吡咯喹啉醌生物合成研究进展

吡咯喹啉醌(pyrroloquinoline quinone,PQQ)是一种多肽修饰类天然产物,是继烟酰胺和核黄素之后第三类辅酶,具有抗氧化、抗衰老、提高免疫力等重要生理功能,在医药、保健等领域具有重要价值.目前,PQQ的大规模制备仍然存在诸多问题,限制了PQQ的广泛应用.当前迫切需求低成本的合成方式,以充分实现其广阔...     

醌蛋白研究进展

醌蛋白又称醌酶,是一类以吡咯喹啉醌(PQQ)或其结构类似物(TTQ、TPQ或LTQ)为辅助因子的氧化还原酶。以PQQ为辅酶的醌蛋白是其中最重要的一类醌蛋白。按其在氧化还原反应过程中包含辅酶的种类和数量可以分为Ⅰ型、Ⅱ型和Ⅲ型等3种型别。Ⅰ型醌蛋白是一类简单的醌蛋白,只含有1分子的PQQ作为辅酶;而Ⅱ型和Ⅲ型醌蛋白含有PQQ和血红素c作为辅酶。Ⅱ型醌蛋白包含1分子PQQ和1分子血红素c,广泛存在于假单胞菌属、丛毛单胞菌属细菌的胞外周质中,参与催化反应过程。Ⅲ型醌蛋白仅存在于醋酸菌属细菌的细胞膜上,由3个亚基组成,即含有1个PQQ/血红素的催化亚单位、1个含有3个血红素c的亚单位和1个不含辅酶的功能未知的亚单位。综述了几种主要醌蛋白的结构、催化机制、电子传递,以及它们的应用情况。     

醌血红素蛋白的电子传递

醌蛋白是以吡咯喹啉醌（PQQ）及其结构类似物为辅酶的一类氧化还原酶。醌血红素蛋白是以PQQ和一个或多个血红素作为辅助因子的醌蛋白,包括醌血红素蛋白醇脱氢酶和醌血红素蛋白胺脱氢酶。简要综述了醌血红素蛋白的结构特点,在分子内从PQQ到血红素的电子传递,以及醌血红素蛋白与蛋白之间的分子间的电子传递。     

吡咯喹啉醌合成及生产工艺研究进展

吡咯喹啉醌(pyrroloquinoline quinone, PQQ)是继烟酰胺和核黄素之后发现的第三类氧化还原酶辅因子,普遍存在于生物体中参与呼吸链电子传递,具有促进线粒体产生、清除自由基、增强细胞代谢和预防心肌损伤等生理功能,在医药、食品和农业领域具有广泛的应用前景。微生物发酵法是PQQ生产的主要方式,解析PQQ生物合成途径及其调控机制,通过代谢工程选育短周期、高产量的生产菌是PQQ工业化的研究方向之一。本文综述了PQQ的合成途径、高产菌株选育以及微生物发酵生产与分离纯化的研发工作,为深入阐释PQQ的生物合成机制和工业化生产菌株的选育提供参考。     

吡咯喹啉醌生物合成研究进展

吡咯喹啉醌(PQQ)是一种较新近发现的氧化还原酶的辅酶,对微生物及动植物均具有重要生理作用。已知能产生PQQ的生物仅限于某些革兰阴性细菌,已分离得到几种不同来源的PQQ生物合成基因,其序列具有一定的保守性。PQQ的生物合成涉及4～7个基因,这些基因一般成簇排列。业已证明,谷氨酸和酪氨酸是PQQ合成的前体物质。对各个基因的功能已有不同程度的了解,但PQQ的生物合成途径还尚未阐明。     

