固定化酵母细胞生产1,6—二磷酸果糖研究

本文研究了固定化酵母细胞制备果糖１,６二磷酸的方法及其生产。用卡拉胶包埋方法固定化酿酒酵母,对含葡萄糖１．０Ｍ,磷酸盐０．８Ｍ的糖磷液,ＰＨ６．５在３７℃下进行磷酸化反应。反复分批转化２０天以上,可达到平均产ＦＤＰＨ４ ２７．５８ｍｇ／ｍｌ。最高为５９．９４ｍｇ／ｍｌ。     

果糖-1,6-二磷酸的酶法测定

前言 果糖-1,6-二磷酸（简称FDP）在临床上有广泛用途,主要是作为治疗心脏缺血症的辅助药物,其工业化生产引起了人们越来越浓厚的兴趣。因此,无论是生产或者临床应用试验中,FDP的含量分析都十分重要。     

1,6-二磷酸果糖与细胞保护

1,6-二磷酸果糖是细胞内糖代谢的中间产物,是能在分子水平上调节细胞代谢中若干酶活性,作为恢复和改善细胞代谢的药物,可通过多方面因素减轻细胞损伤,从而对细胞起保护作用。     

发酵法制备1,6—二磷酸果糖Ⅰ发酵菌株的确定及反应条件的研究

通过比较6株酵母细胞转化1,6－二磷酸果糖的结果,确定酿造酵母Y162作为转化菌株,对其菌株的培养方法,底物反应额的组成及反应条件进行了详细的考察,在最适条件下：pH6.5,蔗糖8％,NaH2PO45％,MgCl2·6H2O0.1％,甲苯4.0％,氯丙嗪0.2％,反应时间为8-10h,湿菌体与反应液的比例为1：4,反应液中FDP的产量可达12．1mg／ml。     

固定化酵母生物合成胞二磷酸胆碱(CDPC)

对固定化酵母生物合成胞二磷酸胆碱反应液浓度进行了研究。通过几组实验,找到了反应液中底物的最佳浓度;磷酸胆碱75μmol/mL,胞苷酸（CMP）10μmol/mL,葡萄糖400μmol/mL,硫酸镁和硫酸锰分别为10μmol/mL和1μmol/mL。可以不使用还原剂。     

紫花苜蓿果糖-1,6-二磷酸醛缩酶基因全长克隆及分析

根据已知的与盐胁迫相关的EST序列,采用SMART RACE方法克隆了紫花苜蓿果糖-1,6-二磷酸醛缩酶(ALD)全长cDNA,命名为MsALD(GenBank accession No.FJ896113).序列分析结果表明,该cDNA全长1 487 bp,包含一个1 194 bp的最大开放阅读框,编码398个氨基酸.经同源比对和进化树分析,MsALD基因编码的氨基酸与红三叶草、马铃薯、烟草等的果糖-1,6-二磷酸醛缩酶(ALD)氨基酸序列一致性高达90%以上,确定其属于第Ⅰ类果糖-1,6-二磷酸醛缩酶.半定量RT-PCR分析表明,MsALD基因可能与紫花苜蓿抗盐机理相关.     

微生物生产二磷酸果糖

1,6-二磷酸果糖（fructose-1,6-diphosphate）简称FDP,又叫“福达平”,系糖代谢的中间产物,溶于水,是人体生命活动所必需的,在细胞代谢过程中起调节剂、生物催化剂和细胞强壮剂的重要作用。已将这种果糖研制成产品用医疗保健,治心脑血管病、糖尿病等重要疾病。这种有价值的老产品找到新的用途,     

1,6-二磷酸果糖的研究进展

１,６—二磷酸果糖（ＦＤＰ）是糖酵解过程中的代谢产物,具有调节糖代谢中若干酶活性之功效,为恢复、改善细胞代谢的分子水平药物。由Ｈａｒｄｅｎ和Ｙｏｕｎｇ［１］于１９０８年首先发现并阐明了一般特性。其后,美国、德国、意大利、日本、芬兰等国的学者分别对ＦＤＰ的制备和应用作了深入而富有成效的研究。［２］［３］［４］［５］１制备方法本世纪初,各国生化学家将注意力集中在糖发酵的机制上。自Ｈａｒｄｅｎ和Ｙ...     

