透性化细胞海藻糖合成酶的制备及其催化合成海藻糖的工艺优化

笔者以麦芽糖为底物,开展经渗透细胞技术处理得到的透性化细胞海藻糖合成酶催化制备海藻糖的新工艺开发。首先,对比不同透性化试剂对异源表达海藻糖合成酶的大肠杆菌工程菌的透性化处理效果,发现使用硫酸粘杆菌素时海藻糖合成酶的催化转化率高达64.6%,是传统超声破碎的2.63倍;其次,通过参数优化获得硫酸粘杆菌素透性化处理的最佳条件,即:1.5 g/L的硫酸粘杆菌素在30℃下处理60 min,得到的海藻糖合成酶活性最高,为5 209 U/mL;最后,利用透性化处理后的大肠杆菌工程菌进行全细胞催化制备海藻糖工艺研究,结果表明:以质量分数25%的麦芽糖为底物,在pH 7.5、30℃条件下,获得海藻糖的转化率为65.6%;进一步通过离心收集细胞进行反复催化试验,在20批次之后,转化率仍维持在62.5%,显示了良好的应用前景。     

亚栖热菌透性化细胞的耦合固定化研究

将海藻酸盐凝胶包埋法与交联法和聚电解质静电自组装覆膜法相耦合,对含有海藻糖合酶活性的亚栖热菌的透性化细胞进行了固定化研究。结果表明,利用重氮树脂和聚苯乙烯磺酸钠对海藻酸凝胶微球交替覆膜,可以显著提高凝胶微球在磷酸盐缓冲液中的稳定性,以碳二亚胺对固定化细胞进行交联处理则可以提高固定化细胞中海藻糖合酶的热稳定性。透性化细胞经包埋－交联－覆膜耦合固定化后,酶活回收率为32％,最适酶反应pH值由6.5左右升至7.0左右,最适反应温度未变,仍为60℃。所得固定化细胞间歇反应时,催化麦芽糖转化为海藻糖的转化率可达60％,重复使用4次（每次50℃、反应24h）,酶活损失小于20％,转化率可保持在50％以上。     

对产胞内脂肪酶的发酵性丝孢酵母超声波透性化处理的条件优化

发酵性丝孢酵母产胞内脂肪酶,为了把细胞整体作为脂肪酶催化剂,需对细胞进行透性化处理.利用超声波进行细胞透性化的适宜条件为:超声波输出功率180 W,每次辐射时间2s(间歇时间5 s),工作总时间1.2 min,菌体浓度40 g/L,此条件下细胞通透性可明显改善,透性化细胞脂肪酶表现出较高活力.     

提高三角酵母细胞DAO表观活力的透性化研究

三角酵母(Trigonopsis variabilis)的D-氨基酸氧化酶(D-amino acid oxidase,DAO,EC1.4.3.3)是一种胞内酶,完整细胞并不呈现酶促活性。为了获得三角酵母细胞的较高表观活力,需对细胞进行处理以改变壁和膜对反应底物和产物的通透性。研究中比较了冻融、丙酮、丁醇和十六烷基三甲基溴化铵(Cetyltrimethylammonium bromide,CTAB)等理化因子的透性化效率,并对丙酮的透性化条件进行了优化。证明丙酮的透性化作用与溶剂浓度、保温时间和温度有关。30%～35%丙酮浓度,4～28℃保温,可得到最大酶活力。透性化所需时间极短,在5min内即可完成。同时,透性化细胞中的DAO比无细胞抽提液中的酶对热更为稳定。用丙酮透性化细胞作为生物催化剂可有效地转化头孢菌素C(Cephalosporin C, CPC)为戊二酰-7ACA(Glutaryl-7-ACA,GL-7ACA)。     

应用细胞透性化技术快速提取氧化葡萄糖杆菌胞内右旋糖酐糊精酶

将细胞透性化技术应用到右旋糖酐酶的提取检测过程中,根据单因素实验和正交试验设计确定了从氧化葡萄糖杆菌(Gluconobacter oxydans)细胞中提取胞内右旋糖酐糊精酶(DDase)的最佳方法:甘氨酸(Gly)质量分数15%,Triton X-100体积分数1%,菌悬液OD600为0.5~6.0,pH为6.81,冰浴处理时间1 h,透性化试剂的提取效率可达到酶活最大值的90%以上。与超声波细胞破碎法相比,该方法条件温和,酶的释放率较高并易于大量试样的平行实验操作。     