食甲基杆菌J1-1吡咯喹啉醌生物合成突变株的筛选和鉴定

目的:通过Tn5转座诱变筛选食甲基杆菌J1-1吡咯喹啉醌(PQQ)生物合成相关基因。方法:构建食甲基杆菌J1-1 Tn5转座突变体库,筛选PQQ合成水平差异明显的突变株,利用质粒拯救法鉴定突变基因,通过基因敲除、回补及过表达进一步研究该基因与PQQ合成的关系。结果:构建了J1-1的Tn5转座突变体库,筛选得到一株PQQ合成水平显著下降的突变株,经鉴定Tn5插入位点为mpq0056基因,该突变株在以甲醇为惟一碳源的培养基中生长速度略慢;敲除J1-1中mpq0056基因后,PQQ的合成水平下降,与Tn5诱变结果一致;回补该基因后,PQQ产量恢复到野生菌水平。结论:mpq0056基因参与了PQQ的生物合成,该基因可能编码分支酸盐裂合酶,并在PQQ生物合成中起重要作用。     

通过转座诱变筛选甲基营养菌吡咯喹啉醌合成缺陷突变株

目的:对电转化等Tn5转座诱变条件进行优化,获得大量甲基营养菌MP688突变株,筛选吡咯喹啉醌(PQQ)合成缺陷突变株,并对失活基因进行鉴定。方法:通过电击方法对MP688株进行Tn5转座诱变;通过检测PQQ产量,选择不产或几乎不产PQQ的突变株,用质粒拯救的方法鉴定突变基因。结果和结论:确定了MP688株电击转化的最优条件,优化了质粒拯救法鉴定突变基因的实验方案,得到了1株PQQ合成明显降低的突变株RM16,并确定了转座子在染色体上的插入位点。     

基于重组大肠杆菌无细胞体系生产吡咯喹啉醌

采用无细胞体系生产吡咯喹啉醌(pyrroloquinoline quinone,PQQ)。首先将肺炎克雷伯氏菌PQQ基因簇pqqABCDEF置于半乳糖苷酶启动子之下,构建表达载体,经转化筛选获得重组大肠杆菌。制备重组菌的细胞匀浆,体外反应后测定PQQ产量。结果显示,与活体重组菌相比,无细胞体系的PQQ产量提高约30%,表明胞内存在PQQ合成的限速反应,而无细胞体系可解除此限速反应。     

吡咯喹啉醌生理医学功效研究进展

吡咯喹啉醌(PQQ)是一种小分子醌类化合物,由某些细菌合成作为细菌脱氢酶氧化还原反应的辅助因子,并广泛存在于各种生物组织中。综述了其在生理医学功效方面的研究进展情况,分析其在神经退行性疾病、心脏病、解毒、消炎、抗癌、预防白内障及骨代谢疾病等方面的临床应用潜力,并对其未来在水生生物生理生态学领域的研究方向进行了展望。     

高产吡咯喹啉醌扭脱甲基杆菌的高通量选育

吡咯喹啉醌(PQQ)是一种细菌脱氢酶的辅酶,具有促进机体生长、调节机体自由基水平等功能,应用于食品、医药等领域。由于化学合成法成本较高,微生物发酵法生产PQQ受到关注。目前,发酵法生产PQQ产量较低,限制了其工业应用。然而,由于对PQQ菌株的合成与调控机制尚缺乏深入理解,以及对野生型菌株缺乏必要的基因工程改造手段,目前采用代谢工程强化PQQ合成菌株还缺乏相关基础。因此,本研究以扭脱甲基杆菌Methylobacterium extorquens I-F2为研究对象,整合常压室温等离子体诱变、流式细胞术分选和高通量筛选策略,对样品制备和流式分选过程进行优化,最终筛选出一株PQQ高产突变菌株1-C6,PQQ产量比出发菌株I-F2提高98.02%。本文所述的流式细胞术结合高通量筛选方法能简单、快速地获得高产突变菌株,相比于基因工程改造和传统筛选方法,具有提升效果明显且易于实施等优势。     

吡咯喹啉醌（PQQ）的研究进展

吡咯喹啉醌是一种与烟酰胺核苷酸、黄素核苷酸不同的新型辅基.近年来,荷兰、日本等学者对它进行了初步研究,而国内研究起步较晚,文章综述了吡咯喹啉醌的发现、分离纯化、鉴定、理化性质以及生理功能,这有利于进一步研究吡咯喹啉醌的分布、产生机理、生物学性质、生理功能及其应用.这将对促进酶学学科的发展具有重要的理论和实践意义.     