丝状真菌转化葡萄糖为果糖-1,6-二磷酸的研究

STUDYONTHEBIOCONVERSIONOFGLUCOSETOFRUCTOSE-1,6-DIPHOSPHATEWITHFILAMENTOUSFUNGICELLMOXiao-Yan;ZHANGu-Yu;XIANGYuHong(DepartmentofBiotechnology,XianJiaotongUniversity,Xian7l0049)80至90年代发现果糖一l,6一二磷酸（Fructose－l,6－diphosphateFDP）在医药、保健品和植物生长调节方面的诸多用途,尤其用于医药备受关注。传统FDP的生产是用面包酵母或啤酒酵母作酶源,通过糖酵解过程而获得（Leisolaetal,1974）,以后有用固定化酵母细胞、固相化多酶系统及嗜热脂肪芽胞杆菌转化葡…     

The Formation of Glucose 1,6-Diphosphate from Fructose 1,6-Diphosphate by Yeast Phosphoglucomutase

It was found that fructose 1,6-diphosphate, the main intermediate of glycolysis, was able to act as a coenzyme of yeast phosphoglucomutase reaction. The mechanism of the coenzymatic activity of fructose 1,6-diphosphate was studied. It was indicated in the fructose 1,6-diphosphate dependent reaction that glucose 1,6-diphosphate was formed by the phosphate-transfer of fructose 1,6-diphosphate to glucose 1-phosphate in the first step, and in the second step the conversion of glucose 1-phosphate to glucose 6-phosphate, the original mutase reaction, occurred in the presence of glucose 1,6-diphosphate. The kinetic constants in the reaction of the first step were determined from the time courses of the fructose 1,6-diphosphate dependent reaction.     

Fructose-1,6-diphosphate as an in vitro and in vivo anti-alcohol agent in the rat

Fructose-1, 6-diphosphate (FDP) decreases the effect of ethanol on Ca++ entry and inhibits the ethanol-stimulated phosphate efflux in rat heart slices. FDP also inhibits the ethanol-stimulated [36Cl-]-uptake by rat brain microvesicles and affects the isolated GABA-receptor in a way opposite to that of ethanol. The in vivo effects of FDP include a dose-dependent decrease in ethanol-induced gastric ulcers and a decrease in the serum transaminase levels raised by chronic ethanol administration. Other central actions of ethanol such as diuresis, narcosis, dependence and withdrawal symptoms are also counteracted by FDP.     

Some effects of fructose-1,6-diphosphate on rat myocardial tissue related to a membrane-stabilizing action

This study aims at elucidating the mechanism of action of extracellular fructose-1,6-diphosphate (FDP). FDP is able to inhibit Ca++ entry into the myocardial tissue with an IC50 value of 11.5 mM and in addition, it is bound by rat heart slices, the binding being activated by Zn and conditions of chemical hypoxia induced by KCN and iodoacetate. The overall effect of extracellular FDP includes an increase of frequency and amplitude of contraction of perfused heart at concentration below 1 mM, and, in general, a stimulation of the oxygen consumption of the tissue. The antihaemolytic effect of FDP suggests its action as a membrane stabilizer. The effects of extracellular FDP on the myocardial cell can be interpreted both on the basis of a limited permeability of the cell membrane to it and as a purely extracellular effect transduced through the cell membrane with a final response consisting of an increase in the intracellular FDP.     

Gas-exchange analysis of chloroplastic fructose-1,6-bisphosphatase antisense potatoes at different air humidities and at elevated CO2

Gas-exchange measurements were performed to analyze the leaf conductances and assimilation rates of potato (Solanum tuberosum L. cv. Desireé) plants expressing an antisense construct against chloroplastic fructose-1,6-bisphosphatase (FBPase, EC 3.1.3.11) in response to increasing photon flux densities, different relative air humidities and elevated CO₂ concentrations. Assimilation rates (A) and transpiration rates (E) were observed during a stepwise increase of photon flux density. These experiments were carried out under atmospheric conditions and in air containing 500 μmol mol⁻¹ CO₂. In both gas atmospheres, two levels of relative air humidity (60–70% and 70–80%) were applied in different sets of measurements. Intercellular CO₂ concentration, leaf conductance, air-to-leaf vapour pressure deficit, and instantaneous water-use efficiency (A/E) were determined. As expected, assimilation rates of the FBPase antisense plants were significantly reduced as compared to the wild type. Saturation of assimilation rates in transgenic plants occurred at a photon flux density of 200 μmol m⁻² s⁻¹, whereas saturation in wild type plants was observed at 600 μmol m⁻² s⁻¹. Elevated ambient CO₂ levels did not effect assimilation rates of transgenic plants. At 70–80% relative humidity and atmospheric CO₂ concentration the FBPase antisense plants had significantly higher leaf conductances than wild-type plants while no difference emerged at 60–70%. These differences in leaf conductance vanished at elevated levels of ambient CO₂. Stomatal response to different relative air humidities was not affected by mesophyll photosynthetic activity. It is suggested that the regulation of stomatal opening upon changes in photon flux density is merely mediated by a signal transmitted from mesophyll cells, whereas the intercellular CO₂ concentration plays a minor role in this kind of stomatal response. The results are discussed with respect to stomatal control by environmental parameters and mesophyll photosynthesis. Received: 24 September 1998 / Accepted: 9 February 1999     

