克拉维酸发酵液中甘油含量的测定

为寻求一种简便易行的克拉维酸发酵液中甘油含量的测定方法,以化学测定方法一高碘酸氧化法对克拉维酸发酵液进行测定。从精密度和回收率实验结果看,该方法可以较精确地测定出发酵液中甘油的含量,平均相对偏差小于5％,适合用来检测克拉维酸发酵过程中甘油的代谢情况。     

基因工程菌高密度发酵液中碳源物质甘油的定量检测及其浓度的优化控制

比较了高碘酸氧化法,铜离子络合法、高压液相色谱法和甘油激酶法四种不同的方法定量测定重组工程菌ＹＫ５３７／ＰＳＢ－ＴＫ高密度发酵液中甘油的浓度,发现甘油激酶法可以排除发酵液中其他物质的干扰,精确测定甘油的含量。在此基础上对发酵培养中甘油的浓度进行了优化,结果表明,甘油浓度控制在较低的水平有利于菌体密度的提高。在５Ｌ自动发酵罐中,控制甘油浓度在５ｇ／Ｌ左右,经２０ｈ的培养,最终细菌密度达到１２０ＯＤ６     

高效液相色谱法测定细菌发酵液中的糖、甘油和乙醇的研究

目的：建立一种用高效液相色谱快速测定细菌发酵液中糖、甘油和乙醇的方法。方法：酌情取细菌发酵液样品适量(1—9mL)于10mL容量瓶中,加入10mg/mL的甘露醇内标物1．0mL,用水定容到刻度,取2mL用4．5μm滤膜过滤,进样2μL分析,糖、甘油和乙醇以Waters Suger—Park—1钙型阳离子交换柱为固定相,0．05g／L EDTA钙钠水溶液为流动相分离,示差折光仪为检测器检测测定。结果：一次进样可同时测定细菌发酵液样品中的糖、甘油和乙醇;方法RDS在0．52％-0．85％之间,标准回收率在99—102％之间,结果满意。结论：该方法简便、快速、结果可靠,为发酵法生产甘油过程中的质量监控提供了方法。     

甘油对粒毛盘菌DP5胞外多酚积累的影响

【目的】探明以甘油为碳源促进粒毛盘菌DP5积累多酚的可能原因。【方法】对碳源种类、甘油浓度、曲酸、抑制剂和前体等对多酚产量和生物量的影响进行分析。【结果】以甘油为碳源,能显著提高粒毛盘菌胞外多酚产量。甘油浓度为20 g/L时,胞外多酚产量最高,达到0.664 g GAE/L,并在发酵液中检测到曲酸,其含量为0.25 g/L。向以蔗糖为碳源的发酵液添加曲酸,胞外多酚含量从0.209 g GAE/L提高至0.376 g GAE/L。以甘油为碳源的发酵液中,酚氧化酶活性较低。粒毛盘菌DP5通过莽草酸途径和聚酮途径合成多酚,甘油有利于莽草酸途径和聚酮途径前体物质的合成。【结论】粒毛盘菌以甘油为碳源合成出曲酸,曲酸抑制多酚向黑色素的转化;甘油促进多酚前体的合成,从而提高了粒毛盘菌胞外多酚的积累量。     

产甘油假丝酵母产甘油关键酶基因在酿酒酵母中的表达

甘油是一种极其理想的耐高渗透压介质。利用PCR方法,从产甘油假丝酵母WL2002-5中扩增出了2个产甘油的关键酶基因GPD和GPP,分别编码3-磷酸甘油脱氢酶（glycerol 3-phosphate dehydrogenase, GPD）和3-磷酸甘油磷酸酶（glycerol 3-phosphate phosphatase, GPP）。利用T-Vector在Escherichia coli JM109中克隆得到大量的GPD和GPP基因,并成功构建了重组质粒pYX212-GPD和pYX212-GPP;通过LiAc转化法将重组质粒导入酿酒酵母Saccharomyces cerevisiae W303-1A。初步实验结果表明：发酵过程中pYX212-GPD/S. cerevisiae W303-1A的生物量高于pYX212-GPP/S. cerevisiae W303-1A和野生型S. cerevisiae W303-1A;发酵72h后,pYX212 GPD/S. cerevisiae W303-1A发酵液中甘油含量大约为12mmol/L,明显高于野生型S. cerevisiae W303-1A的甘油含量,而pYX212-GPP/S. cerevisiae W303-1A与野生型S. cerevisiae W303-1A在甘油含量上相差不大,均只有4mmol/L 左右。     

