宏基因组来源耐Mn2+、热稳定细菌漆酶的分子克隆及酶学特性

【背景】漆酶和锰过氧化物酶(Manganese peroxidase,Mnp)是木质素降解的主要酶,二者有协同效应。Mnp活性依赖于Mn~(2+),而Mn~(2+)是大多数漆酶的抑制剂。【目的】获得耐Mn~(2+)的细菌漆酶用于木质素降解。【方法】构建许昌市某污水河污泥宏基因组文库,通过活性筛选获得细菌漆酶基因lac1542。使用大肠杆菌异源表达Lac1542,研究纯化后的重组蛋白酶学性质并进一步检测了含Lac1542复合酶系降解木质素能力。【结果】测序结果显示lac1542编码一个含513个氨基酸的蛋白。以ABTS为底物Lac1542最适反应pH为4.0,在pH 3.0-6.5范围内酶活性稳定。最适反应温度是75°C,在70°C以下酶活性稳定;100 mmol/L的Mn~(2+)仍能提高酶的活性。动力学参数研究发现,该酶的最适底物顺序为:ABTS丁香醛联氮儿茶酚2,6-DMP愈创木酚。Lac1542/Mnp复合酶系对木质素降解率为47.8%,比单独使用Mnp木质素降解率(22.4%)提高25.4%。Lac1542/Mnp/灰盖鬼伞过氧化物酶(Coprinus cinereus Peroxidase,CIP)复合酶系木质素降解高达71.5%,比Mnp/CIP酶系木质素降解率(48.9%)提高22.6%,加入Lac1542后的复合酶系能明显提高木质素的降解率。【结论】Lac1542的可溶性表达、耐受高浓度Mn~(2+)、热稳定性使得Lac1542可以替代一些经典的真菌漆酶应用于制浆、造纸、纤维素乙醇生产、染料脱色等工业。     

大球盖菇漆酶的分离纯化及酶学性质研究

从大球盖菇Sr-01菌株液体发酵液中分离漆酶,研究温度、p H和金属离子对酶活的影响。采用硫酸铵分级沉淀、Q-Sepharose阴离子交换层析和Superdex 200凝胶过滤层析对大球盖菇漆酶进行分离纯化。以ABTS[2,2-连氮基-双-(3-乙基苯并二氢噻唑啉-6-磺酸)]为底物,分光光度法测定酶活。结果表明,纯化后的漆酶比活力为152.79 U/mg,回收率为35.8%。SDSPAGE显示该漆酶为单体蛋白,相对分子质量约40 k D。该漆酶的最适反应温度和p H分别为35℃和4.0,Mg~(2+)、Cu~(2+)对酶活有激活作用,Fe~(2+)、Cd~(2+)、Hg~(2+)则有显著抑制作用。在最优反应条件下,纯化后的漆酶比活力可达222.93 U/mg。     

短小芽孢杆菌LC01漆酶基因在毕赤酵母中的胞外表达及重组漆酶酶学性质研究

为了获得表达量高、稳定性好及染料脱色效率高的细菌漆酶,通过PCR扩增出短小芽孢杆菌LC01的漆酶基因并构建重组表达载体pPICZαA-lac,转化毕赤酵母菌株SMD1168H后利用甲醇诱导培养重组菌获得重组漆酶,纯化并分析了重组漆酶的性质。重组菌株产漆酶活性在第7天达到最高,为1 390 U/L。纯化的重组漆酶分子量为65 kD,以丁香醛连氮为底物的最适反应温度和pH分别为70℃和6.8。在pH 9.0放置10 d活性没有下降,在70℃保温10 h后仍保留36%的酶活。Al~(3+)、Fe~(3+)和Mn~(2+)完全抑制漆酶活性。在介体乙酰丁香酮参与下该漆酶能够有效脱色RB亮蓝、活性黑5和靛红,在pH 9.0时6 h的脱色率达到了90%以上,表明该重组漆酶能有效应用于染料废水的脱色处理。     