Pyrroloquinoline-quinone and its versatile roles in biological processes

Pyrroloquinoline-quinine (PQQ) was initially characterized as a redox cofactor for membrane-bound dehydrogenases in the bacterial system. Subsequently, PQQ was shown to be an antioxidant protecting the living cells from oxidative damage in vivo and the biomolecules from artificially produced reaction oxygen species in vitro. The presence of PQQ has been documented from different biological samples. It functions as a nutrient and vitamin for supporting the growth and protection of living cells under stress. Recently, the role of PQQ has also been shown as a bio-control agent for plant fungal pathogens, an inducer for proteins kinases involved in cellular differentiation of mammalian cells and as a redox sensor leading to development of biosensor. Recent reviews published on PQQ and enzymes requiring this cofactor have brought forth the case specific roles of PQQ. This review covers the comprehensive information on various aspects of PQQ known till date. These include the roles of PQQ in the regulation of cellular growth and differentiation in mammalian system, as a nutrient and vitamin in stress tolerance, in crop productivity through increasing the availability of insoluble phosphate and as a bio-control agent, and as a redox agent leading to the biosensor development. Most recent findings correlating the exceptionally high redox recycling ability of PQQ to its potential as anti-neurodegenerative, anticancer and pharmacological agents, and as a signalling molecule have been distinctly brought out. This review discusses different findings suggesting the versatility in PQQ functions and provides the most plausible intellectual basis to the ubiquitous roles of this compound in a large number of biological processes, as a nutrient and a perspective vitamin.     

Dietary pyrroloquinoline quinone (PQQ) alters indicators of inflammation and mitochondrial-related metabolism in human subjects

Pyrroloquinoline quinone (PQQ) influences energy-related metabolism and neurologic functions in animals. The mechanism of action involves interactions with cell signaling pathways and mitochondrial function. However, little is known about the response to PQQ in humans. Using a crossover study design, 10 subjects (5 females, 5 males) ingested PQQ added to a fruit-flavored drink in two separate studies. In study 1, PQQ was given in a single dose (0.2 mg PQQ/kg). Multiple measurements of plasma and urine PQQ levels and changes in antioxidant potential [based on total peroxyl radical-trapping potential and thiobarbituric acid reactive product (TBAR) assays] were made throughout the period of 48 h. In study 2, PQQ was administered as a daily dose (0.3 mg PQQ/kg). After 76 h, measurements included indices of inflammation [plasma C-reactive protein, interleukin (IL)-6 levels], standard clinical indices (e.g., cholesterol, glucose, high-density lipoprotein, low-density lipoprotein, triglycerides, etc.) and ¹H-nuclear magnetic resonance estimates of urinary metabolites related in part to oxidative metabolism. The standard clinical indices were normal and not altered by PQQ supplementation. However, dietary PQQ exposure (Study 1) resulted in apparent changes in antioxidant potential based on malonaldehyde-related TBAR assessments. In Study 2, PQQ supplementation resulted in significant decreases in the levels of plasma C-reactive protein, IL-6 and urinary methylated amines such as trimethylamine N-oxide, and changes in urinary metabolites consistent with enhanced mitochondria-related functions. The data are among the first to link systemic effects of PQQ in animals to corresponding effects in humans.     

Intestinal absorption and tissue distribution of [14C]pyrroloquinoline quinone in mice.

