产普鲁兰酶重组大肠杆菌质粒稳定性的研究

通过对产普鲁兰酶的重组大肠杆菌E.coli BL21(DE3)/p ET28a-s-pul菌株在发酵过程中质粒稳定性和普鲁兰酶生成量的考察,发现不同宿主对质粒稳定性及酶活性有重要影响。本文利用E.coli BL21(DE3)p Lys S菌株为宿主,构建重组菌E.coli BL21(DE3)p Lys S/p ET28a-s-pul,通过控制外源蛋白的本底表达,提高了重组菌株的质粒稳定性。优化发酵培养基和发酵条件以后,重组菌产普鲁兰酶能力由480 U/m L提高至627 U/m L,增幅为30.6%。研究结果认为,严格控制外源蛋白的本底表达,是改善重组菌稳定性的重要方法之一。     

基于双酶级联协调表达策略高效催化合成D-甘露醇北大核心CSCD

D-甘露醇(D-mannitol)作为合成抗肿瘤药和免疫刺激剂的重要前体被广泛应用于制药和医疗等行业,酶法合成D-甘露醇反应成本昂贵无法满足工业化生产。本研究首先筛选关键酶获得较优性能的甘露醇脱氢酶Lp MDH和用于辅因子NADH再生的葡萄糖脱氢酶Ba GDH,在大肠杆菌(Escherichia coli)BL21(DE3)中共表达,实现了基于双酶级联反应催化底物D-果糖合成D-甘露醇,D-甘露醇的初步摩尔转化率为59.7%。针对双酶级联催化反应中辅酶再生用酶与催化用酶表达量不协调的问题,通过增加Bagdh拷贝量来提高辅因子循环能力,获得了双酶催化速率平衡的重组大肠杆菌E.coli BL21/pETDuet-Lpmdh-Bagdh-Bagdh。进一步对重组菌的全细胞转化条件进行优化,确定了最适转化条件为反应温度30℃,初始pH值6.5,菌体量OD600=30,底物D-果糖100.0 g/L,辅底物葡萄糖与底物1︰1摩尔当量。于最优转化条件下5 L发酵罐转化24 h,D-甘露醇的最高产量为81.9g/L,摩尔转化率为81.9%。本研究提供了一种绿色、高效生物催化生产D-甘露醇的方法,为实现其规模化生产奠定了基础,同时也对其他相关稀有糖醇的研究具有指导意义。     

重组大肠杆菌全细胞催化高效合成胆绿素北大核心CSCD

胆绿素作为一种重要的保护细胞的抗氧化剂,其传统生产方法主要由胆红素的化学氧化产生,但过程复杂、纯度不高。本研究提出了一种高效、绿色、安全的生产胆绿素的方法。通过比较,筛选得到了破伤风梭状芽孢杆菌(Clostridium tetani)来源的血红素加氧酶(heme oxygenase,HO)基因,并成功构建具备转化血红素合成胆绿素能力的重组大肠杆菌(Escherichiacoli)BL21/pETDuet-hoCt。在pH 7.0、35℃、100 mg/L底物浓度条件下胆绿素产量为32.9 mg/L。为提高还原力,构建了基于谷氨酸脱氢酶(glutamate dehydrogenase,GdhA)的NADPH辅酶再生系统,获得重组菌E.coli BL21/pETDuet-gdhAEc-hoCt,胆绿素产量为71.5 mg/L。此外,通过引入膜表面展示系统,构建重组菌E.coli BL21/pETDuet-gdhAEc-blc/hoCt,缩短转化时间的同时,胆绿素产量进一步得到提高,达到76.3 mg/L,是目前生物法合成胆绿素的最高研究报道。本研究为胆绿素的绿色生产奠定了良好的基础。     

