沼泽红假单胞菌J001产辅酶Q10摇瓶发酵条件优化

本试验对沼泽红假单胞菌J001菌株250ml摇瓶发酵辅酶Q10条件进行了优化。结果表明其最佳初始pH值为6.5-7.5,温度为28-31℃,摇床转速100 r min-1,摇瓶装液量为200ml,接种量达10%时可直接进入对数生长期;碳源以NaAc较好,氮源以(NH4)2SO4和NH4Cl较好,磷源用量对考察指标影响不明显;用中心组合设计响应曲面法对碳氮源用量进行了优化,当NaAc浓度为5.39（g l-1）,(NH4)2SO4浓度为0.385（g l-1）即碳氮比为14/1时,对菌胞生长最有利;当NaAc浓度为5.70（g l-1）,(NH4)2SO4浓度为0.365（g l-1）即碳氮比为15.6/1时,对辅酶Q10产量最有利。     

类球红细菌添加前体物产辅酶Q_(10)的工艺优化

目的:考察摇瓶与发酵罐水平下类球红细菌CU20-9产辅酶Q10添加前体物质的最优配比。方法:通过正交试验优化发酵前体配比,考察摇瓶发酵多种前体物质的添加对辅酶Q10产量的影响,并利用发酵罐进行中试规模实验。结果:优化后发酵前体配比为维生素B1 10 mg/L、乙酸钠100 mg/L、对羟基苯甲酸20 mg/L、花生油0.01%。结论:前体添加及优化配比后,摇瓶及中试发酵水平下辅酶Q10的产量均有显著提高。     

沼泽红假单胞菌去除镉的研究

研究了沼泽红假单胞菌(Rhodopseudomonas palustris)S菌株的生长、Cd2 的去除与供氧光照、碳源、pH及温度的关系。结果表明:在好氧黑暗、苹果酸为碳源、pH7.0和30℃条件下,S菌株对初始浓度为25mg/L的Cd2 去除率最大,达到85%。在最佳条件下,Cd2 初始浓度为10mg/L~25mg/L时,去除率达85%,Cd2 浓度增加到150mg/L时,去除率仅为23%。测定Cd2 在菌体不同部位的分布发现菌体中富集的Cd2 63.5%在细胞壁上,33.5%在细胞原生质体内。同时,结合该菌体细胞超薄切片透射电镜观察证明了S菌株对Cd2 的去除主要在细胞壁上。     

沼泽红假单胞菌富硒发酵条件的研究

通过对沼泽红假单胞菌富硒发酵条件的研究,确定最优的富硒培养方式。采用单一因素变量法,利用微波消解与紫外分光光度法测定沼泽红假单胞菌在不同发酵条件下的生物量变化与富硒效果,确定其最佳培养方式。结果表明,最佳富硒培养条件为:培养温度30℃,环境硒浓度100μg/mL,培养基初始pH为7,硒添加方式应为梯度分次添加,添加时间应为培养周期的第3天、第4天。利用得到的富硒菌种,在最优条件下培养7 d后,测得环境硒浓度下降为10.9μg/mL,硒的转化率为89.1%,菌体富硒量可达到73.54 mg/g(干菌种),其中有机硒含量为97.1%。利用沼泽红假单胞菌生产有机硒具有可行性,富硒菌体可以作为动物饲料添加剂,也可以为人类提供富含有机硒的食品。在以后的实验中,将进一步进行验证和工业化应用。     

施氏假单胞菌PS59产脂肪酶发酵条件及脂肪酶洗涤性能优化北大核心CSCD

旨在对施氏假单胞菌(Pseudomonas stutzeri)PS59产脂肪酶发酵培养基进行单因子优化及响应面分析,选择出最佳的碳氮源,并分别对加酶量、洗涤时间、洗涤温度、洗涤pH和表面活性剂添加量对脂肪酶洗涤效果的影响进行评估,确定各个因素最佳值。结果表明,实验室培养施氏假单胞菌产脂肪酶的最佳碳氮源分别为蔗糖和大豆蛋白胨。优化发酵培养基配方为(g/L):大豆蛋白胨22.39 g,蔗糖10 g,K_2HPO_4·3H_2O 1 g,MgSO_4·7H_2O 0.5 g,无水CaCl_2 0.05 g,橄榄油8.1 g,pH 8.1,培养温度30℃,培养36 h获得平均脂肪酶产量为36.12±1.32 U/mL,相对于起始酶活15.65±4.81 U/mL,产量提高了1.3倍。确定最佳洗涤效果加酶量为100 U,最佳洗涤温度为25℃,最佳洗涤时间为25 min,洗涤pH对该脂肪酶洗涤效果影响不大。在最佳洗涤条件下,加酶洗涤液的洗涤效率可以提高30%-40%。     

