地衣芽孢杆菌产生碱性蛋白酶的动力学研究

应用自动控制发酵设备,首先进行分批发酵试验摸索了地衣芽孢杆菌2709生长与代谢的基本规律。然后采用补料分批发酵方法限制生长基质浓度,测定了一系列(S_I,μ_I)、(μ_j,q_pj)数据,获得K_S、μ_max、α、β等参数的值,并且推导出了细胞生长与产物合成的动力学公式,从而证明了用Monod方程描述地衣芽孢杆菌2709生长速率与基质浓度关系的合理性和合成碱性蛋白酶的发酵属于生长部分关联型。     

菌核菌生长和产生草酸的营养和环境条件研究

本研究探讨了培养基种类、酸碱度、温度对菌核菌(Sclerotinia sclerotiorum)菌丝生长(MG)和草酸累积(OA)的影响及MG和OA之间的动态变化过程。正交试验中,碳源为:琥珀酸钠-葡萄糖、蔗糖-马铃薯汁液和蔗糖的三种培养基中,OA量依次递减且差异显著。MG和OA成负相关关系。培养基的pH值是影响OA的一个主要因子,本试验中pH6最适合菌丝产草酸,而pH5最适合菌丝生长。23℃下OA和MG大于26℃下OA和MG。OA和MG的动态变化过程试验表明,不同培养基的草酸累积速率和最终     

植物细胞离析酶的制备和应用

用 Aspergillus sp．A-19菌经固体发酵研制成一种新的植物细胞离析酶(SeparatasezA—P)。其离析单细胞的酶活力平均为70 767u／g,有效作用的pH在3．0—7．0,温度为20—45℃。发酵培养基配方是麸皮：桔皮粉：(NH₄)₂SO₄(w／w)为100:100:O．63,料水比为1 :2．0,培养适宜条件为25℃、60小时。     

发酵生产十六烷二羧酸的研究

以热带假丝酵母(Candida tropicalis)T_25-14为出发菌株,经紫外线多次诱变,获得生产十六烷二羧酸(DC₁₆)能力比原株提高25％以上的4株突变株。其中UH-3-9突变株经多次复筛,产DC16能力都比T25-14,菌株提高50％以上。经摇瓶条件试验,不加其他生长碳源。只加15％(v／v)正十六烷(nCl6),发酵96h,DC16为48．2g/L,转化率41％,产品纯度95．9％。     

枯草芽孢杆菌中性内切β-甘露聚糖酶的纯化及性质

枯草芽孢杆菌(Bacillus subtilis)BM9602产生的中性内切β甘露聚糖酶(endoβ1,4Dmannan mannanohydrolase,EC,3.2.1.78)经硫酸铵分级沉淀、DEAE纤维素(DE22)离子交换柱层析,得到电泳纯的样品,提纯了455倍,收率为59%。用SDSPAGE测得该酶的分子量为35kD。用PAGEIEF测得其等电点pI为45。酶反应的最适pH为5.8,最适温度为50℃。该酶在pH60～80,50℃以下稳定。金属离子Hg2+和Ag+对酶活性强烈抑制。酶对槐豆胶、羟丙基瓜胶、田菁胶和魔芋粉的Km值分别为38、149、113和24mg/mL,Vmax值分别为245、865、384和198μmol.min-1mg-1。酶水解甘露聚糖为甘露寡糖(不含单糖)。     

黑曲霉FS25产β-葡聚糖酶发酵特性的研究*

黑曲霉 (Aspergillusniger) FS2 5产β 葡聚糖酶最适碳源为大麦粉 ,氮源为玉米浆 ;最佳摇瓶发酵配方为大麦粉 6g ,玉米浆 2g ,(NH₄)₂SO₄0.4g ,FeSO₄·7H₂ O 0.01g ,Na₂ HPO₄·3H₂ O 0.1g,CaCO₃0.5g ,MgSO相似文献   