Pyrroloquinoline quinone (PQQ) functions as a cofactor for prokaryotic oxidoreductases, such as methanol dehydrogenase and membrane-bound glucose dehydrogenase. In animals fed chemically defined diets, PQQ improves reproductive outcome and neonatal growth. Consequently, the present study was undertaken to determine the extent to which PQQ is absorbed by the intestine, its tissue distribution, and route of excretion. About 28 micrograms of PQQ (0.42 microCi/mumol), labeled with 14C derived from L-tyrosine, was administered orally to Swiss-Webster mice (18-20 g) to estimate absorption. PQQ was readily absorbed (62%, range 19-89%) in the lower intestine, and was excreted by the kidneys (81% of the absorbed dose) within 24 hr. The only tissues that retained significant amounts of [14C]PQQ at 24 hr were skin and kidney. For kidney, it was assumed that retention of [14C]PQQ represented primarily PQQ destined for excretion. For skin, the concentration of [14C]PQQ increased from 0.3% of the absorbed dose at 6 hr to 1.3% at 24 hr. Furthermore, most of the [14C]PQQ in blood (greater than 95%) was associated with the blood cell fraction, rather than plasma.     

适应性驯化选育高产吡咯喹啉醌的生丝微菌突变株

吡咯喹啉醌(PQQ)广泛存在于生物体内,具有促进机体生长、维护线粒体功能、促进神经生长因子合成和调节机体自由基水平等生理功能,在医药、食品和化妆品领域具有广阔的应用前景。为提高脱氮生丝微菌Hyphomicrobium denitrificans FJNU-6的PQQ生产性能,文中以高浓度甲醇为拮抗因子进行实验室适应性定向驯化,通过光谱法快速筛选体系,选育PQQ高产正突变株。6轮适应性驯化后,每轮驯化的正向突变率达到90%以上,产量提高1倍的突变株达到10%左右。最后,利用5L发酵罐对突变株FJNU-R8进行分批补料培养,相较于出发菌株,突变株在不同甲醇浓度下pqq和moxF基因簇的表达量较高且差异较小,甲醇消耗和生长速度较慢,PQQ产量达到1 087 mg/L (143 h),单位细胞产量提高了1.42倍,展现出良好的工业应用潜力。文中所述的适应性定向驯化结合快速筛选体系能简单、快速地获得高产PQQ的生丝微菌突变菌株,对其他甲基营养菌高产PQQ突变株的高通量筛选具有借鉴作用。     

Pyrroloquinoline Quinone-Dependent Cytochrome Reduction in Polyvinyl Alcohol-Degrading Pseudomonas sp. Strain VM15C

A polyvinyl alcohol (PVA) oxidase-deficient mutant of Pseudomonas sp. strain VM15C, strain ND1, was shown to possess PVA dehydrogenase, in which pyrroloquinoline quinone (PQQ) functions as a coenzyme. The mutant grew on PVA and required PQQ for utilization of PVA as an essential growth factor. Incubation of the membrane fraction of the mutant with PVA caused cytochrome reduction of the fraction. Furthermore, it was found that in spite of the presence of PVA oxidase, the membrane fraction of strain VM15C grown on glucose without PQQ required PQQ for cytochrome reduction during incubation with PVA. The results provide evidence that PVA dehydrogenase couples with the electron transport chain of PVA-degrading bacteria but that PVA oxidase does not.     