北桑寄生提取液对沼泽红假单胞菌生长的影响

目的研究北桑寄生提取液对沼泽红假单胞菌生长的影响。方法通过添加不同质量浓度的常规培养基和北桑寄生提取液,培养沼泽红假单胞菌,观察活菌量的变化规律、生长曲线及脱氢酶活性。结果在全量培养基和无常规培养基中,北桑寄生提取液浓度分别为(0~6.25)g/L和(0~12.50)g/L,有利于细菌生长。最佳转化和未转化条件下细菌的脱氢酶活性分别增加9.72和7.20倍,活菌数无明显变化。结论低质量浓度北桑寄生促进沼泽红假单胞菌生长,反之则表现为抑制作用。高浓度北桑寄生提取液可以增强脱氢酶活性。     

基因工程菌Pseudomonas sp.B4的构建及其产邻苯二酚发酵条件的初步研究

利用PCR技术以Pseudomonas sp. B3-1基因组DNA为模板,扩增出2.9kb编码苯甲酸双加氧酶基因簇benABC。将该基因簇连接于pLAFRJ载体,电转化至E．coli DH5α,再通过三亲本结合法导入野生菌株Pseudomonas sp. B3-1中,得到了一株邻苯二酚产量提高的基因工程菌,命名为Pseudomonas sp.B4。发酵条件优化表明,当苯甲酸钠浓度为6.0 g/L,聚蛋白胨浓度为2.0 g/L,温度为32℃以及pH值为6.0时,工程菌在200rpm旋转摇床发酵36小时后,邻苯二酚产量达到0.7 mg/ml,比优化前提高了20％。     

沼泽红假单胞菌染色体的提取

近年来,由于工业产生的有机物对环境的污染越来越严重,推动了人们对能降解芳香物的沼泽红假单胞菌(Ｒ.ｐａｌｕｓｔｒｉｓ)的研究兴趣[1-8]。大多数芳香族化合物是严重危害人类健康的污染物质,为从基因水平研究沼泽红假单胞菌对芳香族化合物的生物降解机制,需要找到一种提取染色体的理想方法。泽沼红假单胞菌质膜构造比较特殊,且菌体富含蛋白质、色素、多糖等,给染色体提取造成一定难度。在本工作中采用了几种方法进行染色体的提取。1　材料与方法11　菌株来源:Ｙ6为本实验室分离、鉴定并保存的沼泽红假单胞菌。12　菌体培养121　沼泽红假单…     

5种光合细菌种间原生质体融合及优良农用融合子的筛选鉴定

处于对数生长期的光合细菌球形红假单胞菌(Rhodopseudomonassphaeroides)、沼泽红假单胞菌(Rhodopseudomonaspalustris)、嗜酸红假单胞菌(Rhodopseudomdnasacidophila)、深红红螺菌(Rhodospirarubrum)、万尼氏红微菌(Rhodomocrobiumvannielii),经溶菌酶(3mg/L)处理50min后,获得了它们的菌体形成的原生质体,其再生率分别为80%、71%、82%、61%、74%.取等量的亲本菌株在35%的PEG(MW6000)诱导下两两融合5min,共10种组合.其融合率为球×沼2.5×10-4、球×嗜2.1×10-4、球×深2.0×10-4、球×万2.1×10-4、沼×嗜2.8×10-4、沼×深2.4×10-4、沼×万2.6×10-4、嗜×深2.0×10-4、嗜×万2.3×10-4、深×万2.4×10-4.经影印法鉴定:形成的融合子可以分别生长于以相应的有机物为唯一碳源的培养基上,所有融合子体积均相当于两亲本株体积之和,融合子菌落形态特征介于两亲本株之间.从中随机挑选100个融合子,以辣椒苗作为靶标植物,从上述融合子中筛选到了1株具有显著促进作物生长、提高抗病性的融合子.     

