粪肠球菌精氨酸脱亚胺酶酶学性质研究

经硫酸铵分级沉淀、Q-Sepharose Fast Flow阴离子交换层析、SephadexG-75凝胶柱层析从NJ402自溶细胞超声破碎液中提纯得到精氨酸脱亚胺酶(ADI), 纯化倍数为34.5, 活力回收率为31.4％, 经SDS-PAGE以及Native-PAGE测定结果表明, ADI亚基分子量约为46 kD, 该酶非变性情况下的分子量约为190 kD左右, 该酶为同四聚体结构。酶学性质研究结果表明：ADI催化最适温度和最适pH分别为50℃和6.5, 在45℃以下和pH 5~8之间有很好的稳定性。ADI是L-型脱亚胺酶, 具有严格的光学选择性, 适当浓度的Mn2+、Mg2+、Co2+对ADI催化活力的促进作用较大, 高浓度的Zn2+和Co2+对酶有一定程度的抑制作用, L-瓜氨酸对酶无抑制作用而L-鸟氨酸却表现出较强的抑制作用。ADI在最佳催化条件下作用于L-精氨酸的米氏常数为3.2686 mmol/L, 最大反应速度为2.44 μmol/min。     

精氨酸脱亚胺酶发酵过程特性研究

精氨酸脱亚胺酶有较好的体内体外肿瘤生长抑制作用。通过对粪肠球菌（Enterococcus faecalis）NJ402菌株产精氨酸脱亚胺酶的发酵特性的研究,建立起代谢物的过程变化与精氨酸脱亚胺酶产生机理的内在联系。NJ402菌株生长过程中碳源物质代谢产生乳酸导致发酵体系pH的下降,而培养基中L-精氨酸的脱亚胺作用有利于发酵体系pH的稳定和菌体生长。进一步的研究表明,低pH生长环境有利于NJ402菌株产精氨酸脱亚胺酶,且精氨酸脱亚胺酶的产生与能量代谢无关。NJ402菌株产精氨酸脱亚胺酶受底物L-精氨酸的诱导,但该诱导作用受菌体生长体系pH的调控,即精氨酸脱亚胺酶的产生是低pH生长环境与L－精氨酸共同作用的结果。     

重组钝齿棒杆菌全细胞转化生产L-瓜氨酸条件优化

实现了精氨酸脱亚胺酶(ADI)首次在钝齿棒杆菌Corynebacterium crenatum SYPA 5-5中的高效表达。通过Ni-NTA亲和层析纯化得到纯化ADI,经SDS-PAGE测定其分子量约为46.8 k Da,酶学性质研究发现ADI的最适催化温度为37℃,最适pH为6.5,ADI在最佳催化条件下作用于L-精氨酸的米氏常数为12.18 mmol/L,最大反应速率为0.36μmol/(min·mL)。优化了重组菌全细胞转化产L-瓜氨酸的工艺条件,在最优条件下可一次转化300 g/L L-精氨酸,转化速率达8 g/(L·h)。进行重组菌5 L罐发酵并进行罐上全细胞转化300 g/L L-精氨酸,一批菌体可进行多次转化,累计产量达1 900 g以上。     

精氨酸脱亚胺酶在大肠杆菌中的分泌型表达

将变形假单胞菌的精氨酸脱亚胺酶(ADI)编码基因arc A克隆至具有阿拉伯糖启动子的分泌型表达载体pBAD/gⅢB中,经鉴定得到重组质粒pBAD-ADI。将重组质粒转化大肠杆菌TOP10F'后进行诱导表达,分别考察了不同诱导物L-arabinose浓度、诱导温度、诱导时间对重组蛋白表达的影响,最适诱导条件为L-arabinose浓度0.002%(w/v),25℃下诱导5 h,全细胞的酶活为68 mU/mL(指单位发酵液体积,下同)。采用Osmotic Shock法使ADI从胞周质释放出来,经检测分泌到胞周质的重组蛋白活性为53 mU/mL,细胞内的酶活为34 mU/mL。SDS-PAGE分析显示,重组蛋白大小约为46 kD。     

精氨酸脱亚胺酶活性以及瓜氨酸转化研究

目前,L-瓜氨酸的主要生产方法为化学法水解L-精氨酸.利用精氨酸脱亚胺酶可以直接转化L-精氨酸获得L-瓜氨酸,条件温和、效率高、环境友好,因而在工业化生产中具有很好的应用前景.本研究将优化后的单增李斯特菌(Listeriamonocytogenes)精氨酸脱亚胺酶(ADI)基因序列分别克隆到相应的载体pET-21a、pWB980及pAO815中,并分别转化大肠杆菌BL21(DE3)、枯草芽胞杆菌WB600及毕赤酵母GS115中构建表达菌株.发现大肠杆菌能高效表达ADI.经过5L发酵罐验证,发酵后酶活力单位可达200 000～270 000 U·L-1.经过验证,该酶可以很好地应用于L-瓜氨酸生产.在7 920～17 600 U· L-1的ADI存在条件下,5.5h内,可使95％的浓度为94～258 g·L-1的精氨酸转化为瓜氨酸.该方法具备良好的工业化应用前景.     