人工合成的黑曲霉NRRL3135菌株植酸酶基因在毕赤酵母系统中的高效表达

本文以黑曲霉(Aspergillus niger)NRRL3135菌株植酸酶基因为对象,通过基因人工合成的方法去除了该基因的内含子与信号肽编码序列,换用在毕赤酵母(Pichia pastoris)中使用频率较高的密码子以优化其表达。该人工合成植酸酶基因(PhyA-as)以N端融合的方式正确插入到毕赤酵母表达载体pPICZαA。通过电击将重组表达载体整合入酵母染色体DNA中得到重组转化子。SDSPAGE结果与表达产物酶学性质研究表明植酸酶得到分泌表达,且与天然产物性质基本一致。筛选得若干株高产基因工程菌,其中SPANⅢ菌株达到了在摇床培养条件下,每毫升发酵液产生165000u植酸酶的水平,基本满足工业化生产的要求。     

聚球藻7002在光生物反应器中的光自养培养

通过对聚球藻7002在光生物反应器中的培养,研究了光强在聚球藻7002培养液中的衰减规律,得到了培养过程光强随藻细胞浓度和光程距离变化的关系式,即I=I₀exp［-(-0.0239+0.0777OD₇₅₀)·L］。并对培养过程特性及培养温度、外加CO₂浓度和光照强度对藻细胞生长的影响进行了较为详细的研究,得到了反应器中较为适宜的聚球藻7002的培养条件,藻细胞培养密度达到3.4g/L(干重),体积产率达到0.57g/(L·d)的较高水平。     

利用重组Pichia pastoris生产腺苷甲硫氨酸

为改造甲醇利用型酵母Pichia pastoris来生产腺苷甲硫氨酸（SAM,S-adenosylL-methionine）,我们将一个带有SAM合成酶基因的胞内表达质粒转化入Pichia pastoris菌株GS115,经过G418抗性筛选得到一株有两个基因拷贝的转化子。该菌在含有甲醇和甲硫氨酸的培养基中生长5d后,其细胞内的SAM的产量比原始菌株提高了30余倍。对该菌生产SAM的培养基中的碳源与氮源进行了优化,结果显示碳源的控制对该菌SAM产量的影响很大。在试管水平,该菌在含有0.75％的L-methionine并且碳源和有机氮源经过一定程度优化的培养基中,生长6d后SAM产量达到1.58g/L。     

小球藻病毒基因调控序列在大肠杆菌和真核藻中的调控活性研究

以小球藻病毒腺嘌呤甲基转移酶基因(amt)和主要外壳蛋白VP54基因的5′上游调控序列构建大肠杆菌和真核藻转化载体。以P_RP_L及CaMV35S启动子为阳性对照,研究了小球藻病毒来源的两种调控序列在E.coli和真核藻细胞中的启动活性。发现P_AMT在4种E.coli菌株中都具有极强的调控活性,启动Luc基因表达而产生的酶活性高于P_RP_L 50～400倍;P_VP54在DH5α中也具有较强的启动活性。同时PAMT在两种小球藻中启动GUS基因瞬时表达的能力也明显高于CaMV35S启动子,表明它们有可能在真核藻类遗传转化中具有很好的应用前景。     

Control of intracellular pH. Predominant role of oxidative metabolism, not proton transport, in the eukaryotic microorganism Neurospora

