头孢菌素脱乙酰酶的重组表达及固定化

本文构建了利用trp启动子表达头孢菌素脱乙酰酶(CAH)的重组大肠杆菌DH5α-pCAH。重组菌在7L发酵罐(装液量2L)中发酵28 h,发酵液OD_(600)达到27,产酶313 kU/L发酵液,粗略估算重组蛋白占细胞总蛋白的70%。发酵生产的重组CAH粗酶液经过硫酸铵分级沉淀分离纯化和超滤除盐浓缩两步操作,纯化倍数为1.44,总酶活回收率56%,聚丙烯酰胺凝胶电泳检测纯化后蛋白没有明显杂蛋白条带出现。纯化后的CAH共价结合固定在环氧基载体LX-1000EP(c)上,通过对固定化条件的优化最终得到固定化酶比活443 U/g。该固定化酶重复催化50 mL 5%7-ACA底物100次后,酶活没有降低。     

微囊载体固定化多酶的研究进展

多酶催化是利用多种生物酶构建反应体系或网络,在生物体外实现化学品的合成.在生物制造过程中,多酶的共固定化有利于提高酶的稳定性和重复使用率,更利于多酶间的协同催化.在精准调控下,多酶固定化载体的微囊材料有望实现多酶协同催化性能的最大化.本文中,笔者分析了微囊体系的特点,综述了微囊材料及其固定化多酶的优缺点,总结了微囊多酶...     

大孔树脂固定化脂肪酶及在微水相中催化合成生物柴油的研究

以不同大孔树脂吸附法固定化假丝酵母99_125脂肪酶,在微水有机相中的应用表明非极性树脂NKA是最佳的固定化载体。分别以正庚烷及磷酸盐缓冲液作为固定化介质,发现在正庚烷介质中树脂NKA的固定化效率能够达到98.98%,与采用磷酸盐缓冲液作为介质相比,固定化酶的水解活力和表观酶活回收率分别提高了4.07和3.43倍。考察了在微水相中固定化酶催化合成生物柴油的催化性能,结果表明,在给酶量为1.92∶1(初始酶粉与树脂的质量比),pH值为7.4,体系水含量为15%(水与油的质量比),反应温度为40℃条件下,固定化酶具有最佳的催化能力;以正庚烷为介质固定化脂肪酶催化合成生物柴油,采用三次流加甲醇的方式,单批转化率最高达到97.3%,连续反应19批以后转化率仍保持为70.2%。     

固定化脂肪酶催化毛油合成生物柴油

本研究开发了一种用石油醚提取毛油的工艺,研究了以提取的毛油和甲醇为原料,用固定化Candida sp.99-125脂肪酶催化合成脂肪酸甲酯(FAMEs)的可行性。同时考察了磷脂对固定化酶活性、反应起始速率、固定化酶使用批次的影响以及毛油和精炼油对固定化酶使用批次等的影响。研究结果表明,用磷脂质量分数为1%的石油醚悬液浸泡过的脂肪酶比仅用石油醚浸泡过的脂肪酶初始转酯化速率显著下降。当大豆油中无磷脂时,15min时FAMEs的产率为26.2%;磷脂质量分数为5%时,FAMEs降为12.4%。当大豆油中磷脂质量分数小于1%时,固定化酶使用10个批次,FAMEs产率无明显变化。固定化脂肪酶催化石油醚浸提得到的大豆和小桐子毛油,经过10个批次反应FAMEs产率都保持在70%以上,该固定化酶直接催化毛油生产生物柴油具有良好的工业化前景。     

多级孔金属-有机框架固定化酶的研究进展

金属-有机框架(metal-organic frameworks, MOFs)作为酶固定化的优良载体,为生物催化反应提供优越的物理和化学保护。近年来,多级孔金属-有机框架(hierarchical porous metal-organic frameworks, HP-MOFs)由于其独特的结构优势,在固定化酶方面显示出更大的潜力。到目前为止,已经开发了各类具有原生多级孔或缺陷多级孔的HP-MOFs用于酶的固定化研究,并且使得固定化酶在催化活性、稳定性和重复利用性等方面得到了显著增强。本文系统总结了HP-MOFs用于固定化酶的各种策略,介绍了HP-MOFs固定化酶(enzyme@HP-MOFs)在催化合成、生物传感、生物医药等领域的最新应用进展。最后,讨论并展望了HP-MOFs固定化酶这一领域所面临的挑战和机遇。     

