亮白曲霉乳糖酶基因在毕赤酵母中的高效分泌表达及酶学性质研究

将克隆的亮白曲霉 (Aspergilluscandidus)乳糖酶基因lacb′插入到毕赤酵母 (Pichiapastoris)高效表达载体pPIC9中 ,与分泌信号肽序列α_因子融合 ,通过同源重组将lacb′整合到酵母染色体上。通过SDS_PAGE检测和表达产物的酶活性筛选 ,得到重组转化子 ,证明乳糖酶获得有效分泌和高效表达。表达的乳糖酶为糖蛋白 ,表观分子量为 130kD ,脱糖基后的蛋白分子量下降为 110kD。经过 5L小罐高密度发酵 ,重组酵母中酶蛋白表达量为 6mg mL发酵液 ,每毫升发酵液中乳糖酶的活力为 36 0 0U ,高于目前国内外报道的水平。进一步研究了表达产物的酶学性质 ,该酶最适pH为 5 . 2 ,最适反应温度 6 0℃ ,比活性为 70 6 . 5± 2 . 6U mg ,Km为 1 7mmol L ,Vmax为 3. 3μmol min。与米曲霉ATCC 2 0 4 2 3的乳糖酶相比 ,该乳糖酶具热稳定性强、比活性高、pH范围宽等特点。     

黑曲霉纤维素酶的纯化及酶学性质研究

黑曲霉(Aspergillusniger)固态发酵后粗酶液经硫酸铵盐析,2次SephadexG-200柱层析后可提纯8倍左右.CMC酶最适作用温度为60℃,最适作用pH为3.5,30℃～70℃区间酶活力较稳定,在pH3.0～5.0范围内,50℃保温30min能保持80%的酶活力.CMC酶的Km、Vmax值分别为7.69%CMCg/ml、0.33mg/ml·     

嗜热脂肪芽孢杆菌羧酸酯酶的异源表达及酶学性质研究

运用生物信息学技术从嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus) CICC 20156中克隆获得羧酸酯酶基因,构建黑曲霉和毕氏酵母表达质粒,将重组质粒分别转化毕氏酵母GS115和黑曲霉pyrG基因缺陷株M54．SDS-PAGE和Western blot检测显示：携带His标记的外源蛋白在转化真菌宿主中均获得了高效分泌性表达,毕氏酵母和黑曲霉表达的外源蛋白分子质量均约为29 ku,蛋白质浓度分别为30.7 mg/L和15.3 mg/L．生物学活性测定表明,毕氏酵母与黑曲霉表达的羧酸酯酶单位蛋白酶活分别为22 671 U/mg和21 438 U/mg．酶学性质研究显示,两种表达系统表达的重组羧酸酯酶的酶学特性基本一致,它们在40～70℃范围内均显示较好的酶活性,最适反应温度为60℃．70℃处理30 min,毕氏酵母和黑曲霉表达重组羧酸酯酶残余酶活分别为 76.7%和67.6%,显示出良好的热稳定性．在pH 6.5～8.5的范围内显示较高酶活性,最适pH为8.0．上述研究首次实现了具有良好热稳定性的嗜热脂肪芽孢杆菌羧酸酯酶在黑曲霉和毕氏酵母中高效异源分泌性表达,其中毕氏酵母羧酸酯酶的产量要高于黑曲霉的酶产量,但考虑到重组黑曲霉表达外源性蛋白无需使用任何诱导剂,黑曲霉菌表达热稳定性羧酸酯酶可能具有更好的应用前景．     

天然巴曲酶的酶学性质研究

目的 研究巴西矛头蝮蛇蛇毒中纯化的天然巴曲酶的酶学性质.方法 测定不同的温度、pH值和金属离子等条件对重组定点突变巴曲酶活性的影响.结果 实验表明,该酶在温度30～40℃,pH 6.5～9.0活性较为稳定;其最适反应温度为37℃,pH为7.5;Mg2+、K+和Ca2+离子对酶活有激活作用,而Cu2+、Fe2+和Zn2+离子对该酶有抑制作用.结论 从蛇毒提取的天然巴曲酶酶学性质比较稳定.     

黑曲霉β-葡萄糖苷酶的酶学特性研究

研究黑曲霉β-葡萄糖苷酶的酶学特性,采用酶学研究方法,通过硫酸铵沉淀、Sephadex G-25脱盐和Sephadex G-100纯化了β-葡萄糖苷酶,并进行了黑曲霉β-葡萄糖苷酶的最适反应温度、最适pH、热稳定性、pH稳定性及米氏常数等特性研究,采用SDS-PAGE凝胶电泳测定了分子量。研究表明,β-葡萄糖苷酶的最适反应温度为70℃、最适反应pH为4.5;在40、50和60℃下较稳定,80℃以上稳定性差;β-葡萄糖苷酶在pH为3、7、8、9的缓冲液中的稳定性很差,在pH为4、5、6的缓冲液中稳定性较好,其中在pH为5时,稳定性最好;酶的Km=41.67 mmol/L,Vmax=23.81 U/L;其分子量为65.2 ku。β-葡萄糖苷酶在饲料工业具有良好的应用前景。     

