构建重组枯草芽孢杆菌催化制备D-对羟基苯甘氨酸

目的: 在Bacillus subtilis中表达异源D-海因酶基因(hyd)和D-氨甲酰水解酶基因(adc),构建重组细胞作为催化剂,用于生产D-对羟基苯甘氨酸(D-HPG)。方法: 构建hyd表达质粒,考察培养基中二价金属离子对D-海因酶活性的影响。过表达acoR基因,考察AcoR蛋白胞内水平与P_acoA-hyd基因拷贝数的关系。筛选表达adc基因的启动子,构建hyd和adc基因共表达质粒,考察双酶活性菌株的催化特性。结果: 成功构建了海因酶表达质粒pHPS和pUBS,培养基中添加0.8mmol/L的MnCl₂·4H₂O,使168N/pUBS菌株的D-海因酶活性达到956U/gDCW。整合表达P_cdd-acoR基因,使LSL02/pUBS菌株的D-海因酶活性达到1 470U/gDCW。单拷贝P_AE-adc基因的表达水平相对最高。双酶共表达质粒pUBSC被成功构建,菌株LSL02/pUBSC的最适催化温度为40℃45℃,催化活性能够持续12h,当底物起始浓度为20g/L时,反应12h生成的D-HPG达到14.32g/L,转化率达到95%,收率超过80%。结论: 构建具有D-海因酶和D-氨甲酰水解酶双酶活性的重组Bacillus subtilis作为全细胞催化剂,用于海因酶法生产D-HPG,具有技术上的可行性和优势。     

海因酶研究新进展

海因酶在生产手性药物中间体D-氨基酸上具有广泛的应用价值。全面综述了海因酶的研究历史、分类、进化和酶学性质;总结了产海因酶的茵种筛选和产酶条件、D-海因酶的纯化、微生物海因酶基因的克隆及其在大肠杆茵中的表达等技术;阐述了酶法合成D-(-)-对羟基苯甘氨酸的工艺条件。     

微生物酶法制备D-对羟基苯甘氨酸的研究进展

D-对羟基苯甘氨酸(D-p-HPG)是半合成青霉素和头孢霉素的重要前体。综述了微生物酶法生产D-对羟基苯甘氨酸的方法种类及其优缺点并对酶的来源即菌种的选育方法做了总结。此外,还简介了基因工程领域内的研究状况。     

代谢工程改造酿酒酵母合成植物萜类D-柠檬烯的策略

植物萜类化合物是以异戊二烯为结构单位的一大类植物天然的次生代谢产物。D-柠檬烯属于单萜类化合物,由于它具有抑菌、增香、抗癌、止咳、平喘等多种功能,已被广泛应用于食品、香料、医疗等行业。目前D-柠檬烯的工业生产主要是从植物的果皮或者果肉中提取的,但提取方法存在着分离纯化复杂、产率低、能耗大等缺点。而本世纪初合成生物学技术的兴起,为微生物异源合成天然活性化合物带来了全新的理念与工具,打破了物种间的界限,使微生物异源合成D-柠檬烯成为现实。构建定向、高效的异源合成D-柠檬烯的微生物细胞工厂,实现微生物发酵法替换传统的植物提取法,具有重要的经济与社会效益。本文主要回顾了近几年利用代谢工程改造酿酒酵母异源合成萜类化合物取得的成就,阐述了以酿酒酵母作为底盘微生物,利用代谢工程和合成生物学的手段构建高产D-柠檬烯的合成策略。     

过氧化物酶体内的D-双功能蛋白质

D-双功能蛋白质是1996年发现的哺乳动物过氧化物酶体内的一种酶,广泛分布于全身各组织,参与过氧化物酶体β-氧化反应,特异地催化D-3-羟脂酰辅酶A的脱水与脱氢,在脂肪酸分解、胆汁酸合成等代谢中具有重要作用。D-双功能蛋白质缺陷病人通常在出生后6月至2岁之间死亡。     