沼泽红假单胞菌对微囊藻毒素的降解

以铜绿微囊藻为材料,通过固相萃取-高效液相色谱方法(SPE-HPLC),研究了沼泽红假单胞菌对微囊藻毒素MC-LR的降解作用。结果表明:沼泽红假单胞菌在厌氧、光照强度2000lx、35℃、pH7.0、乙酸钠为碳源、菌液初始浓度OD680为0.325和初始MC-LR为3mg.L-1时,6d降解率为36.5%,12d达到最高,降解率为78.7%。此降解条件和蓝藻水华爆发的环境条件基本一致,因此该菌株在水华爆发季节消除水中的微囊藻毒素方面具有应用潜力。     

Coenzyme Q10 production by Sphingomonas sp. ZUTE03 with novel precursors isolated from tobacco waste in a two-phase conversion system

Coenzyme Q10 (CoQ10) is a widely used supplement in heart diseases treatment or antioxidative dietary. The microbial production of CoQ10 was enhanced by addition of solanesol and novel precursors recovered from waste tobacco. The novel precursors were separated by silica gel and identified as alpha-linolenic acid (LNA) and butylated hydroxytoluene (BHT) based on the effect on CoQ10 production and GC-MS. The effects of novel precursors on CoQ10 production by Sphingomonas sp. ZUTE03 were further evaluated in a two-phase conversion system. The precursor's combination of solanesol (70 mg/l) with BHT (30 mg/l) showed the best effect on the improvement of CoQ10 yield. A maximal CoQ10 productivity (9.5 mg l-1 h-1) was achieved after 8 h conversion, with a molar conversion rate of 92.6% and 92.4% on BHT and solanesol, respectively. The novel precursors, BHT and LNA in crude extracts from waste tobacco leaves, might become potential candidates for application in the industrial production of CoQ10 by microbes.     

Biosynthesis of Territrems by Aspergillus terreus

Different radioactive precursors were added to 8-day potato-dextrose liquid cultures of Aspergillus terreus 23-1. Territrems were isolated from chloroform extracts of the cultures at day 14 and purified by thin-layer chromatography and high-pressure liquid chromatography. The territrem B obtained was treated with alkaline hydrogen peroxide, and 3, 4, 5-trimethoxy benzoic acid was isolated from an ethyl acetate extract of the reaction mixture and purified by thin-layer chromatography and high-pressure liquid chromatography. By comparison of the specific radioactivities of territrem B and its cleaved aromatic product (disintegrations per minute per micromole of compound), it was demonstrated that the radioactivity of territrem B was located mainly on its aromatic moiety when [U-C]shikimate, l-[methyl-C]methionine, and l-[methyl-H]methionine were precursors; however, the radioactivity of territrem B was located mainly on its nonaromatic moiety when [2-C]mevalonate was the precursor. Mevinolin, a specific inhibitor of beta-hydroxyl beta-methyl glutaryl coenzyme A reductase, was shown to inhibit production of territrems by A. terreus 23-1. When [U-C]acetate was used as a precursor, mevinolin inhibited the incorporation of radioactive carbon into territrem but mevinolin did not inhibit incorporation of radioactive carbon from [2-C]mevalonate into territrem.     

Influence of medium composition on the cephalosporin C production with a highly productive strain Cephalosporium acremonium.