化学比色法测定发酵液中L-缬氨酸的研究

应用“化学比色法”对发酵液中L -缬氨酸进行了定量分析研究 ,确立了L -缬氨酸定量测定条件和计算方法。以高速氨基酸分析仪测定结果为参比 ,用化学比色法测定结果的准确度比纸层析 -色斑洗脱比色法高 ,且应用化学比色法测定L-缬氨酸的含量较为方便。     

甘油影响红曲菌产色素的碳源选择性探究

红曲菌(Monascus spp.)是我国重要的药食同源微生物,红曲色素(Monascus pigments,MPs)是其主要次级代谢产物之一。有研究表明,甘油可促进红曲菌产MPs,但作用机制不明。以丛毛红曲菌(Monascus pilosus)MS-1为实验菌株,考察甘油与葡萄糖或蔗糖复合对红曲菌产MPs的影响。在不含碳源的合成培养基中,将甘油与葡萄糖或蔗糖复合,采用分光光度法和高效液相色谱法等分析MPs的产量和组分、生物量及发酵液pH。当甘油与葡萄糖复合,添加甘油后发酵液pH、生物量无显著变化(P0.05),总色价显著降低(P0.05)。当2 g/L或40 g/L甘油与蔗糖复合,发酵液pH显著降低而生物量及总色价显著增加(P0.05)。当40 g/L甘油与蔗糖复合时,总色价是仅以蔗糖为碳源时的16.5倍,且MPs同系物数量明显增多(P0.05)。在合成培养基条件下,甘油促进红曲菌产MPs具有碳源种类的选择性。该结果可为研究甘油影响红曲菌产MPs的作用机制提供参考,为甘油用于MPs生产提供依据。     

酶电极法快速测定甘油含量的研究

利用酶固定化技术,以甘油激酶（GK）、甘油-3-磷酸氧化酶（GPO）为反应酶,研究GK、GPO的固定化方法及固定化模式,制备甘油酶膜、甘油酶电极,并利用其测定甘油含量。结果表明,GK、GPO按1：1比例固定化时,酶电极电流信号最高;最高效固定模式为：GK固定于核微孔膜,共价偶联GPO固定于Biodyne膜,形成共价双酶膜,进而组装为甘油酶电极。性能研究表明,甘油酶电极最适pH值为7.0,最佳温度为28~32℃;最佳实验条件下,线性范围为0.05~9.00 g/L;回收率为98.4%~102.4%,稳定性高,相对标准偏差（RSD）<5%;测定结果与高效液相色谱法、高碘酸氧化法比较,无明显差异（P>0.05）,且该方法操作简单,专一性强,检测快速,适于实际生产中甘油的实时定量及监控。     

高效液相法测定蜂蜜中甘油的含量

建立了以混合溶剂直接提取测定蜂蜜中甘油含量的方法.采用ZORBAX Carbohydrate a-nalysis(4.6 mm×250 mm 5-Micro)色谱柱,以乙腈/水(80:20,v/v)为流动相,示差检测器,使用乙腈/甲醇/水混合作为溶剂快速检测甘油.结果表明,应用此法的甘油浓度在10.0 mg/L～250...     

一株以甘油为底物产二羟丙酮(DHA)微生物菌种的筛选及鉴定

甘油为微生物可利用的理想碳源,从自然界筛选出21株以甘油为唯一碳源产二羟丙酮（DHA）的菌株,经初步发酵测定发酵液中DHA含量,其中菌株6-8DHA产量最高达6.4g／L。对其进行常规生理生化鉴定实验,并结合16S rDNA基因分析,比对结果表明,菌株6-8与Acinetobacter sp.相似性最高,达99．7％,在细菌分类学上属于假单胞茵目莫拉茵科不动杆菌属。将其命名为Acinetobactersp.6-8。     

Biochemical indices of erythrocytes cryopreserved with protection by 1,2-propanediol and glycerin

Based on the data concerning the content of ATP, 2,3-DFG, K+, and Na+ in depreserved red cells stored at 4 degrees C in resuspending media TsNIIGPK 8b and 8v it has been established that blood preserved under protection of 1,2-propanediol is comparable with that preserved under glycerin protection as regards its quality and is even better from the standpoint of some other indicators. Medium TsNIIGPK 8v may be used as resuspending medium for blood exposed to low temperature preservation under protection of 1,2-propanediol.     