基于双酶级联协调表达策略高效催化合成D-甘露醇北大核心CSCD

D-甘露醇(D-mannitol)作为合成抗肿瘤药和免疫刺激剂的重要前体被广泛应用于制药和医疗等行业,酶法合成D-甘露醇反应成本昂贵无法满足工业化生产。本研究首先筛选关键酶获得较优性能的甘露醇脱氢酶Lp MDH和用于辅因子NADH再生的葡萄糖脱氢酶Ba GDH,在大肠杆菌(Escherichia coli)BL21(DE3)中共表达,实现了基于双酶级联反应催化底物D-果糖合成D-甘露醇,D-甘露醇的初步摩尔转化率为59.7%。针对双酶级联催化反应中辅酶再生用酶与催化用酶表达量不协调的问题,通过增加Bagdh拷贝量来提高辅因子循环能力,获得了双酶催化速率平衡的重组大肠杆菌E.coli BL21/pETDuet-Lpmdh-Bagdh-Bagdh。进一步对重组菌的全细胞转化条件进行优化,确定了最适转化条件为反应温度30℃,初始pH值6.5,菌体量OD600=30,底物D-果糖100.0 g/L,辅底物葡萄糖与底物1︰1摩尔当量。于最优转化条件下5 L发酵罐转化24 h,D-甘露醇的最高产量为81.9g/L,摩尔转化率为81.9%。本研究提供了一种绿色、高效生物催化生产D-甘露醇的方法,为实现其规模化生产奠定了基础,同时也对其他相关稀有糖醇的研究具有指导意义。     

内消旋-二氨基庚二酸脱氢酶不对称合成非天然手性D-氨基酸的研究进展

内消旋-二氨基庚二酸脱氢酶不对称合成非天然的手性D-氨基酸是目前生物催化领域的研究热点。内消旋-二氨基庚二酸脱氢酶具有优良的立体选择性,利用其进行酶催化不对称合成光学纯的手性D-氨基酸,被广泛用于医药、食品、化妆品、精细化学品等领域。为了促进生物催化法在合成手性D-氨基酸方向的进一步发展,本文对内消旋-二氨基庚二酸脱氢酶催化合成D-氨基酸的现状进行了综述。重点介绍了Corynebacterium glutamicum、Ureibacillus thermosphaericus、Symbiobacterium thermophilum来源的内消旋-二氨基庚二酸脱氢酶在新酶的挖掘、催化性能、晶体结构解析、分子改造、功能与催化机制、合成D-氨基酸新途径等方面的研究进展,并对内消旋-二氨基庚二酸脱氢酶的未来研究方向及策略进行了展望。本综述将进一步加深人们对内消旋-二氨基庚二酸脱氢酶的认识,也为具有挑战性的生物合成任务提供信息借鉴。     

D-天冬酰胺和D-高丝氨酸的制备研究

在以L-天冬氨酸为原料制备D-天冬氨酸的基础上,设计了D-天冬酰胺和D-高丝氨酸的合成新方法。即以L-天冬氨酸为原料,经酯化、消旋、拆分后得到D-天冬氨酸甲酯;D-天冬氨酸甲酯盐酸盐氨解、精制可得到D-天冬酰胺,总收率为49.9%,光学纯度达到99%以上;由D-天冬氨酸甲酯经还原、精制可得到D-高丝氨酸,总收率为64.7%,旋光纯度达到99%以上。     

重组共表达大肠杆菌细胞从D-葡萄糖一锅法生产D-甘露糖

【背景】D-甘露糖的酶促转化方法已受到相当大的关注。【目的】研究D-葡萄糖异构酶(D-glucoseisomerase,D-GIase)和D-来苏糖异构酶(D-lyxoseisomerase,D-LIase)共表达于大肠杆菌细胞生产D-甘露糖的工艺条件。【方法】将D-GIase和D-LIase基因片段合成后酶切连接到载体p CDFDuet-1上,构建p CDFDuet-Acce-DGI/Peba-DLI重组质粒并导入到大肠杆菌BL21(DE3)中共表达,通过摇瓶培养得到产D-GIase和D-LIase的菌体,测定该共表达细胞体系的反应条件。【结果】添加1 mmol/L Co~(2+),共表达体系酶的最适温度和p H分别为70°C和6.0。以浓度分别为100、300、500 g/L的D-葡萄糖为底物生产D-甘露糖,平衡后D-甘露糖质量浓度分别为13.8、38.1、62.6 g/L,相应的转化率分别为13.8%、12.7%、12.5%,D-葡萄糖、D-果糖和D-甘露糖的平衡比约为50:37.5:12.5。【结论】D-GIase和D-LIase在大肠杆菌细胞中组成的共表达体系通过一锅法可利用D-葡萄糖为底物生产D-甘露糖。     

