and poly(3-hydroxypropionate-co-4-hydroxybutyrate) copolymer by recombinant Escherichia coli

Conversion of 3-hydroxypropionate (3HP) from 1,3-propanediol (PDO) was improved by expressing dehydratase gene (dhaT) and aldehyde dehydrogenase gene (aldD) of Pseudomonas putida KT2442 under the promoter of phaCAB operon from Ralstonia eutropha H16. Expression of these genes in Aeromonas hydrophila 4AK4 produced up to 21 g/L 3HP in a fermentation process. To synthesize homopolymer poly(3-hydroxypropionate) (P3HP), and copolymer poly(3-hydroxypropionate-co-3-hydroxybutyrate) (P3HP4HB), dhaT and aldD were expressed in E. coli together with the phaC1 gene encoding polyhydroxyalkanoate (PHA) synthase gene of Ralstonia eutropha, and pcs' gene encoding the ACS domain of the tri-functional propionyl-CoA ligase (PCS) of Chloroflexus aurantiacus. Up to 92 wt% P3HP and 42 wt% P3HP4HB were produced by the recombinant Escherichia coli grown on PDO and a mixture of PDO+1,4-butanediol (BD), respectively.     

吸附剂及培养基组成对香荚兰细胞悬浮培养产生香兰素的影响

本文报道了培养基的组成及添加吸附剂对香荚兰细胞悬浮培养产生香兰素的影响。结果表明,添加吸附剂活性炭及XAD-2后,香荚兰细胞产生的香兰素含量明显增加,而且活性炭的效果优于XAD-2;香荚兰细胞在全组成的MS培养基中香兰素含量均低于由矿物质盐组成培养基中的含量。可以考虑采用二步培养法来培养香荚兰细胞及产生香兰素。     

陕西食源性沙门氏菌耐药及相关基因

【目的】研究食源性沙门氏菌对常用抗生素的药敏性及相关耐药基因,更好的了解耐药性的产生和传播途径,确保食品安全。【方法】使用the Clinical and Laboratory Standards Institute推荐的琼脂稀释法测定沙门氏菌的药敏性,PCR和基因序列测定方法确定耐药沙门氏菌中整合子及其携带的耐药基因、与头孢菌素抗性相关的基因、沙门氏菌基因岛及与氟喹诺酮类抗生素耐药相关的基因突变。【结果】359株沙门氏菌中,67%的菌株对磺胺甲恶唑产生抗性,对甲氧苄啶/磺胺甲恶唑、四环素、卡那霉素、萘啶酮酸、氨苄西林、阿莫西林/克拉维酸、链霉素、氯霉素和庆大霉素、环丙沙星、头孢曲松、头孢西丁和头孢哌酮的耐药率分别为58%、56%、37%、35%、33%、32%、29%、26%、21%、16%、9%和8%。284株耐药菌中,79%的菌株可抗至少1种抗生素,25.9%可抗10种以上抗生素,2.5%可抗14种抗生素。耐药的Ⅰ类整合子以1.4kb最为常见,携带的耐药基因有aadA1、aadA2、aadA5、tetR、blaPSE-1、blaDHA-1、blaVEB-1、dhfrⅠ、dhfrⅤ、dhfrⅦ和dhfr17等。62株耐头孢曲松和/或头孢哌酮的沙门氏菌中,blaTEM和blaCMY-2基因的检出率分别为51.6%和56.5%。13.6%的沙门氏菌中检出了沙门氏菌基因岛。35株耐氟喹诺酮类抗生素的沙门氏菌的gyrA、parC和parE基因中共检出68个点突变,gyrA基因中常见突变为Ser83Phe、Ser83Tyr、Asp87Gly和Asp87Asn,parC基因中为Ser80Arg。parE基因中检出了Lys441Ile、Lys428Gln、Asp494Asn、Lys428Gln和Gly442Ser突变,这些点突变均为首次在食源性沙门氏菌中检出。【结论】陕西食源性沙门氏菌耐药状况严重,整合子、沙门氏菌基因岛和β-内酰胺酶编码基因的存在及解旋酶和拓扑异构酶基因突变是导致沙门氏菌耐药的重要机制。     