Optical resolution of glycerin sulfides and glycerin selenides on the chiral stationary phase of cellulose‐tris(3,5‐dimethylphenylcarbamate)

Cellulose‐tris(3,5‐dimethylphenylcarbamate) was prepared after a reported method and was coated onto an aminopropylated mesopore spherical silica gel. The final product was used as a chiral stationary phase of high performance liquid chromatography for the enantioseparation of a series of glycerin sulfides and glycerin selenides. Mixtures of hexane and 2‐propanol were used as mobile phases. The effects of 2‐propanol concentration in the mobile phase on the retention and resolution were investigated. Some enantiomers of the glycerin monosulfides and monoselenides could be separated satisfactorily, but none of the disulfides could be separated. The structural features of the solutes that influence chiral separation were discussed. Chirality 11:598–601, 1999. © 1999 Wiley‐Liss, Inc.     

Studies on purification of 1,3-propanediol by molecular distillation

The separation of 1,3-propanediol using molecular distillation has been studied. The effects of operating temperature and feed flow rate through a sequential distillation strategy were investigated. The optimal experimental temperature was at 70°C for separating 1,3-propanediol and the by-product 2,3-butanediol. Meanwhile, the volume flow rate was 10 mL/min. As a result, the recovery of 1,3-propanediol and 2,3-butanediol were 87.6 and 87.5%, respectively. Furthermore, the integrated separation characteristic of 1,3-propanediol was evaluated through macrolevel and micro-level models. The separation factors of 1,3-propanediol versus 2,3-butanediol and glycerol were 0.11 and 1.07, respectively, affirming that the separation of 1,3-propanediol by molecular distillation was feasible.     

Evaluation of liquid extraction potentials for downstream separation of 1,3-propanediol

The application of liquid-liquid extraction for the downstream separation of 1,3-propanediol from dilute aqueous solutions has been tested. Preliminary solvent screening was performed using the group contribution method. Experimental verification of the predictions revealed that the distribution of 1,3-propanediol into organic solvents is not good enough to make simple extraction efficient.     

1,3-丙二醇发酵液后提取技术研究进展

1,3-丙二醇是一种重要的化工原料,以甘油或葡萄糖为原料发酵法制备1,3-丙二醇具有原料可再生、反应条件温和等优点,是近年来国内外的研究热点。由微生物发酵获得的1,3-丙二醇发酵液是含多种强极性的醇及盐类的稀溶液,这使得采用传统的分离方法难以经济、有效地的将1,3-丙二醇从发酵液中纯化出来,后提取过程成为发酵法工业化生产1,3-丙二醇的瓶颈。1,3-丙二醇后提取过程主要包括微生物菌体等高分子物质的去除,盐的去除、回收,有机物的纯化和水的去除。以下对应用于以上分离过程的技术的研究进展进行讨论,提出在该领域应该重视的发展方向。     

Isolation and characterization of microorganisms able to produce 1,3-propanediol under aerobic conditions

Microbial fermentation under strictly anaerobic conditions has been conventionally used for the production of 1,3-propanediol, a key raw material required for the synthesis of polytrimethylene terephthalate (PTT) and other polyester fibers. In the current study, we have identified eight strains of microorganism which are able to produce 1,3-propanediol under aerobic condition. Those strains were isolated from garden soil, which were enriched by culturing in LB medium with glycerol added under aerobic condition. The identities of those strains were established based on their 16S rRNA sequences and physiological characteristics. Results indicated 6 strains are Citrobacter freundii and 2 strains are Klebsiella pneumoniae subsp Penumoniae. One of Klebsiella pneumoniae subsp Penumoniae strains, designated as TUAC01, demonstrated comparable levels of 1,3-propanediol oxidoreductase, glycerol dehydratase and glycerol dehydrogenase activity to the anaerobic microorganisms described in the literature. Accordingly, in larger scales (5 l) fed-batch culture the TUAC01 strain showed a remarkable 1,3-propanediol producing potency under aerobic conditions. 60.1 g/l of 1,3-propanediol was yield after 42 h incubation in an agitating bioreactor; and in air-lift bioreactor 66.3 g/l of 1,3-propanediol was yield after 58.5 h incubation. The aerobic ferment process, reduced the product cost and made the biological method of 1,3-propanediol production more attractive.     