聚苯乙烯树脂固定化D-海因酶的初步研究

D-海因酶广泛用于D-氨基酸的制备研究和生产中,目前已有许多固定化D-海因酶及含D-海因酶细胞的研究报道。尝试了不同功能基团的聚苯乙烯树脂进行D-海因酶的固定化,结果表明功能基为伯氨基和仲氨基效果较好,并选取聚苯乙烯树脂D92进行了固定化D-海因酶的研究。采用该树脂制备固定化酶的最优条件是：酶质量浓度6mg／mL、温度25℃、固化时间12h。所得固定化酶的最适作用温度45℃,最适作用pH为8.5,且作用温度及适宜pH较广,Km为游离酶的1,8倍,且储存稳定性、操作稳定性较好。45℃下半衰期为11d。     

Adsorptive removal of inhibitory by-product in the enzymatic production of optically active D-p-hydroxyphenylglycine from 5-substituted hydantoin

Summary One-step enzymatic production of D-p-hydroxyphenylglycine (D-HPG) from 5-substituted hydantoin was carried out using a bacterium Agrobacterium sp I-671 which possesses D-hydantoinase and N-carbamoylase. From the inhibition study, it was found that N-carbamoylase was severely inhibited by ammonium ions which is co-produced with D-HPG. In order to increase the conversion yield of D-HPG, simultanious removal of inhibitory by-product from reaction mixture was carried out using the specific adsorbents for ammonium ions. The conversion yield of D-HPG reached about 98% in the presence of adsorbents in 27 hours, while 50% conversion was observed in the absence of adsorbents.     

One-step production of D-p-hydroxyphenylglycine by recombinant Escherichia coli strains

The gene encoding D-hydantoinase from Agrobacterium radiobacter NRRL B11291 was successfully cloned by use of polymerase chain reaction. A positive clone was scored, and its nucleotide sequence was further analyzed. The analysis by deleting various lengths of nucleotides from the amino terminus of the open reading frame revealed the putative regions for promoter and RBS site. By highly expressing both D-hydantoinase and carbamoylase, recombinant Escherichia coli strains were able to convert DL-hydroxyphenyl hydantoin (DL-HPH) to D-p-hydroxyphenylglycine (D-HPG) with a conversion yield of 97%, accounting for productivity 5 times higher than that obtained by A. radiobacter NRRL B11291. Immobilizing the recombinant cells with kappa-carrageenan could also achieve a conversion of 93%, while A. radiobacter NRRL B11291 attained 20% within the same period of reaction time. These results illustrate the feasibility in employing recombinant E. coli to accomplish one-step conversion of DL-HPH to D-HPG. In the process of improving D-HPG production, D-hydantoinase activity was increased 2.57-fold but carbamoylase activity remained constant, which resulted in only a 30% increase in the reaction rate. It suggests that carbamoylase is the step setting the pace of the reaction. Since the reaction substrate is highly insoluble, achieving sufficient agitation appears to be an important issue in this heterogeneous system. This view is further supported by the study on repeated use of cells, which shows that to reach a conversion of more than 90% free cells can be recycled six times, whereas immobilized cells can be used only twice. In conclusion, the poor reusability of immobilized cells is due to the fouling on the gel surface.     