Cephalosporin production by a highly productive Cephalosporium acremonium strain was carried out and optimized by fed-batch operation in a 40 l stirred tank reactor using a complex medium containing 30-120 g l-1 peanut flour. The concentrations of cephalosporin C (CPC) and its precursors: penicillin N (PEN N), deacetoxy cephalosporin C (DAOC), and deacetyl cephalosporin C (DAC) were monitored with an on-line HPLC. The concentrations of amino acids valine (VAL), cysteine (CYS), alpha-amino adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV), were determined off-line by HPLC. The RNA content and dry weight of the sediment as well as the oxygen transfer rate (OTR) and the CO2 production rate (CPR) were used to calculate the cell mass concentration (X). The influences of peanut flour (PF) and the on-line monitored and controlled medium components: glucose (GLU), phosphate, methionine (MET) as well as the dissolved oxygen (DOC) on the cell growth, the product formation, and the pathway of cephalosporin C biosynthesis were investigated and evaluated. When the glucose fed-batch cycle was optimized and oxygen transfer limitation was avoided (DOC greater than 20% of the saturation value), high process performance (103.5 g l-1 X, 11.84 g l-1 CPC, a maximum CPC productivity of 118 mg l-1 h-1, and the whole concentration of the beta-lactam antibiotics CPC, DAC, DAOC, PEN N 17.34 g l-1) was achieved by using 100 g l-1 PF in the medium with the optimum concentration of phosphate (260-270 mg l-1) and a low glucose concentration (less than 0.5 g l-1). The cultivations with different medium concentrations demonstrated that the product formation was directly proportional to the cell mass concentration. On the average, the cell mass-based yield coefficient of CPC: YCPC/X amounted to 0.115 g CPC per g cell mass.     

Biosynthesis of ethylene from methionine in aminoethoxyvinylglycine-resistant avocado tissue

This study was conducted to determine if aminoethoxyvinylglycine (AVG) insensitivity in avocado (Persea americana Mill., Lula, Haas, and Bacon) tissue was due to an alternate pathway of ethylene biosynthesis from methionine. AVG, at 0.1 millimolar, had little or no inhibitory effect on either total ethylene production or [(14)C] ethylene production from [(14)C]methionine in avocado tissue at various stages of ripening. However, aminoxyacetic acid (AOA), which inhibits 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (the AVG-sensitive enzyme of ethylene biosynthesis), inhibited ethylene production in avocado tissue. Total ethylene production was stimulated, and [(14)C]ethylene production from [(14)C]methionine was lowered by treating avocado tissue with 1 millimolar ACC. An inhibitor of methionine adenosyltransferase (EC 2.5.1.6), l-2-amino-4-hexynoic acid (AHA), at 1.5 millimolar, effectively inhibited [(14)C]ethylene production from [(14)C]methionine in avocado tissue but had no effect on total ethylene production during a 2-hour incubation. Rates of [(14)C]AVG uptake by avocado and apple (Malus domestica Borkh., Golden Delicious) tissues were similar, and [(14)C]AVG was the only radioactive compound in alcohol-soluble fractions of the tissues. Hence, AVG-insensitivity in avocado tissue does not appear to be due to lack of uptake or to metabolism of AVG by avocado tissue. ACC synthase activity in extracts of avocado tissue was strongly inhibited (about 60%) by 10 micromolar AVG. Insensitivity of ethylene production in avocado tissue to AVG may be due to inaccessibility of ACC synthase to AVG. AVG-resistance in the avocado system is, therefore, different from that of early climacteric apple tissue, in which AVG-insensitivity of total ethylene production appears to be due to a high level of endogenous ACC relative to its rate of conversion to ethylene. However, the sensitivity of the avocado system to AOA and AHA, dilution of labeled ethylene production by ACC, and stimulation of total ethylene production by ACC provide evidence for the methionine --> SAM --> ACC --> ethylene pathway in avocado and do not suggest the operation of an alternate pathway.     

Association of colony morphology with coenzyme Q(10) production and its enhancement from Rhizobium radiobacter T6102W by addition of isopentenyl alcohol as a precursor

Rhizobium radiobacter T6102 was morphologically purified by the aniline blue agar plates to give two distinct colonies; white smooth mucoid colony (T6102W) and blue rough colony (T6102B). The coenzyme Q(10) (CoQ(10)) was produced just by T6102W, showing 2.0 mg/g of CoQ(10) content, whereas the T6102B did not produce the CoQ(10). All of the used CoQ(10) biosynthetic precursors enhanced the CoQ(10) production by T6102W. Specifically, the supplementation of 0.75 mM isopentenyl alcohol improved the CoQ(10) concentration (19.9 mg/l) and content (2.4 mg/g) by 42% and 40%, respectively.     