Escherichia coli str.K-12 substr.MG1655海藻糖酶Tre F的重组表达及性质研究

海藻糖酶(Trehalase)是一种海藻糖水解酶,能够特异性的将海藻糖分解为两分子的葡萄糖。为了将Escherichia coli str.K-12 substr.MG1655的海藻糖酶基因Tre F在E.coli BL21(DE3)中重组表达和应用,该研究通过PCR扩增获得E.coli str.K-12 substr.MG1655的海藻糖酶基因Tre F,构建了基因工程菌E.coli BL21(DE3)/p ET-24a(+)-Tre F。对重组菌进行摇瓶发酵,25℃,IPTG浓度为0.4 mmol/L时,摇瓶发酵诱导24 h时得到最高酶活为107 U/m L。进一步研究了海藻糖酶的酶学性质,发现该海藻糖酶的最适p H为7.0,最适温度为50℃;此外,将该酶应用于海藻糖的水解,起始海藻糖浓度为300 g/L,初始p H 7.0、反应温度30℃,加酶量为84 U/g,反应36 h,葡萄糖转化率可达98.4%。该研究是首次将E.coli str.K-12 substr.MG1655海藻糖酶基因Tre F在E.coli BL21(DE3)宿主中重组表达的报道。     

过量表达烟酸单核苷酸腺苷酰转移酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因(ldhA)和丙酮酸-甲酸裂解酶的编码基因(pflB)的发酵生产丁二酸的潜力菌株。厌氧条件下NADH不能及时再生为NAD+,引起胞内辅酶NAD(H)的不平衡,最终导致厌氧条件下菌株不能利用葡萄糖生长代谢。nadD为催化NAD(H)合成途径中烟酸单核苷酸(NaMN)生成烟酸腺嘌呤二核苷酸(NaAD)的烟酸单核苷酸腺苷酰转移酶(Nicotinic acid mononucleotide adenylyltransferase,NAMNAT)的编码基因,通过过量表达nadD基因能够提高NAD(H)总量与维持合适的NADH/NAD+比例。文中构建了重组菌E.coli NZN111/pTrc99a-nadD,在厌氧摇瓶发酵过程中通过添加终浓度为1.0 mmol/L的IPTG诱导表达,重组菌E.coli NZN111/pTrc99a-nadD中NAD+和NADH的浓度分别比宿主菌E.coli NZN111提高了3.21倍和1.67倍,NAD(H)总量提高了2.63倍,NADH/NAD+从0.64降低为0.41,使重组菌株恢复了厌氧条件下生长和代谢葡萄糖的能力。重组菌与对照菌相比,72 h内可以消耗14.0 g/L的葡萄糖产6.23 g/L的丁二酸,丁二酸产量增加了19倍。     

吡咯喹啉醌产生菌筛选方法建立及菌种筛选

吡咯喹啉醌(PQQ)是一种氧化还原酶的辅酶,具有多种生理功能。扩增得到大肠杆菌葡萄糖脱氢酶(GDH)基因,并利用表达载体pET28a在E.coli BL21(DE3)中进行了表达。纯化了可溶性表达产物,并建立了基于GDH的重组酶法分析PQQ的方法。确定了甲基营养菌筛选模型,从2000余份土样中分离得到一株PQQ高产生菌MP606,在未经培养条件优化及诱变选育的条件下PQQ产量达113mg/L。从该菌培养液中制备得到了产物的结晶,HPLC分析、特征光谱分析以及酶法分析均证实该产物为PQQ。扩增并分析了MP606的16S rDNA序列,结果显示该菌16S rDNA序列与12种甲基营养菌都具有95%以上同源性,其中与食甲基菌属两菌株的16S rDNA序列同源性达99%。     

大肠杆菌BL21(DE3)膜组分相关基因的敲除对重组蛋白胞外分泌的影响

摘要:【目的】探究Escherichia coli BL21(DE3)中膜组分相关的脂多糖合成基因waaF或msbB的敲除对重组蛋白胞外分泌的影响。【方法】运用Red重组技术将E.coli BL21 (DE3)染色体上的基因waaF或msbB敲除,构建敲除菌株E.coli BL21(ΔwaaF)、E.coli BL21(ΔmsbB)。将本实验室保存的带有β-呋喃果糖苷酶(β-fructofuranosidase,β-FFase)、青霉素G 酰化酶(penicillin G acylase,PGA)基因的重组质粒pET-ffase、pET-pga分别转入敲除菌株及出发菌株中,构建工程菌株E.coli BL21(ΔmsbB)/pET-ffase、E.coli BL21(ΔwaaF)/pET-ffase、E.coli BL21(DE3)/pET-ffase、E.coli BL21(ΔmsbB)/pET-pga、E.coli BL21(ΔwaaF)/pET-pga、E.coli BL21(DE3)/pET-pga。最后通过摇瓶发酵研究敲除菌株对β-FFase、PGA胞外分泌的影响。【结果】当诱导表达4 h,以出发菌株E.coli BL21(DE3)为宿主时,β-呋喃果糖苷酶β-FFase的胞外分泌量占总表达量的2.6%,以敲除菌株ΔmsbB为宿主时,胞外分泌量达到19.7%,而以敲除菌株ΔwaaF为宿主时,胞外分泌量达到50.9%。另外,当诱导表达24 h,以敲除菌株ΔwaaF为宿主时,青霉素G酰化酶PGA的胞外酶活是出发菌株中的4.1倍,达到1708 U/L。【结论】本研究成功构建了敲除菌株ΔmsbB和ΔwaaF,ΔmsbB能明显增强β-FFase的胞外分泌,而ΔwaaF对β-FFase和PGA的胞外分泌均有显著的强化作用。     