纳米生物催化的研究现状和发展态势

纳米生物催化领域包括:(ⅰ)利用纳米技术或纳米材料调控生物催化剂的效率;(ⅱ)直接利用纳米材料或技术实现生物催化功能,并拓展生物催化在非友好环境及疾病诊疗中的应用.纳米生物催化已成为纳米生物学重要的研究领域,主要涉及纳米载体固定化酶和纳米材料人工模拟酶(纳米酶).一方面,可以借助纳米技术或材料所具有的特殊纳米效应来增强生物催化剂的效率和稳定性.另一方面,从模拟酶的理念出发,借助纳米材料自身所具有的催化能力,直接实现对生化反应的催化,这类具有酶学特性的纳米酶被视为新一代人工模拟酶.近年来,基于纳米载体固定化酶和纳米酶技术的纳米生物催化已在疾病诊断和治疗、化工制药、环境处理等领域得到了广泛研究,并展示了其具有重要的应用价值.本文简要综述了纳米载体固定化酶和纳米酶的发展历程及应用进展.     

漆酶的固定化及其在生物传感器方面的应用

漆酶是一类含铜的多酚氧化酶,它能够催化许多酚类和非酚类物质的氧化.固定化漆酶能改善漆酶的稳定性,实现酶制剂的重复连续使用,具有重要意义.该文综述了漆酶固定化的各种方法,阐述了漆酶相关活性、机械性能和功能等内容,并对漆酶固定化在生物传感器方面的应用作了介绍.     

大孔树脂固定化磷脂酶D

磷脂酶D(PhospholipaseD,EC3.1.4.4,PLD)是催化磷酸酯键水解和碱基交换反应的一类酶的总称.利用PLD的转碱基作用是目前催化合成磷脂酰丝氨酸(PS)的最佳途径.本实验以5种大孔树脂为载体固定化磷脂酶D(PLD)进行了研究.以酶回收率为主要指标,选择了最佳载体和优化了固定化条件.结果表明:非极性阳离子交换树脂H103是最佳固定化载体;其最优固定化条件:加酶液量1.2 mL,固定时间80 min,pH 6.0柠檬酸-柠檬酸钠的缓冲液浓度为10 mmol/L.最佳固定化条件下,固定化之后的PLD比游离PLD酶活提高了三倍.     

Eupergit C250L固定化D-海因酶及其催化性质

D-海因酶是海因酶法制备D-氨基酸的关键酶。利用Burkholderic cepecia1003菌发酵产酶,所得海因酶纯化后,以Eupergit C250L为载体进行共价固定化。分别考察了酶液蛋白浓度、固定化时间对蛋白固定量和酶活回收率的影响以及固定化前后海因酶催化性质的变化。结果表明:较高的酶液蛋白浓度和较长的固定化时间均有助于改善海因酶的固定化效果;固定化可显著提高海因酶的最适作用温度,但对其最适作用pH影响不大;固定化后海因酶对D,L-BH和MH的米氏常数均有较大幅度的降低。固定化酶反应器的实验表明:40℃下,底物(D,L-BH)1.0 g.L-1,体积流速1.0 mL.min-1,经21 h转化,产物N-Phe质量浓度可达0.47 g.L-1,转化率达43.21%。     

DA-F127水凝胶包埋固定化含腈水合酶细胞

腈水合酶是一类可催化腈类化合物转化生成相应酰胺类物质的酶。含腈水合酶的游离细胞催化水合反应存在酶容易失活、细胞无法重复利用、分离纯化困难等缺陷,细胞固定化技术可有效解决这些问题。为探索合适的固定化方法,以含腈水合酶的重组E. coli细胞为研究对象,以固定化酶活回收率和批次反应情况为评价指标,筛选比较了几种常用的包埋固定化方法。结果表明,DA-F127水凝胶包埋固定化细胞不仅具有较高的酶活回收率,而且稳定性也很好。对该方法进行了固定化条件和操作稳定性优化,当DA-F127浓度为15%、UV光源距离为20cm、光照时间为6min、菌体含量为20mg/g固定化细胞时,酶活回收率为89. 74%,并且可以催化9批次150g/L的3-氰基吡啶完成转化,第九批次转化率可达98. 26%。与游离细胞催化过程相比,单位质量游离细胞的烟酰胺产量提高了12倍,具有良好的工业应用前景。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

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     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.