莱氏野村菌产几丁质酶条件及酶学性质研究

对莱氏野村菌(Nomuraea rileyi)菌株CQ031021产几丁质酶条件及酶学性质进行了研究。结果表明:该菌株最适产酶碳源为2.0%(W/V)葡萄糖,氮源为1.2%(W/V)复合氮源(蛋白胨、牛肉膏按1∶1的比例),接种量为孢悬液2mL(1×107个/mL),培养温度28℃,培养液初始pH6.0,培养时间6d;一定浓度的吐温-80对几丁质酶活性有促进作用,而SDS有抑制作用;粗酶液最适反应温度50℃,最适pH6.0,在40℃以下及pH5.5～6.5范围内酶活力较稳定。     

黑曲霉CICIM F0410中α-葡萄糖苷酶的酶学性质研究

研究了黑曲霉(Aspergillus niger)CICIMF0410发酵粗酶液中α-葡萄糖苷酶的酶学性质,发现该酶最适反应温度为55℃,最适反应pH值5.0,在65℃以下保持酶活力相对稳定,且能在pH值(4.0～6.5)范围内保持相对较高的酶活力。以该菌株的染色体DNA为模板,PCR扩增出大小为3.1kb的片段,经序列比对发现与NCBI上已公布的黑曲霉(Aspergillus niger)CBS513.88(登录号XP_001402053)序列有2个碱基的区别:2272(A→G),3098(T→G),导致2个氨基酸残基的变化:687(Asn→Ser)、983(Ser→Arg)。     

棘孢木霉几丁质酶tachi2 基因的原核表达及酶学性质研究

目的:实现棘孢木霉(Trichoderma asperellum)几丁质酶基因tachi2的原核高效表达,研究几丁质酶Tachi2的酶学性质.方法:利用PCR技术扩增得到几丁质酶基因tachi2,将其克隆到原核表达载体pEHISTEV中,测序后,转化大肠杆菌BL21感受态细胞,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导后进行Tachi2蛋白的纯化和复性.用纯化的目的蛋白Tachi2进行几丁质酶酶学性质的研究.结果:tachi2基因在重组大肠杆菌中正确表达,其主要以包涵体形式存在;重组蛋白Tachi2分子量约为44kDa,经过纯化和复性后得到的Tachi2有较高的几丁质酶活性.该酶的最适温度为40℃,最适pH值为7.0,几丁质酶在40℃以下比较稳定、pH 6～9时酶有较高活性,受Cu2和Zn2+的强烈抑制.结论:成功实现了棘孢木霉几丁质酶基因tachi2的原核高效表达,表达纯化了重组蛋白,明确了几丁质酶Tachi2的酶学性质,为该几丁质酶的进一步开发利用和深入研究奠定了基础.     

嗜热枯草芽孢杆菌普鲁兰酶基因的表达与重组酶的性质

克隆嗜热枯草芽孢杆菌WY-34普鲁兰酶基因并在大肠杆菌中进行表达,对重组酶进行纯化和酶学性质研究,根据枯草芽孢杆菌的普鲁兰酶蛋白序列,设计PCR引物对WY-34的普鲁兰酶基因进行克隆及异源表达.对表达蛋白的最适pH、pH稳定性及最适温度、温度稳定性等特性进行研究,并测定重组普鲁兰酶的底物特异性.将普鲁兰酶基因pluA克隆及分析序列后,发现基因长度为2.2 kb,编码718个氨基酸,在大肠杆菌中异源表达.通过Ni-IDA亲和层析一步纯化得到比活力为93.2 U/mg的纯酶,SDS-PAGE和凝胶层析测定的分子量分别为76.2 kD和74.3 kD.酶学性质研究表明,该酶的最适温度为40℃,在温度不高于45℃条件下稳定;最适pH为6.0,同一温度下pH 6.0-9.0范围内处理30 min可以保持80％以上的酶活力,此酶对普鲁兰糖有很强的底物特异性.此重组普鲁兰酶的酶学性质表明此酶具有一定的工业化应用价值.     

蜂房哈夫尼菌植酸酶基因appA的克隆、表达及酶学性质研究

从蜂房哈夫尼菌(Hafniaalvei)中克隆获得一个植酸酶编码基因appA,该基因全长1335bp,编码444个氨基酸,其中前33个氨基酸为信号肽,成熟蛋白的理论分子量为45.2kD。将基因appA克隆到大肠杆菌E.coli表达载体pET-22b( ),并在大肠杆菌中表达,表达产物具有植酸酶活性。对表达的酶蛋白进行纯化,并初步研究了该酶的酶学性质,结果表明:酶的作用最适pH值为4.5;在pH2.0~10.0范围内,酶活性保留80%以上;酶的作用最适温度为60℃;酶的比活性为356.7U/mg,酶动力学分析表明其Km为0.49mmol/L,Vmax为238U/mg;该酶对胰蛋白酶和胃蛋白酶有一定的抗性。该研究为哈夫尼菌属来源植酸酶的首次报道。     

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.     

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.     

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