D194G突变对meso-2,3-丁二醇脱氢酶催化特性的影响

目的:比较来源于Enterobacter aerogenes CICC10293和Bacillus subtilis的meso-2,3-丁二醇脱氢酶(E. a-BDH和D194G B. s-BDH)活性和动力学参数,分析D194氨基酸对BDH催化特性的影响。方法:利用E. coli BL21(DE3)原核表达E. a-BDH和D194G B. s-BDH,经HiTrap Q FF阴离子交换柱和Superdex 75凝胶柱纯化后,用MALDI-TOF MS确定其分子质量;检测NADH/NAD⁺氧化还原的吸光度变化确定BDH活性、辅酶和底物的特异性、最适pH、温度及动力学参数。结果:重组表达E. a-BDH和D194G B. s-BDH是同源四聚体蛋白,基因序列有两处碱基不同(g.27A/T和g.581A/G),其中g.581A/G导致BDH的一处氨基酸发生改变(p.D194G)。D194G B. s-BDH的活性约为E. a-BDH的2.3%,并且丧失了氧化meso-2,3-丁二醇的能力。二者均以乙偶姻/NADH为最适底物,但D194G B. s-BDH的K_m是E. a-BDH的5.63倍。结论:D194G氨基酸突变降低了BDH的活性。     

乙醇脱氢酶与葡萄糖脱氢酶偶联催化制备(S)-1-(2,6-二氯-3-氟苯基)乙醇

(S)-1-(2,6-二氯-3-氟苯基)乙醇是抗癌药物克唑替尼的手性合成前体,可由2,6-二氯-3-氟苯乙酮经乙醇脱氢酶催化还原制备,还原中所需的还原型辅酶Ⅱ再生是该反应的技术瓶颈.本研究构建重组大肠杆菌E.coli BL21-ADH和E.coli BL21-GDH,实现了葡萄糖脱氢酶和乙醇脱氢酶的共表达,并进行偶联转化.结果表明,当在反应温度为30℃,pH为7的条件下,(S)-l-(2,6-二氯-3-氟苯基)乙醇的产量达到最高,在投料量为6％时,该体系转化率为93.75％.     

重组大肠杆菌生物转化甘油生产3-羟基丙酸

目的:以甘油为底物构建高效的3-羟基丙酸生产菌株。方法:以自身携带乙醛脱氢酶的E.coli BL21(DE3)plysS作为宿主,异源表达源自Klebsiella pneumoniae的甘油脱水酶基因dhaB。结果:重组菌E.coli HP获得的甘油脱水酶比活力在1.0mmol/L IPTG的诱导下达到了77.2 U/mg,摇瓶条件下,3-HP的最大产量为5.44 g/L,摩尔转化率为53%,该产量比目前报道的最高水平(4.4 g/L)提高了23.6%。结论:重组菌株E.coli HP实现了甘油向3-羟基丙酸(3-HP)的高效生物转化。     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

鸡传染性法氏囊病病毒研究进展

传染性法氏囊病(Infection bursal disease, IBD)是由鸡传染性法氏囊病毒(Infectious bursal disease virus, IBDV)引起的鸡和火鸡的一种高度接触性传染病,给世界各国的禽养殖业带来了巨大损失.自IBDV发现至今新的变异株不断出现,分子结构的改变导致病毒致病力的改变及宿主对疫苗应答的改变,使得传统的疫苗已不能控制其流行,因此各国学者对其基因组结构和功能进行了广泛深入的研究,并积极研制新型有效的疫苗以达到防治的目的.     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).