海藻糖合成酶活性受固定化处理过程影响的研究*

在麦芽糖苷基海藻糖合成酶(MTSase)和麦芽糖苷基海藻糖水解酶(MTHase)双酶的作用下,淀粉可转化为海藻糖,但是其转化率较低。中采用多种固定化载体进行酶固定化研究,发现通过经戊二醛与壳聚糖交联后的载体与酶液作用,可吸附与海藻糖合成无关的杂酶和杂质,从而提高海藻糖合成酶的活性。通过比较固定化过程中与反应条件中多个因素的影响,得到了如下最佳作用条件：将酶液与经3％戊二醛交联18h后的滤纸作用18h,再与10％的淀粉溶液反应9h,与未经固定化作用比较,海藻糖的产率提高10倍,达到27．22g／L转化率从5．33％提升到54．43％。     

吸水链霉菌发酵生产谷氨酰胺转胺酶的补料研究

在吸水链霉菌(Streptomyces hygroscopicus)分批发酵研究的基础上,通过在菌体生长阶段指数流加葡萄糖,进行高细胞密度培养,获得了较高的菌体量;待菌体生长进入产酶期后,通过补加氮源,为产酶提供充足的氮源,其中通过流加蛋白质氮源,可以减少蛋白酶对成熟MTG的分解,促进产酶。结果表明,8~16 h采用较高的的比生长速率(0.15 h-1),后期降低比生长速率(0.10 h-1),此时得到的菌体量较高,可达到36 g/L,比分批发酵下的菌体量提高了80%。同时在培养基中添加50g/L的豆饼粉,最终酶活可达到5.79U/ml,提高了83%。     

海藻糖生产过程中产酶发酵条件的研究

研究了产酶的培养基组分和比例以及最佳培养条件对微球菌生产麦芽寡糖基海藻糖合成酶(MTSase)和麦芽寡糖基海藻糖海藻糖水解酶(MTHase)的影响,得到最优培养基组成为：葡萄糖2．0％,酵母膏2．0％,蛋白胨1．0％,磷酸氢二钾0．1％,硫酸镁0．05％;优化后的培养条件为：以15％的接种量接种至250mL的锥形瓶中,装液量为50mL,初始pH值7．5～8．5,培养温度为30℃,摇床培养4d。经优化后菌体干重由原来的1．938g／L增加到18．5g／L,生物量几乎增长了10倍;而酶活也由原来的30．64U／g增加到206．11U／g,酶活提高了接近7倍。     

三角酵母整细胞酶促转化头孢菌素C为戊二酰—7—氨基头孢烷酸

研究了利用含D－氨基酸氧化酶(D-amino acid oxidase,DAO EC1.4.3.3）的透性化三角酶母多倍体FA10（Trigonopsis variabilis FA10）细胞酶促转化头孢菌素C（cephalosporin C,CPC）为戊二酰－7－氨基头孢烷酸（Glutaryl-7-ACA,GL-7ACA）的反应过程和细胞中同时存在的过氧化氢酶（Catalase,CAT）通过水解H2O2而对转化反应产生的干扰作用及其对策。实验证明适量添加外源H2O2（6％）或在反应体系中加入过氧化氢酶抑制剂NaN3(0.13mg/mL )可使GL－7ACA生成率分别为73.0％和70.1％。如果将透性化的FA10细胞在pH10.5-11.0,20℃条件下保温30min,CAT被不可逆性完全钝化,以无过氧化氢酶的FA10细胞进行CPC的酶促转化反应GL－7ACA的生成率可达84％。     

