D-乳酸生产菌菊糖芽孢乳杆菌来源的D-乳酸脱氢酶同工酶

【目的】菊糖芽孢乳杆菌(Sporolactobacillus inulinus)作为典型的同型发酵产D-乳酸的优势菌株,能够高效生产高纯度的D-乳酸。该菌株发酵受到多方面环境因素影响。糖代谢的关键酶例如葡萄糖激酶、磷酸果糖激酶、丙酮酸激酶以及乳酸脱氢酶均为由葡萄糖代谢成为乳酸的关键酶,该菌中相关代谢酶的研究是发酵调控至关重要的基础。分析S.inulinus的基因组表明有3个推测为D-乳酸脱氢酶的基因,其中已有报道研究了1个双功能蛋白[bifunctional protein(BP)]。本研究分别克隆并解析了另2个D-乳酸脱氢酶同工酶的性质。【方法】本研究以S.inulinus Y2-8基因组DNA为模板,克隆得到2个D-ldh基因(dldh、dhdh),经测序分别为D-乳酸脱氢酶[D-lactic acid dehydrogenase(DLDH)]和D-羟基酸脱氢酶[D-isomer specific 2-hydroxyacid dehydrogenase(DHDH)]的基因。构建的重组菌表达蛋白DLDH,DHDH均具有催化丙酮酸生成D-乳酸的功能。【结果】重组菌表达的蛋白经镍柱亲和层析达到电泳纯。SDS-PAGE分析表明DLDH的表观分子量为37 k Da,DHDH的表观分子量为39 k Da。此外,DLDH以丙酮酸为底物时Km值为(0.58±0.04)mmol/L,对底物有较高的亲和力,最适反应温度为35°C,最适p H为6.5;而DHDH以丙酮酸为底物时Km值为(1.70±0.08)mmol/L最适反应温度为30°C,最适p H为7.5。另有报道的BP以丙酮酸为底物时Km值为(3.40±0.02)mmol/L,最适反应温度为30°C,最适p H为5.5。【结论】根据对底物丙酮酸的亲和力,最适温度及最适p H,推测DLDH是乳酸发酵中产D-乳酸的主导催化剂。结合相关酶学性质的分析可为今后的发酵调控提供理论依据。     

左旋多巴的合成与提取

左旋多巴 (L DOPA)是治疗帕金森病的有效药物。L DOPA的生产方法有化学合成、从植物中提取和微生酶物转化等 ,其中利用微生物的酪氨酸酚解酶以邻苯二酚、丙酮酸和氨为底物合成L DOPA被证明是一种最经济且最有前途的方法。应用基因工程技术构建高效菌株。左旋多巴的提取有多种方法 ,其中向反应体系中加入晶种使多巴从反应体系中析出 ,除去菌体和杂质 ,再进行重结晶可得到纯度较高的多巴是一种很好的方法。     

创新霉素产生菌吲哚丙酮酸甲基转移酶的研究

在创新霉素产生菌济南游动披线菌的无细胞提取物中检测到吲哚丙酮酸甲基转移酶活性,并进行了分离提取。该酶能利用S-腺苷-L-甲硫氨酸对吲哚丙酮酸进行甲基化,它可能作用于创新霉素中间体的甲基化。经过硫酸铵分部盐析和DEA F一纤维素柱层析,得到了纯化60倍的甲基转移酶,比活0．66mu／ms。酶的最适底物是吲哚丙酮酸,最适pH7．5,对于底物s一腺苷一L一甲硫氨酸和吲哚丙酮酸的米氏常数(KⅢ)分别是4×10-,mol／L和1．8×10-7moI／L。用Sephadcx G-150凝胶过滤测得分子量是55000士5000道尔顿。     

烟草吡哆胺 丙酮酸转氨酶的部分纯化与表征

通过冷冻干燥、DEAE Sepharose Fast Flow阴离子交换柱层析、Sephadex G 100凝胶过滤柱层析,SP葡聚糖凝胶C 25阳离子交换柱层析等分离纯化技术,对烟草吡哆胺 丙酮酸转氨酶进行分离纯化。采用苯肼衍生化方法检测活性,并对其基本酶学性质进行分析。结果显示：该酶被纯化了92.34倍;最适温度为70 ℃,最适pH为9.0。在pH7.0～9.0内稳定且热稳定性较好,80 ℃保温3 h仍有51.55%的酶活力;在最适反应条件下,测得反应底物吡哆胺和丙酮酸的Km值分别为6.337 mmol?L 1和0.867 mmol?L 1。该结果为进一步研究烟草体内VB6代谢机制奠定了基础。     

离体小麦、向日葵叶片及子叶中γ-氨基丁酸的代谢

离体小麦、向日葵叶片及子叶,在预照光5小时条件下可以利用外源γ-氨基丁酸,形成丙氨酸和谷氨酰胺。小麦叶片中γ-氨基丁酸的脱氨过程主要是γ-氨基丁酸-丙酮酸转氨酶的作用。磷酸钙胶的吸附作用可以将此酶的比活力提高到5.6倍。此转氨酶酶促反应的最适 pH为8.9,在最初反应1小时之内,反应的速度与酶的浓度和反应时间成直线关系,对底物γ-氨基丁酸的米氏常数 Km 为2.6×1~(-3)M。讨论了转氨酶在γ-氨基丁酸代谢中的作用。     