A novel thin-layer chromatography method to screen 1,3-propanediol producers

To date, there is no established protocol for the screening of 1,3-propanediol producers. The proposed method has a wide applicability to harness the commercial potential of microorganisms which produce 1,3-propanediol as the end product. Glycerol fermentation broth of 50 bacteria spotted on thin-layer chromatography plates and run by appropriate solvent systems followed by colour development using vanillin reagent gave different coloured spots with most of the compounds present in the fermentation broth. The appearance of a purple-coloured spot of 1,3-propanediol with a retention factor (R _f) of 0.62 forms the basis for the selection of 1,3-propanediol producers. Apart from being a rapid detection system the proposed method is pH independent and its authenticity was reconfirmed by HPLC.     

一步法产1,3-丙二醇酿酒酵母基因工程菌的构建

1,3-丙二醇(1,3-PD)是一种重要的化工原料,发酵法生产1,3-PD是一条新颖且具有潜在竞争力的生产途径。本研究在前期工作的基础上,将分别来源于大肠杆菌和肺炎克雷伯氏菌的基因片段yqhD和dhaB串联表达,构建重组表达载体pYX212-zeocin-pGAP-yqhD-pGAP-dhaB;并得到重组酿酒酵母(Saccharomyces cerevisiae)W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB。该重组菌和对照S.cerevisiae分别以葡萄糖为底物摇瓶发酵72h后,重组酿酒酵母发酵液中1,3-PD含量约为1.5g/L;而对照菌株不产1,3-PD。以上结果表明本研究在国内首次成功构建了直接以葡萄糖为底物发酵生产1,3-PD的酿酒酵母基因工程菌。为进一步将dhaB、yqhD基因导入其他以葡萄糖为底物高产甘油的酵母宿主中表达,获得以葡萄糖为底物一步法发酵高产1,3-丙二醇工程菌打下了坚实的基础。     

Lipase catalysed synthesis of propanediol monoesters in biphasic aqueous medium

The 1,3-regiospecific lipase from Candida deformanscatalysed the esterification of oleic acid and propanediol in biphasic aqueous/lipid medium without organic solvent. The highest conversion of oleic acid into 1,2-propanediol ester was 74% in 24 h with 6.25 mol/l 1,2-propanediol and 0.08 mol/l oleic acid, and produced 100% monoester. The esterification of 1,3-propanediol converted up to 98% of oleic acid into esters in 24 h (with 7.5 mol/l 1,3-propanediol and 0.08 mol/l oleic acid) and formed 35-90% monoester depending on 1,3-propanediol initial concentration (2.5-10 mol/l).     

利用PDOR同工酶基因yqhD对产1,3-丙二醇克雷伯氏杆菌进行基因工程改造

由于Klebsiella pneumoniae 1,3-丙二醇合成途径中,加强甘油脱水酶基因表达,导致因NADH供应不足使3-羟基丙醛累积,并对菌体生长及1,3-丙二醇合成造成负面影响。为改善Klebsiella pneumoniae 1,3-丙二醇合成途径,本文利用PCR技术从大肠杆菌(Escherichia coli)中扩增出以NADPH 为辅酶的1,3-丙二醇氧化还原酶同工酶编码基因yqhD,从克雷伯氏杆菌中扩增出2.66kb的甘油脱水酶基因（dhaB）,构建了产1,3-丙二醇关键酶基因的串联载体pEtac-dhaB-tac-yqhD,并将其转入到野生克雷伯氏杆菌(Klebsiella pneumoniae)中,重组载体得到了表达。通过初步发酵,重组后的克雷伯氏杆菌产量比原始菌高20%左右,副产物中乙酸和丁二醇分别下降30%左右。