Factors relevant in bacterial pyrroloquinoline quinone production

Quinoprotein content and levels of external pyrroloquinoline quinone (PQQ) were determined for several bacteria under a variety of growth conditions. From these data and those from the literature, a number of factors can be indicated which are relevant for PQQ production. Synthesis of PQQ is only started if synthesis of a quinoprotein occurs, but quinoprotein synthesis does not depend on PQQ synthesis. The presence of quinoprotein substrates is not necessary for quinoprotein and PQQ syntheses. Although the extent of PQQ production was determined by the type of organism and quinoprotein produced, coordination between quinoprotein and PQQ syntheses is loose, since underproduction and overproduction of PQQ with respect to quinoprotein were observed. The results can be interpreted to indicate that quinoprotein synthesis depends on the growth rate whereas PQQ synthesis does not. In that view, the highest PQQ production can be achieved under limiting growth conditions, as was shown indeed by the much higher levels of PQQ produced in fed-batch cultures compared with those produced in batch experiments. The presence of nucleophiles, especially amino acids, in culture media may cause losses of PQQ due to transformation into biologically inactive compounds. Some organisms continued to synthesize PQQ de novo when this cofactor was administered exogenously. Most probably PQQ cannot be taken up by either passive diffusion or active transport mechanisms and is therefore not able to exert feedback regulation on its biosynthesis in these organisms.     

Adduct formation of pyrroloquinoline quinone and amino acid

When pyrroloquinoline quinone (PQQ) is mixed with an amino acid, a corresponding Schiff base PQQ adduct is readily formed between carbonyl groups of PQQ and the primary amino group. A potent growth stimulating effect for microorganisms was observed with the PQQ adduct when it was administered in a culture medium. Although PQQ itself shows a marked growth stimulating effect, PQQ adducts appeared to be more active than authentic PQQ when compared on a molar basis. Conversely, unlike authentic PQQ, PQQ adducts were shown to be less active (greater than or equal to 100-fold) as the prosthetic group for a quinoprotein apo-glucose dehydrogenase when examined by holoenzyme formation by exogenous addition of PQQ or PQQ adducts. These observations suggested that PQQ adduct formation readily occurs during isolation procedures for PQQ from biological materials or PQQ - chromophore from quinoproteins. Therefore, the presence of such adducts gives a PQQ estimation much lower than theoretically expected. As an example, formation, isolation and characterization of PQQ - serine are described.     

Factors relevant in bacterial pyrroloquinoline quinone production.

Quinoprotein content and levels of external pyrroloquinoline quinone (PQQ) were determined for several bacteria under a variety of growth conditions. From these data and those from the literature, a number of factors can be indicated which are relevant for PQQ production. Synthesis of PQQ is only started if synthesis of a quinoprotein occurs, but quinoprotein synthesis does not depend on PQQ synthesis. The presence of quinoprotein substrates is not necessary for quinoprotein and PQQ syntheses. Although the extent of PQQ production was determined by the type of organism and quinoprotein produced, coordination between quinoprotein and PQQ syntheses is loose, since underproduction and overproduction of PQQ with respect to quinoprotein were observed. The results can be interpreted to indicate that quinoprotein synthesis depends on the growth rate whereas PQQ synthesis does not. In that view, the highest PQQ production can be achieved under limiting growth conditions, as was shown indeed by the much higher levels of PQQ produced in fed-batch cultures compared with those produced in batch experiments. The presence of nucleophiles, especially amino acids, in culture media may cause losses of PQQ due to transformation into biologically inactive compounds. Some organisms continued to synthesize PQQ de novo when this cofactor was administered exogenously. Most probably PQQ cannot be taken up by either passive diffusion or active transport mechanisms and is therefore not able to exert feedback regulation on its biosynthesis in these organisms.     

Pyrroloquinoline quinone: excretion by methylotrophs and growth stimulation for microorganisms

A marked excretion of pyrroloquinoline quinone (PQQ) by methylotrophs into the culture medium was observed when incubation was prolonged to the late stationary phase. When the organisms were growing vigorously in the early exponential phase, accumulation of PQQ was repressed at a low level. Some evidence was obtained that the excretion of PQQ is related to turnover of quinoproteins of the organisms. The growth stimulation of microorganisms by PQQ was demonstrated using Acetobacter aceti. The presence of PQQ even at the pg/ml level in the culture medium stimulated the bacterial growth by reducing the lag time. The growth stimulating effect of PQQ was observed only by the reduction of the lag time but not by increase in either the subsequent growth rate or the total cell yield. The results indicated that PQQ must have an important role in the initiation of cell reproduction.