粪便微生物宏基因组来源的热稳定性邻苯二酚1,2-双加氧酶异源表达及酶学性质

【目的】克隆倭蜂猴粪便微生物宏基因组的邻苯二酚1,2-双加氧酶基因cat PLCgl,并对该酶进行异源表达及酶学特性研究。【方法】利用宏基因组高通量测序技术获得cat PLCgl,并对其氨基酸序列进行分析。将cat PLCgl重组到载体p EASY-E2中并转化到大肠杆菌BL21(DE3)中异源表达,研究其酶学性质。【结果】cat PLCgl全长852 bp,G+C含量48%,编码283个氨基酸,理论分子量为33.56 k D。重组Cat PLCgl酶学性质分析显示最适作用p H为7.0,其中在p H 7.0–10.0范围内处理1 h后,酶活剩余90%以上;最适作用温度为40°C,在25°C和40°C条件下稳定性较好,耐受210 h酶活性几乎不变。重组酶在最适条件下的动力学参数K_m、V_(max)和k_(cat)分别为24.9μmol/L、8.3 mmol/(min·g)和13.7 s~(-1);Fe~(2+)、Hg~(2+)、Cu~(2+)、Triton X-100、SDS、Ag+强烈抑制该酶活性,而其它金属离子及有机试剂影响较小。【结论】从倭蜂猴粪便微生物宏基因组中克隆得到邻苯二酚1,2-双加氧酶基因cat PLCgl,并对重组Cat PLCgl酶学性质进行研究,该酶具有较好的热稳定性和耐碱性,在降解环境中的邻苯二酚和生产顺,顺-己二烯二酸方面具有应用潜力。     

Acetone-soluble cellulose acetates prepared by one-step homogeneous acetylation of cornhusk cellulose in an ionic liquid 1-allyl-3-methylimidazolium chloride (AmimCl)

Cellulose samples extracted from cornhusk have been successfully acetylated in an ionic liquid 1-allyl-3-methylimidazolium chloride (AmimCl). Without using any catalyst, cornhusk cellulose acetates (CCAs) with the degree of substitution (DS) in a range from 2.16 to 2.63 were prepared in one-step. Under the homogeneous state, the DS value of CCAs was easily controlled by the acetylation time. The obtained CCAs were characterized by means of FT-IR, ¹³C NMR, DSC, TGA, and a mechanical test. The NMR results showed that the distribution of the acetyl moiety among the three OH groups of the anhydroglucose unit shows a preference at the C₆ position. The CCAs exhibited good solubility in some organic solvents, such as acetone and DMSO. The cast CCA films from their acetone solutions had good mechanical properties. At the end of each acetylation of cornhusk cellulose, the ionic liquid AmimCl could be effectively recovered. Therefore, this study presents a promising approach and “green process” to make use of crop by-products.     

Predictive value of circulating miR-328 and miR-134 for acute myocardial infarction

MicroRNA (miRNAs) is demonstrated to be present in the blood of humans and has been increasingly suggested as a novel biomarker for various pathological processes in the heart, including myocardial infarction, myocardial remodeling and progression to heart failure. In this study, we aim to evaluate the diagnostic and prognostic value of circulating miR-328 and miR-134 in patients with acute myocardial infarction (AMI). Circulating levels of miR-328 and miR-134 were detected by quantitative real-time PCR in plasma samples from 359 AMI patients and 30 healthy volunteers. Concentrations of high-sensitivity cardiac troponin T (hs-cTnT) were measured using electrochemiluminescence-based methods. MiRNAs were assessed for discrimination of a clinical diagnosis of AMI and for association with primary clinical endpoint defined as a composite of cardiogenic death and development of heart failure within 6 months after infarction. Results showed that levels of plasma miR-328 and miR-134 were significantly higher in AMI patients than in healthy controls. Receiver operating characteristic curve analyses showed significant diagnostic value of miR-328 and miR-134 for AMI. However, neither of them was superior to hs-cTnT for the diagnosis. Additionally, increased miRNA levels were strongly associated with increased risk of mortality or heart failure within 6 months for miR-328 (OR 7.35, 95 % confidence interval 1.07–17.83, P < 0.001) and miR-134 (OR 2.28, 95 % confidence interval 1.03–11.32 P < 0.001). In conclusion, circulating miR-328 and miR-134 could be potential indicators for AMI, and the miRNA levels are associated with increased risk of mortality or development of heart failure.     

Inhibition of severe acute respiratory syndrome virus replication by small interfering RNAs in mammalian cells

Severe acute respiratory syndrome (SARS) is an acute respiratory infectious disease that spread worldwide in early 2003. The cause was determined as a novel coronavirus (CoV), SARS-associated CoV (SARS-CoV), with a single-stranded, plus-sense RNA. To date, no effective specific treatment has been identified. To exploit the possibility of using RNA interference as a therapeutic approach to fight the disease, plasmid-mediated small interfering RNAs (siRNAs) were generated to target the SARS-CoV genome. The expression of siRNAs from two plasmids, which specifically target the viral RNA polymerase, effectively blocked the cytopathic effects of SARS-CoV on Vero cells. These two plasmids also inhibited viral replication as shown by titer assays and by an examination of viral RNA and protein levels. Thus, our results demonstrated the feasibility of developing siRNAs as effective anti-SARS drugs.     

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。