利用基因工程菌HC01固定化细胞转化生产D-对羟基苯甘氨酸

对一菌两酶工程菌HC01转化底物DL-对羟基苯海因(DL-HPH)的最适条件及其细胞固定化进行了研究,HC01游离细胞转化DL-HPH的最适条件为40°C、pH7.5。通过对固定化细胞酶活力测定,确定细胞固定化的最优条件为海藻酸钠浓度2.5%、细胞浓度0.029g/mL、钙离子浓度3%。固定化HC01的热稳定性比游离细胞高5°C,二价金属离子Mn2+、Mg2+、Cu2+、Co2+和Ni2+在浓度为0.1mmol/L时对固定化细胞中D-海因酶(HYD)和N-氨甲酰-D-氨基酸酰胺水解酶(CAB)两酶的活力无显著影响,Mn2+和Mg2+可分别使游离细胞中CAB活力提高至原来的2.1和2.7倍。在氮气保护下,当初始pH为9.0、转化温度为40°C、转速为80r/min,利用固定化HC01转化30g/L的DL-HPH时,36h后转化率可达97%左右,产物D-HPG经纯化后光学纯度达到99.7%,得率可达85%。     

Variation in Constraint Versus Positive Selection as an Explanation for Evolutionary Rate Variation Among Anthocyanin Genes

It has been argued that downstream enzymes in metabolic pathways are expected to be subject to reduced selective constraint, while upstream enzymes, particularly those at pathway branch points, are expected to exhibit more frequent adaptive substitution than downstream enzymes. We examined whether these expectations are met for enzymes in the anthocyanin biosynthetic pathway in Ipomoea. Previous investigations have demonstrated that downstream enzymes in this pathway have substantially higher rates of nonsynonymous substitution than upstream enzymes. We demonstrate here that the difference in rates between the most upstream enzyme (CHS) and the two most downstream enzymes (ANS and UFGT) is explained almost entirely by differences in levels of selective constraint. Adaptive substitutions were not detected in any of these genes. Our results are consistent with suggestions that constraint is greater on enzymes with greater connectivity.     

氮沉降增加对森林凋落物分解酶活性的影响

氮沉降增加对森林凋落物分解酶产生的影响在世界范围受到关注。综述了凋落物分解酶的种类、影响酶的因素、酶的生态学意义和土壤酶研究技术的研究发展趋势。根据森林凋落物底物性质的不同,将凋落物分解酶分为纤维素分解酶类、木质素分解酶类、蛋白水解酶类和磷酸酶类。目前普遍认为,氮沉降增加,磷酸酶类活性随之增加,其它三类酶活性未呈现规律性变化。此外,还对氮沉降增加与土壤酶之间关系的研究前景进行了探讨。     

Metabolic capacity of nasal tissue: interspecies comparisons of xenobiotic-metabolizing enzymes

High levels of xenobiotic-metabolizing enzymes occur in the nasal mucosa of all species studied. In certain species, including rats and rabbits, unique enzymes are present in the nasal mucosa. The function of these enzymes is not well understood, but it is thought that they play a role in protecting the lungs from toxicity of inhalants. The observation that several nasal xenobiotic-metabolizing enzymes accept odorants as substrates may indicate that these enzymes also play a role in the olfactory process. Xenobiotic-metabolizing enzymes were found in the nasal cavity around 15 years ago. Since that time, much has been learned about the nature of the enzymes and the substrates they accept. In the present review, this information is summarized with special attention to species differences in xenobiotic-metabolizing enzymes of the nasal cavity. Such differences may be important in interpreting the results of toxicity assays in animals because rodents are apparently more susceptible to nasal toxicity after exposure to inhalants than are humans.     

Biochemical and genetic analysis of the biosynthesis, sorting, and secretion of Dictyostelium lysosomal enzymes

Dictyostelium discoideum is a useful system to study the biosynthesis of lysosomal enzymes because of the relative ease with which it can be manipulated genetically and biochemically. Previous studies have revealed that lysosomal enzymes are synthesized in vegetatively growing amoebae as glycosylated precursor polypeptides that are phosphorylated and sulfated on their N-linked oligosaccharide side-chains upon arrival in the Golgi complex. The precursor polypeptides are membrane associated until they are proteolytically processed and deposited as soluble mature enzymes in lysosomes. In this paper we review biochemical experiments designed to determine the roles of post-translational modification, acidic pH compartments, and proteolytic processing in the transport and sorting of lysosomal enzymes. We also describe molecular genetic approaches that are being employed to study the biosynthesis of these enzymes. Mutants altered in the sorting and secretion of lysosomal enzymes are being analyzed biochemically, and we describe recent efforts to clone the genes coding for three lysosomal enzymes in order to better understand the molecular mechanisms involved in the targeting of these enzymes.