利用巨大芽孢杆菌酶系合成L-丙氨酸和L-缬氨酸

本文研究了利用巨大芽孢杆菌ATCC_(39118)酶系合成氨基酸,同时也研究了丙氨酸脱氢酶、缬氨酸脱氢酶及葡萄糖脱氢酶的提纯工艺。所获得的AlaDH、ValDHc和GlcDH的比活性分别为11.2u/mg,7.8u/mg和23.0u/mg。为了进一步探讨由α-酮酸酶法转化成氨基酸的最适条件,我们对以上三种酶的主要性质,包括稳定性,最适pH、动力学常数、底物专一性及底物和产物对酶的抑制作用等进行了测定。同时用粗酶提取液和纯酶进行了由丙酮酸合成L-丙氨酸,由α-酮异戍酸合成L-缬氨酸的批量实验,在转化中葡萄糖脱氢酶作为NADH的再生酶。结果粗酶提取液催化L-丙氨酸产量的克分子转化率为80％,而纯酶催化的克分子转化率增加到92％。L-缬氨酸产量的克分子转化率也类似(93％)。     

植物乳杆菌ZS2058生物转化共轭亚油酸的反应动力学

摘要：【目的】对本实验室从泡菜中筛选到的植物乳杆菌ZS2058完整细胞生物转化共轭亚油酸的反应动力学进行研究。【方法】探讨底物浓度、细胞浓度、反应体系pH值等因素对生物转化共轭亚油酸反应速度的影响,并通过双倒数和Hanes-Woolf作图法拟合反应初始阶段的速度方程。【结果】生物转化共轭亚油酸时存在明显的底物抑制现象,当亚油酸浓度为0.4 mg/mL时产c9, t11-共轭亚油酸的反应速度达最大值15.99 μg/(mL?h);反应速度随细胞浓度增加而上升,当细胞浓度为5×1010 cfu/mL时反应速度达到最高;最适pH值和最适反应温度分别为6.5和40 ℃。利用双倒数和Hanes-Woolf作图法求得米氏常数和最大反应速度,在低底物浓度下,反应初始阶段的反应规律与经典的米氏方程相符,而在高底物浓度下,存在明显的底物抑制现象。【结论】通过对植物乳杆菌ZS2058完整细胞催化合成共轭亚油酸各因素的考察,在得到最佳反应条件的同时建立了不同底物浓度范围内的反应速度方程,这对于实现共轭亚油酸的生产和研究其生理功能具有十分重要的理论价值。     

海藻酸钠-壳聚糖固定化木瓜蛋白酶催化内吗啡肽的合成

反应体系以乙腈作为有机介质,在微水有机溶剂体系中以Boc-Trp-OH和Phe-NH2为底物,用海藻酸钠 壳聚糖固定化木瓜蛋白酶催化合成Trp-Phe-NH2时,产率为27.8%．在这一合成反应中,对pH值、离子强度、溶液含量、反应温度、酶用量和反应时间进行正交试验,证明pH是本合成过程的最重要影响因素．反应体系以乙腈为有机介质,在微水有机溶剂体系中以 Boc-Tyr-Pro-OMe和Trp-Phe-NH2为底物,用IPSAC催化合成Tyr-Pro-Trp-Phe-NH2,产率为35~2%．     

聚丙烯腈纤维固定化青霉素酰化酶合成头孢氨苄的研究

将巨大芽孢杆菌胞外青霉素酰化酶通过共价键结合到聚丙烯腈纤维的衍生物上。制成的丝状固定化青霉素酰化酶表现活力达 1 5 3U g(湿重 )。固定化酶合成头孢氨苄的最适pH为 6 5 ,最适温度为 40℃。 7 ADCA的投料浓度以 4%为好 ,7 ADCA与PGME的投料量比率为1∶2 ,最佳用酶量为 1 70U g 7 ADCA。在pH6 5、温度 3 0℃时 ,固定化酶对 7 ADCA的表观米氏常数K7 ADCA为 0 1 6 2mol L ,对PGME的表观米氏常数KPGME为 0 3 6 4mol L ,最大反应速度Vmax为0 0 4 6 2mol·L- 1·min- 1,用固定化酶合成头孢氨苄 ,使用 5 0次保留酶活力 83 9%     

枇杷果实多酚氧化酶活性影响因素的研究

研究了温度、pH、几种还原剂对枇杷果实多酚氧化酶活性的影响。结果表明:以邻苯二酚为作用底物,该酶的最适温度为25℃、最适pH为6.3,抗坏血酸、L-半胱氨酸、亚硫酸钠等还原剂对该酶活性有明显的抑制作用。     

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.