结核分枝杆菌FtsZ的异源表达及酶学性质研究

作为细菌分裂所必需的微管蛋白类似物,丝状温度敏感蛋白Z(filamentous temperature-sensitive protein Z,FtsZ)被认为是一个具有潜力的药物作用新靶点。为了构建高纯度的FtsZ重组蛋白分离纯化体系,探讨其酶学性质,该研究利用大肠杆菌BL21异源表达结核分枝杆菌FtsZ重组蛋白,通过Ni亲和层析柱和G-50层析柱纯化目的蛋白,采用孔雀石绿法和90°光散射法测定FtsZ重组蛋白的GTP酶活和蛋白聚集。研究结果表明:成功获得具有生物学活性的结核分枝杆菌FtsZ重组蛋白,其相对分子质量约为49kD;该酶最适反应温度为37℃,最适pH为6.8,金属离子Mg~(2+)和K~+对FtsZ重组蛋白酶活具有促进作用,有机溶剂DMSO和TritonX-100的体积分数分别高于0.1%和0.005%时对酶活有显著抑制作用(P0.05)。此外,FtsZ重组蛋白在加入底物GTP诱导后,快速聚集。本实验利用基因工程技术成功获得具有生物活性的FtsZ重组蛋白,并明确了该蛋白质的酶学性质,为其进一步的研究和应用奠定了基础。     

奇异变形杆菌JN458 D-乳酸氧化酶的性质研究北大核心

[目的]对奇异变形杆菌JN458 D-乳酸氧化酶进行酶学性质研究。[方法]克隆D-乳酸氧化酶基因(lod D),构建重组质粒在大肠中表达,生物信息学初步分析D-乳酸氧化酶氨基酸序列,镍柱纯化该酶并研究其性质。[结果]生物信息学分析该酶C-末端含膜结合结构域,所以是膜蛋白,属于以醌作为电子受体的D-乳酸氧化酶。最适温度60℃,50℃下保持稳定1 h,中高温适应性较好。最适pH 7,pH 7~9下保持稳定1 h。以D-乳酸为底物时Km值为0.61 mmol/L,对D-乳酸有很高的亲和力。Fe^(2+)、Mn^(2+)和Mg^(2+)在1~6 mmol/L的浓度内能提高酶活,其中Fe^(2+)效果最好。[结论]lod D在大肠中实现了高效表达,使新型D-乳酸氧化酶的应用有了理论依据。     

一色齿毛菌漆酶的酶学特性及染料脱色研究

染料由于具有复杂的化学结构通常难以降解。本文从白腐菌一色齿毛菌LS0547中纯化出胞外漆酶并用于染料脱色实验。SDS-PAGE结果显示纯化的漆酶分子量大小为63.7kDa。漆酶氧化底物ABTS的最适pH为2.2,最适温度为50℃。叠氮钠可强烈抑制漆酶活性,半胱氨酸和二硫苏糖醇可部分抑制漆酶活性。漆酶氧化ABTS,丁香醛连氮和2,6-二甲氧基苯酚的米氏常数分别为0.217,0.306和0.199mmol/L。粗酶和纯化的漆酶用于不同化学结构的染料的脱色研究,结果表明一色齿毛菌纯化漆酶可快速对RB亮蓝进行脱色,偶氮胭脂红和结晶紫的脱色效果低于RB亮蓝,测试的三种染料均可在没有介体存在的条件下被漆酶脱色,显示出一色齿毛菌漆酶在染料废水处理中的应用前景。     

褐藻胶裂解酶基因的克隆表达及重组酶酶学性质

【目的】克隆交替假单胞菌(Pseudoalteromonas sp.)BYS-2的褐藻胶裂解酶基因,实现其在大肠杆菌细胞中异源表达,对分离纯化的重组酶进行酶学性质研究。【方法】以交替假单胞菌BYS-2菌株基因组DNA为模板,克隆得到褐藻胶裂解酶基因alg738,构建重组基因工程菌BL21(DE3)/p ET22b-alg738,诱导表达,表达产物通过Ni-NTA树脂纯化后进行酶学性质研究。【结果】重组酶的最适反应p H为8.0,在p H 6.0-9.0范围内37°C保温1 h仍能保持84%以上的相对酶活力,具有较好的p H稳定性;最适反应温度为45°C,热稳定性实验显示在37°C下保温60 min其残余酶活力仍达66.6%;在5 mmol/L浓度下,Na~+、Mg~(2+)、Mn~(2+)对该酶具有明显的促进作用,Ni~(2+)、Co~(2+)、Cu~(2+)、Hg~(2+)、Zn~(2+)、EDTA、β-巯基乙醇、SDS具有明显的抑制作用。动力学参数Km、Vmax分别为1.11 g/L和0.011 g/(L·min),底物特异性分析表明该重组酶为偏好聚甘露糖醛酸钠(Poly M)裂解作用的双功能酶。【结论】重组褐藻胶裂解酶具有良好的酶学特性,为褐藻胶裂解酶的开发应用打下基础。     