豇豆几丁质酶部分酶学特性的研究

该文测定了纯化的豇豆几丁质酶部分酶学特性。结果表明,该酶在pH5-8,温度低于60℃的范围内稳定性较好,酶活力最适pH为65,最适温度为50℃。10mmol/L浓度的Hg2 、Mn2 、Mg2 、Co2 等金属离子对酶活力有一定抑制作用,其中Hg2 离子抑制率最高(6883%)。Km(胶状几丁质)值为1662mg/ml;以SDS-PAGE电泳和SephadexG-100柱层析两种方法分别测得分子量为34kD、325kD;IEF电泳测得等电点为83。     

Secretory expression of arginine deiminase in E.coli

将变形假单胞菌的精氨酸脱亚胺酶(ADI)编码基因arcA克隆至具有阿拉伯糖启动子的分泌型表达载体pBAD/gⅢ B中,经鉴定得到重组质粒pBAD-ADI.将重组质粒转化大肠杆菌TOP10F’后进行诱导表达,分别考察了不同诱导物L-arabinose浓度、诱导温度、诱导时间对重组蛋白表达的影响,最适诱导条件为L-arabinose浓度0.002％ (w/v),25℃下诱导5h,全细胞的酶活为68 mU/mL(指单位发酵液体积,下同).采用Osmotic Shock法使ADI从胞周质释放出来,经检测分泌到胞周质的重组蛋白活性为53 mU/mL,细胞内的酶活为34 mU/mL.SDS-PAGE分析显示,重组蛋白大小约为46 kD.     

木霉GXC产β-葡聚糖酶条件和酶学性质

研究了木霉GXC产β-葡聚糖酶的条件.结果表明,最适产酶碳源为麸皮,氮源为硫酸铵;产酶的最适条件为初始pH为4.0～5.0,30℃培养44h.粗酶液经硫酸铵沉淀、Sephadex G-25、Sephadex G-100和DEAE-Sehadex A-50柱层析得到纯β-葡聚糖酶,SDS-PAGE凝胶电泳显示一条带,测得分子量为35kD.该酶最适反应pH5.0,最适反应温度为60℃,在40℃以下、pH4.0～5.0酶活力相对稳定.5.0mmol/L以下的Ca2+、Zn2+和Fe2+,以及10.0mmol/L以下的Co2+对酶活力有激活作用;而Cu2+和Fe3+具有抑制作用.     

利用定点突变法研究精氨酸脱亚胺酶活性的影响机制

精氨酸脱亚胺酶(ADI)是一种针对精氨酸缺陷型癌症(如:肝癌、黑素瘤)的新药,目前处于临床三期试验。文中通过定点突变技术分析了精氨酸脱亚胺酶的特定氨基酸位点对酶活力的影响机制。针对已报道的关键氨基酸残基A128、H404、I410,采用QuikChange法进行定点突变,获得ADI突变株M1(A128T)、M2(H404R)、M3(I410L)和M4(A128T/H404R)。将突变株在大肠杆菌BL21(DE3)中进行重组表达,并对纯化获得的突变蛋白进行酶学性质研究。结果表明,突变位点A128T和H404R对ADI最适pH的提高,生理中性(pH 7.4)条件下的酶活力和稳定性的提高,以及Km值的降低均具有显著的作用。研究结果为阐明ADI的酶活力影响机制和蛋白质的理性改造提供了一定的依据。     

鲫鱼酸性磷酸酶酶学特性及不同效应物对酶活力的影响

经NaAc-HAc缓冲液(pH5.0)抽提,正丁醇处理,硫酸铵分级沉淀,DEAE-32离子交换层析,SephadexG-150凝胶过滤纯化,从鲫鱼内脏中分离纯化出电泳纯的酸性磷酸酶。该酶提纯倍数为30.82,比活力195.06U/mg。研究表明,该酶催化对硝基苯磷酸二钠水解反应,最适pH4.8,pH小于4和大于7时不稳定;最适温度45℃,温度高于50℃不稳定;米氏常数为0.23mmol/L,利用SDS-PAGE测定酶亚基分子量为33.3kD。化学修饰剂SUAN、PMSF、DTT、NBS对该酶活力影响不大,BrAc和IAc有明显抑制作用。金属离子对该酶催化活力有不同影响,Na+、K+、Ni2+、Co2+影响不显著,Mg2+、Ca2+、Ba2+、Mn2+有激活作用,Ag+、Cu2+、Pb2+、Cd2+有抑制作用,其中Mg2+、Ca2+、Pb2+、Cd2+对鲫鱼酸性磷酸酶荧光光谱的影响表明金属离子对酶活力的影响与酶构象改变有关。       

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     

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.     

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.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

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).     

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.