A study of the sulphur amino acids of rat tissues

1. In a study of the metabolism of l-[(35)S]methionine in vivo, the labelled sulphur compounds of rat liver and brain were separated first by ion-exchange chromatography into two fractions containing (i) free sulphur amino acids such as methionine, cystathionine, cyst(e)ine and homocyst(e)ine and (ii) glutathione. 2. Two-dimensional paper chromatography with butan-1-ol-acetic acid or propionic acid-water in the first direction and 80% acetone or acetone-ethyl methyl ketone-water in the second direction was found superior to other solvent systems for separating the sulphur amino acids. 3. At 10min. after injection of [(35)S]methionine only a small part of the (35)S was found combined in free methionine or other free sulphur amino acids. 4. Evidence was obtained of the presence of adenosyl[(35)S]methionine and adenosyl[(35)S]homocysteine in perchloric acid extracts of rat liver and brain. 5. The trans-sulphuration pathway was active in brain as well as in liver.     

The role of valine in the biosynthesis of penicillin N and cephalosporin C by a Cephalosporium sp

1. The production of penicillin N, but not that of cephalosporin C, was inhibited by the addition of d-valine to suspensions in water of washed mycelium of Cephalosporium sp. 8650. The production of cephalosporin C was selectively inhibited by gamma-hydroxyvaline. 2. l-[(14)C]Valine was taken up rapidly and virtually completely by suspensions of washed mycelium but d-[(14)C]valine and alpha-oxo[(14)C]-isovalerate were taken up relatively slowly. 3. Part of the l-valine was rapidly degraded in the mycelium and part was incorporated into protein. Turnover of the valine in the amino acid pool was estimated to occur in 10-17min. 4. No detectable amount of l-[(14)C]valine was converted into the d-isomer in the mycelium. alpha-Oxo[(14)C]isovalerate was rapidly converted into l-[(14)C]valine in mycelium and mycelial extracts. 5. d-[(14)C]Valine was partially converted into the l-isomer in the mycelium and (14)C from d-valine was incorporated into protein. 6. The labelling of penicillin N and cephalosporin C by (14)C from l-[(14)C]valine was consistent with the view that l-valine is a direct precursor of C(5) fragments of both antibiotics and that any intermediates involved are present in relatively small pools in rapid turnover. 7. Labelling of the antibiotics with (14)C from d-[1-(14)C]valine appeared to occur after the latter had been converted into the l-isomer. Unlabelled d-valine did not decrease the efficiency of incorporation of (14)C from l-[1-(14)C]valine. 8. Intracellular peptide material which contained, among others, residues of alpha-aminoadipic acid, cysteine and valine, was rapidly labelled by (14)C from l-[1-(14)C]valine in a manner consistent with it being an intermediate in the biosynthesis of one or both of the antibiotics. 9. Labelling of penicillin N from l-[1-(14)C]valine occurred more rapidly than that of cephalosporin C. However, the effects of d-valine and gamma-hydroxyvaline on antibiotic production and the course of labelling of the antibiotics from l-[(14)C]valine could not readily be explained on the assumption that penicillin N was a precursor of cephalosporin C.     

Effect of allylamine antimycotic agents on fungal sterol biosynthesis measured by sterol side-chain methylation

Sterol side-chain (C-24) methylation was assayed by incorporation of radioactivity from [Me-14C]methionine into the ergosterol fraction in cells of the pathogenic fungi Candida albicans, Candida parapsilosis and Trichophyton mentagrophytes. Methylation at C-24 occurred after nuclear demethylation in all cases. The method was used to measure ergosterol biosynthesis inhibition by the allylamine antimycotics naftifine and SF 86-327, which are known to block squalene epoxidation. In C. albicans cells treated with SF 86-327 (1 mg l-1) to fully inhibit squalene epoxidation, C-24 methylation continued for several hours at about 40% of the control rate. This residual biosynthesis was probably due to methylation of endogenous sterol precursors. The degree of residual biosynthesis in the three fungi correlated well with their susceptibility to SF 86-327. The highly susceptible dermatophyte T. mentagrophytes had negligible residual sterol biosynthesis. These differences were not due to inhibition of methionine uptake. For naftifine (100 mg l-1) there was evidence of a second inhibitory action in C. albicans. A cell-free assay indicated that this was due to direct inhibition of the C-24 methyltransferase.