发酵性丝孢酵母胞内脂肪酶与蛋白酶性质的研究

为了探讨发酵性丝孢酵母胞内脂肪酶和蛋白酶的潜在应用,通过超声波破碎细胞获得胞内酶,研究了温度、pH、金属离子、有机溶剂、表面活性剂、蔗糖、淀粉、酪蛋白对粗酶液的酶活力的影响.研究结果表明,两种酶的最适反应条件均为55 ℃、pH中性;5 mmol/L的金属离子Ca2+、Mn2+降低了脂肪酶活力,而提高了蛋白酶的活力;20％ (v/v)甲醇、乙醇、异丙醇、正己烷、甲苯对脂肪酶均具有激活作用,其中正己烷激活作用最大;而所试有机溶剂均严重抑制蛋白酶活力;0.01％(v/v) TritonX-100和蔗糖7.5％ (w/v)对脂肪酶和蛋白酶均具有激活作用,0.5％ (w/v)可溶性淀粉和1％ ～2.5％ (w/v)酪蛋白均能提高脂肪酶活力且降低蛋白酶活力.这些特性使发酵性丝孢酵母胞内脂肪酶和蛋白酶应用于洗涤剂具有可能性.     

Selective release of superoxide dismutase from Saccharomyces cerevisiae with isopropanol

Summary A new and inexpensive approach by which the release and purification of intracellular protein can be carried out simultaneously has been developed. Alcoholic solvents were used to selectively release superoxide dismutase (SOD) from Saccharomyces cerevisiae. 90%(v/v) isopropanol was used to treat the cells for 120 minutes and then 50 mM phosphate buffer(pH7.0) was added after removal of the solvent by filtration. The recovery of SOD was up to 90% , while the specific activity of SOD increased 25 times compared to traditional methods such as ultrasonic method.     

Activity of ethanol-stressed Oenococcus oeni cells: a flow cytometric approach

Aims:  To study the effect of ethanol on Oenococcus oeni activity at the single cell level.
Methods and Results:  The active extrusion of the fluorescent probe carboxy fluorescein (cF) was used to assess the metabolic activity of ethanol-stressed O. oeni cells. Subsequent flow cytometric analysis revealed that O. oeni cells extrude the accumulated cF upon energizing with l -malic acid. However, O. oeni cells exposed to 12% (v/v) ethanol for 1 h showed a decreased capacity for active extrusion of cF. Moreover, two subpopulations could be distinguished, one of which being able to extrude cF and the other one remaining cF fluorescent. Growing cells in the presence of 8% (v/v) ethanol resulted in robust cells that maintained the capacity to actively extrude cF after being exposed to 12% (v/v) ethanol, which in turn correlated with the high levels of ATP observed in these ethanol stressed, malolactic fermentation (MLF) performing cells.
Conclusion:  From our results, it becomes evident that active extrusion of cF can be used to assess malolactic activity in O. oeni .
Significance and Impact of the Study:  The present study provides information for the development of a rapid method to assess the malolactic activity of individual O. oeni cells performing MLF during wine production.     

Beneficial effect of intracellular free high-mannose oligosaccharides on cryopreservation of mammalian cells and proteins

The cryoprotective effect of intracellular free high-mannose oligosaccharides (HMOS) on mammalian cells and proteins was examined by monitoring PC-12 cell viability and assaying protein kinase C (PKC)-epsilon activity. 1-Deoxymannojirimycin, an inhibitor of alpha-mannosidase, to cause an increase in intracellular free HMOS, significantly rescued PC-12 cells with 2-h freezing insult at -15 degrees C in a concentration (1-50mM)- and pretreatment time (48-72h)-dependent manner, as compared with unpretreated cells; full rescue from freezing injury was obtained with 1-deoxymannojirimycin at more than 25mM for 48-h pretreatment and more than 3mM for 72- and 96-h pretreatment. For PC-12 cells pretreated with 1-deoxymannojirimycin at 1mM for 72h, thawed cell viability after more than 8-w cryopreservation at -80 degrees C in 10% (v/v) dimethyl sulfoxide was much higher than that for cells without pretreatment. PKC-epsilon activity was well preserved after 16-h cryopreservation at -20 degrees C in the presence of mannose 9-N-acetylglucosamine 2 (Man9-GlcNAc2) (1 mM), an HMOS, while the activity was reduced to 15% without Man9-GlcNAc2. Collectively, the results of the present study suggest that intracellular free HMOS is a key molecule to protect mammalian cells and proteins from freezing injury; in other words, HMOS could be a new target for cryopreservation of mammalian cells and proteins.     