Biological evaluation and SAR analysis of novel covalent inhibitors against fructose-1,6-bisphosphatase

Fructose-1,6-bisphosphatase (FBPase) is an attractive target for affecting the GNG pathway. In our previous study, the C128 site of FBPase has been identified as a new allosteric site, where several nitrovinyl compounds can bind to inhibit FBPase activity. Herein, a series of nitrostyrene derivatives were further synthesized, and their inhibitory activities against FBPase were investigated in vitro. Most of the prepared nitrostyrene compounds exhibit potent FBPase inhibition (IC₅₀ < 10 μM). Specifically, when the substituents of F, Cl, OCH₃, CF₃, OH, COOH, or 2-nitrovinyl were installed at the R₂ (meta-) position of the benzene ring, the FBPase inhibitory activities of the resulting compounds increased 4.5–55 folds compared to those compounds with the same groups at the R₁ (para-) position. In addition, the preferred substituents at the R₃ position were Cl or Br, thus compound HS36 exhibited the most potent inhibitory activity (IC₅₀ = 0.15 μM). The molecular docking and site-directed mutation suggest that C128 and N125 are essential for the binding of HS36 and FBPase, which is consistent with the C128-N125-S123 allosteric inhibition mechanism. The reaction enthalpy calculations show that the order of the reactions of compounds with thiol groups at the R₃ position is Cl > H > CH₃. CoMSIA analysis is consistent with our proposed binding mode. The effect of compounds HS12 and HS36 on glucose production in primary mouse hepatocytes were further evaluated, showing that the inhibition was 71% and 41% at 100 μM, respectively.     

Structure and Activity of the Metal-independent Fructose-1,6-bisphosphatase YK23 from Saccharomyces cerevisiae

Fructose-1,6-bisphosphatase (FBPase), a key enzyme of gluconeogenesis and photosynthetic CO₂ fixation, catalyzes the hydrolysis of fructose 1,6-bisphosphate (FBP) to produce fructose 6-phosphate, an important precursor in various biosynthetic pathways. All known FBPases are metal-dependent enzymes, which are classified into five different classes based on their amino acid sequences. Eukaryotes are known to contain only the type-I FBPases, whereas all five types exist in various combinations in prokaryotes. Here we demonstrate that the uncharacterized protein YK23 from Saccharomyces cerevisiae efficiently hydrolyzes FBP in a metal-independent reaction. YK23 is a member of the histidine phosphatase (phosphoglyceromutase) superfamily with homologues found in all organisms. The crystal structure of the YK23 apo-form was solved at 1.75-Å resolution and revealed the core domain with the α/β/α-fold covered by two small cap domains. Two liganded structures of this protein show the presence of two phosphate molecules (an inhibitor) or FBP (a substrate) bound to the active site. FBP is bound in its linear, open conformation with the cleavable C1-phosphate positioned deep in the active site. Alanine replacement mutagenesis of YK23 identified six conserved residues absolutely required for activity and suggested that His¹³ and Glu⁹⁹ are the primary catalytic residues. Thus, YK23 represents the first family of metal-independent FBPases and a second FBPase family in eukaryotes.     

酵母蛋白质YJL084c的磷酸化分析

PCL6、PCL7(PAP1)和PHO80为酵母蛋白激酶PHO85的细胞周期蛋白因子 ,同属于PHO80亚类 ,它们在蛋白质序列上有较高的同源性 ,彼此在功能上有一定程度的重叠。YLR190w为先前鉴定的PCL7 PHO85的一个底物 ,根据BLAST比较 ,YJL0 84c与YJLR190w在一段区域内有一定的同源性 ,并且根据已公布公共数据库 ,YJL0 84c是PCL6的一个结合蛋白。利用双杂交系统分析了YJL0 84c与这三个细胞周期蛋白因子之间的相互作用 ,表明PCL7也可以结合YJL0 84c。利用免疫共沉淀证实它们在体内的相互作用 ;通过体外GST沉降分析 ,验证它们的体外相互作用。YJL0 84c的体外翻译产物可以被PCL7 PHO85复合物磷酸化 ,进一步分别表达纯化YJL0 84c的氨基末端、中间和羧端三部分的GST融合蛋白片段 ,PCL7 PHO85复合物可以磷酸化其中间部分 ,而对N末端和C末端则未表现出磷酸化。以该中间片段蛋白质为底物 ,进一步研究高低磷条件对其磷酸化的影响。PCL7 PHO85对YJL0 84c的磷酸化受到无机磷条件的明显影响 :高磷条件下 ,磷酸化程度高 ;而低磷条件下磷酸化程度低。有意思的是 ,PCL6 PHO85免疫沉淀复合物也表现出同样的磷酸激酶特征。于是分析PHO81与这三个细胞周期因子之间相互作用。此外 ,还构建了Yjl0 84c的缺失株 ,分析了缺失的可能影响。