马铃薯α-淀粉酶在毕赤酵母中的表达、纯化及其酶学性质的研究

采用RT-PCR法扩增马铃薯夏波蒂的α-淀粉酶成熟肽基因,将其亚克隆至毕赤酵母表达载体pPIC9k上,SacII线性化重组表达载体,电击转化毕赤酵母GS115感受态细胞,构建重组酵母GS115/pPIC9k-amy,利用锥虫蓝法筛选获得高活性转化子(GSamyA5),以终浓度为0.5%甲醇诱导该重组菌表达α-淀粉酶,通过Ni~(2+)-NTA agarose亲和层析纯化,并对其酶学性质进行研究。结果表明:该酶的最适反应温度为45℃,40~50℃酶活较稳定,保温50 min,残留相对活力达92.6%;最适反应pH值为6.0,并在pH 6.0~7.0范围内酶活保持稳定。Ca~(2+)、K~+可促进酶反应,以Ca~(2+)影响为最,相对酶活力提高到125%;Cu~(2+),Fe~(2+),Fe~(2+),Zn~(2+)对该酶有显著抑制作用;Mn~(2+),Mg~(2+)对酶有微弱抑制作用,Li~+、Na~+对酶活影响不大。     

嗜热厌氧乙醇杆菌α-葡萄糖苷酶基因的表达及酶学性质的研究

用PCR方法从嗜热厌氧乙醇杆菌(Thermoanaerobacter ethanolicus)JW200中扩增出编码a-葡萄糖苷酶的基因,将其克隆到大肠杆菌(Escherichia coli)表达载体pTrc99A上并获得表达a-葡萄糖苷酶的大肠杆菌重组菌。重组菌经IPTG诱导表达,SDS-PAGE检测出蛋白相对分子量约89kDa,经阴离子交换层析和凝胶层析纯化后的a-葡萄糖苷酶最适反应温度为70℃,最适反应pH为5～5.5,且在pH 5.5～6.5之间有较高的稳定性。重组a-葡萄糖苷酶在70℃下105 min后酶活仍达到80%。     

溶葡球菌酶在乳酸克鲁维酵母中重组表达、诱变、优化及酶学研究

根据模仿葡萄球菌(Staphylococcus simulans)的溶葡球菌酶基因序列以及乳酸克鲁维酵母密码子偏好性设计引物扩增溶葡球菌酶基因表达片段,构建溶葡球菌酶(lysostaphin,Lys)基因表达载体(p KLAC1-Lys),转化乳酸克鲁维酵母(K.lactis GG799),实现了Lys基因的分泌表达。对重组菌株(K.lactis GG799/p KLAC1-Lys)进行NTG随机化学诱变,优化表达条件,筛选获得高表达菌株,并通过Ni-NTA亲和层析纯化蛋白并研究其酶学性质。结果表明:通过诱变重组溶葡球菌酶乳酸克鲁维菌株,Lys酶比活性提高了约5.2倍(约8 000U/L)。最适接种量为40g/L,诱导过程中每24h添加一次终浓度为20g/L的半乳糖和NH_4NO_3可提高酶比活性,最适表达p H为7.0~7.5,最适反应p H为7.0~8.0,最适反应温度为37℃。实验表明,低于40℃,p H 3~6之间时,重组溶葡球菌酶较稳定。Sr~(2+)对其酶活性有明显的促进作用,Ba~(2+)、Ca~(2+)、Zn~(2+)、Cu~(2+)、Mn~(2+)、Mg~(2+)对其有明显的抑制作用。     

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.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

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.