L-asparaginase release from Escherichia coli cells with K2HPO4 and Triton X100

A method to release L-asparaginase (EC 3.5.1.1) from ATCC Escherichia coli 11303 cells by chemical permeabilization was studied. It was found that a combination of K2HPO4 and Triton X100 was effective. The influences of K2HPO4 concentration, Triton concentration, E. coli cell concentration and pH on the release of enzyme and proteins were investigated in detail. Experimental results showed that 12.5% (w/v) K2HPO4, 2% (w/v) Triton X100 and 3 x 10(8) cells/mL made the amount of enzyme released over 70%. L-Asparaginase in K2HPO4 and Triton solution could remain stable at least for 24 h. The release effect of K2HPO4 and Triton X100 used simultaneously was better than that of K2HPO4 and Triton X100 used separately in succession. Electron microscopy indicated that the chemical treatment altered the surface structure of E. coli cells but did not break them. As the method does not produce a large amount of cell fragments and the amount of enzyme released is relatively high, it can be thought to be an valuable and economic method to release intracellular enzyme.     

Simultaneous determination of 19 intracellular nucleotides and nucleotide sugars in Chinese Hamster ovary cells by capillary electrophoresis

Twelve nucleotides and seven nucleotide sugars in Chinese Hamster ovary (CHO) cells were determined by capillary electrophoresis (CE). The CE operating conditions of buffer pH value, ion strength, capillary temperature, polymer additive and cell extraction method were investigated. Optimum separation was achieved with 40 mM sodium tetraborate buffer (pH 9.5) containing 1% (w/v) polyethylene glycol (PEG) at a capillary temperature of 22 degrees C. Acetonitrile and chloroform were used for intracellular extraction. This method can be used to monitor intracellular carbohydrate metabolism.     

Optimizing high-throughput metabolomic biomarker screening: a study of quenching solutions to freeze intracellular metabolism in CHO cells

Metabolomics is a rapidly emerging tool for studying and optimizing both media and bioprocess development for culturing recombinant mammalian cells that are used in protein production processes. Quenching of the cells is crucial to fix their metabolic status at the time of sampling. Three precooled quenching solutions were tested for their ability to fix the metabolic activity of CHO cells: phosphate-buffered saline (PBS) (pH 7.4; 0.5°C), 60% methanol with 70?mM HEPES (pH 7.4; -20°C), and 60% methanol with 0.85% (w/v) ammonium bicarbonate (AMBIC) (pH 7.4; -20°C). The metabolic activity of the sampled CHO cells was assessed by determining the intracellular levels of ATP using a bioluminescence assay and selected metabolites with LC-MS/MS. We found the precooled PBS (pH 7.4; 0.5°C) to be the optimal quenching reagent for fixing intracellular metabolism. Importantly, the structural integrity of the cell membrane was maintained and highest yields were obtained for intracellular levels of ATP as well as for 18 out of 28 intracellular metabolites. In contrast to the previously reported studies, buffered methanol quenching was not applicable for suspension cultured CHO cells as cellular membrane integrity was affected. We recommend that the cells are quenched and washed simultaneously to keep the sampling time to a minimum and to prevent any further metabolic activity in the cells. We observed that additional washing steps are not required. Our analyses suggest that methanol as quenching solution, even in combination with a buffer substance, appears not suitable for quenching sensitive mammalian cells. The protocol we report herein is a simple cell sampling method that enables high-throughput metabolomic analyses and is suitable for a large number of samples.     