Recessed-tip microelectrodes were used to measure internal pH (pHi) in the fungus Neurospora, and to examine the response of pHi to several kinds of stress: changes of extracellular pH (pHo), inhibition of the principal proton pump in the plasma membrane, and inhibition of respiration. Under control conditions, at pHo = 5.8, pHi in Neurospora is 7.19 +/- 0.04. Changes of pHo between 3.9 and 9.3 affect pHi linearly but with a slope of only approximately 0.1 unit pHi per unit pHo, stable pHi being reached within 3 min of changed pHo. Despite a postulated high passive permeability of the Neurospora membrane to protons (Slayman, 1970), neither active nor passive H+ transport appears critical to pHi because (alpha) specific inhibition of the proton pump by orthovanadate has little effect on pHi, and (b) cytoplasmic acidification produced by respiratory blockade is unaffected by the size or direction of proton gradient. To convert measured changes in pHi into net proton fluxes, intracellular buffering capacity (beta i) was measured by the weak acid/weak base technique. At pHi = 7.2, beta i was (-) 35 mmol H+ (liter cell water)-1 (pH unit)-1, but beta i increased substantially in both the acid and alkaline directions, which suggests that amino acid side chains are the principal source of buffer.     

Fluorescence lifetime-based pH mapping of tumors in vivo using genetically encoded sensor SypHerRed

Changes in intracellular pH (pHi) reflect metabolic states of cancer cells during tumor growth and dissemination. Therefore, monitoring of pHi is essential for understanding the metabolic mechanisms that support cancer progression. Genetically encoded fluorescent pH sensors have become irreplaceable tools for real-time tracking pH in particular subcellular compartments of living cells. However, ratiometric readout of most of the pH probes is poorly suitable to measure pH in thick samples ex vivo or tissues in vivo including solid tumors. Fluorescence lifetime imaging (FLIM) is a promising alternative to the conventional fluorescent microscopy. Here, we present a quantitative approach to map pHi in cancer cells and tumors in vivo, relying on fluorescence lifetime of a genetically encoded pH sensor SypHerRed. We demonstrate the utility of SypHerRed in visualizing pHi in cancer cell culture and in mouse tumor xenografts using fluorescence lifetime imaging microscopy and macroscopy. For the first time to our knowledge, the absolute pHi value is obtained for tumors in vivo by an optical technique. In addition, we demonstrate the possibility of simultaneous detection of pHi and endogenous fluorescence of metabolic cofactor NADH, which provides a complementary insight into metabolic aspects of cancer. Fluorescence lifetime-based readout and red-shifted spectra make pH sensor SypHerRed a promising instrument for multiparameter in vivo imaging applications.     

Metabolic regulation of neutrophil spreading, membrane tubulovesicular extensions (cytonemes) formation and intracellular pH upon adhesion to fibronectin

Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na(+) and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl(-) efflux through chloride channels and Na(+) influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.     

Na+-independent Cl(-)-HCO-3- exchange in Madin-Darby canine kidney cells. Role in intracellular pH regulation

The role of plasma membrane Cl(-)-HCO-3-exchange in regulating intracellular pH (pHi) was examined in Madin-Darby canine kidney cell monolayers. In cells bathed in 25 mM HCO-3, pH 7.4, steady state pHi was 7.10 +/- 0.03 (n = 14) measured with the fluorescent pH probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Following acute alkaline loading, pHi recovered exponentially in approximately 4 min. The recovery rate was significantly decreased by Cl- or HCO-3 removal and in the presence of 50 microM 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS). Na+ removal or 10(-3) M amiloride did not inhibit the pHi recovery rate after an acute alkaline load. Following acute intracellular acidification, the pHi recovery rate was significantly inhibited by 10(-3) M amiloride but was not altered by Cl- removal or 50 microM DIDS. At an extracellular pH (pHo) of 7.4, pHi remained unchanged when the cells were bathed in either Cl- free media, HCO-3 free media, or in the presence of 50 microM DIDS. As pHo was increased to 8.0, steady state pHi was significantly greater than control in Cl(-)-free media and in the presence of 50 microM DIDS. It is concluded that Madin-Darby canine kidney cells possess a Na+-independent Cl(-)-HCO-3 exchanger with a Km for external Cl- of approximately 6 mM. The exchanger plays an important role in pHi regulation following an elevation of pHi above approximately 7.1. Recovery of pHi following intracellular acidification is mediated by the Na+/H+ antiporter and not the anion exchanger.     