Active control of the nucleation temperature enhances freezing survival of multipotent mesenchymal stromal cells

Cryopreservation is a technique that has been extensively used for storage of multipotent mesenchymal stromal cells (MSCs) in regenerative medicine. Therefore, improving current cryopreservation procedures in terms of increasing cell viability and functionality is important. In this study, we optimized the cryopreservation protocol of MSCs derived from the common marmoset Callithrix jacchus (cj), which can be used as a non-human primate model in various pathological and transplantation studies and have a great potential for regenerative medicine. We have investigated the effect of the active control of the nucleation temperature using induced nucleation at a broad range of temperatures and two different dimethylsulfoxide concentrations (Me₂SO, 5% (v/v) and 10%, (v/v)) to evaluate the overall effect on the viability, metabolic activity and recovery of cells after thawing. Survival rate and metabolic activity displayed an optimum when ice formation was induced at −10 °C. Cryomicroscopy studies indicated differences in ice crystal morphologies as well as differences in intracellular ice formation with different nucleation temperatures. High subzero nucleation temperatures resulted in larger extracellular ice crystals and cellular dehydration, whereas low subzero nucleation temperatures resulted in smaller ice crystals and intracellular ice formation.     

Production of trehalose by permeabilized Micrococcus QS412 cells

Permeabilized Micrococcus QS412 cells were used to produce trehalose from starch through catalysis of maltooligosyl trehalose synthase and maltooligosyl trehalose trehalohydrolase in the cells. The permeabilized cells could omit the enzyme purification and simplify the immobilization of intracellular enzymes. The reagent, reagent dosage and time of cell permeabilization treatment were determined. The maximum trehalose biosynthesis activity was obtained after the cells were treated with 5% (w/v) of toluene at 30 °C for 40 min. Reaction conditions of trehalose synthesis of permeabilized cells were optimized. The yield of trehalose was up to 188 mg/g wet permeabilized cells in pH 8.0, 100 mmol/l phosphate buffer at 30 °C after 12 h reaction. Batch reactions showed that the permeabilized cells could be reused for 16 cycles in the biosynthesis reaction. The total trehalose yield was up to 2.5 g/g wet permeabilized cells. Development of permeabilized cells provide a new cheaply alternative technology for trehalose production.     

Influence of Water on the Primary Photosynthetic Activity of Rhodospirillum Rubrum in Reverse Micelles

The effect of water on the primary photosynthetic activity of purple bacterium Rhodospirillum rubrum was studied in Hexadecane-Tween-Spane (HTS)- and phospholipid (PLC)-reverse micelles. Reverse micelles offer the possibility of modulating the amount of water to which enzymes and multienzymatic complexes are exposed. Fast bacteriochlorophyll (BChl) fluorescence induction kinetics and reaction centre absorption changes at 820 nm were used as an assay for the functional transfer of bacterial cells into HTS-reverse micelles and bacterial photosynthetic complexes (BPC) into PLC-reverse micelles. Both the bacterial cells and BPC showed an increase in the rate of primary photosynthetic activity by increasing the concentration of water in the reverse micelles. The bacterial cells could be kept viable for many hours in HTS-reverse micelles in presence of 6% (v/v) water. NMR studies indicated that the photosynthetic activity was affected by the availability of water in reverse micelles. The bacterial cells in HTS or BPC in PLC reverse micelles could be used to further understand the influence of water on the organisation and function of photosynthetic complexes. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effect of water-miscible solvents on the Δ1-dehydrogenase activity of free and PAAH-entrapped Arthrobacter simplex

Summary The effect of water-miscible cosolvents on biotransformations of poorly water-soluble substrates by immobilized cells was investigated, using ¹-dehydrogenation of hydrocortisone by Arthrobacter simplex as a model. Criteria for solvent selection on the basis of retention of enzymic activity were postulated and tested. Diols were considered to be the most suitable group of solvents. Substrate solubility increased tenfold in 30% (v/v) ethylene glycol, but reaction rates were significantly slower in such solutions. This was mainly caused by a decrease of oxygen solubility in the presence of the cosolvent and conformational changes imposed on the intracellular enzyme by cosolvent molecules penetrating the cell. The inhibition could be eliminated by the addition of an artificial electron acceptor, phenazine methosulphate (PMS). Reaction rates faster than those for substrate suspensions (no cosolvent added) could thus be achieved. Immobilization of Arthrobacter simplex in cross-linked polyacrylamide hydrazide gave high retentions of activity. PMS exhibited toxic effects on the entrapped cells, leading to reduced activity after extended use.