Relationship between acid tolerance, cytoplasmic pH, and ATP and H+-ATPase levels in chemostat cultures of Lactococcus lactis.

The acid tolerance response (ATR) of chemostat cultures of Lactococcus lactis subsp. cremoris NCDO 712 was dependent on the dilution rate and on the extracellular pH (pHo). A decrease in either the dilution rate or the pHo led to a decrease in the cytoplasmic pH (pHi) of the cells, and similar levels of acid tolerance were observed at any specific pHi irrespective of whether the pHi resulted from manipulation of the growth rate, manipulation of the pHo, or both. Acid tolerance was also induced by sudden additions of acid to chemostat cultures growing at a pHo of 7.0, and this induction was completely inhibited by chloramphenicol. The end products of glucose fermentation depended on the growth rate and the environmental pHo of the cultures, but neither the spectrum of end products nor the total rate of acid production correlated with a specific pHi. The rate of ATP formation was not correlated with pHi, but a good correlation between the cellular level of H+-ATPase and pHi was observed. Moreover, an inverse correlation between the cytoplasmic levels of ATP and pHi was established. Each pHi below 6. 6 was characterized by unique levels of ATR, H+-ATPase, and ATP. High levels of H+-ATPase also coincided with high levels of acid tolerance of cells in batch cultures induced with sublethal levels of acid. We concluded that H+-ATPase is one of the ATR proteins induced by acid pHi through growth at an acid pHo or a slow growth rate.     

Evidence of alpha 1-adrenergic----protein kinase C----Na+/H+ antiporter-dependent increase in pinealocyte intracellular pH. Role in the adrenergic stimulation of cGMP accumulation

The regulation of intracellular pH (pHi) in isolated rat pinealocytes was studied using the fluorescent pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Resting pHi was 7.09 when the extracellular pH (pHe) was 7.2. Treatment of pinealocytes with the physiological regulator of pineal function, norepinephrine, resulted in a concentration-dependent increase in pHi. Further analysis indicated that norepinephrine is probably acting via an alpha 1-adrenergic----[Ca2+]i----Ca2+/phospholipid- dependent protein kinase (protein kinase C) mechanism to activate the Na+/H+ antiporter, thereby causing cytoplasmic alkalization. A potential influence of cytosolic alkalization on the responsiveness of cyclic nucleotides to adrenergic agonists was also studied. Five analogs of the antiporter inhibitor amiloride reduced norepinephrine stimulation of cGMP accumulation with the same relative potency as they act on the antiporter. In contrast, although inhibitory effects of these compounds on cAMP accumulation were detectable, they occurred at 10-100-fold higher concentrations, and the relative potency of these inhibitors did not indicate they were acting via the antiporter. These findings provide evidence that 1) alpha 1-adrenergic receptor activation increases pinealocyte pHi through Ca2+----protein kinase C-dependent activation of the Na+/H+ antiporter; and 2) norepinephrine stimulation of cGMP accumulation is pHi-dependent. It would appear that alpha 1-adrenergic regulation of pHi via the Na+/H+ antiporter may be of general importance in the control of cGMP accumulation.     

Fluorescence emission spectroscopy of 1,4-dihydroxyphthalonitrile. A method for determining intracellular pH in cultured cells.

We have developed new methodology for measuring intracellular pH (pHi) in cultured cell monolayers and epithelia by analyzing the emission spectra of the trapped fluorescent pH probe, 1,4-dihydroxyphthalonitrile (1,4-DHPN). This compound is unique since both its acid and base forms possess different fluorescence emission characteristics that can be used to quantitate pHi. The fluorescence difference spectrum between an acid and alkaline solution of 1,4-DHPN has a maximum at 455 nm and a minimum at 512 nm. By determining the ratio of the intensity at these two wavelengths as a function of pH, a calibration curve was constructed. Since the two intensities are determined simultaneously, the measurement is independent of dye concentration, bleaching, and intensity fluctuation of the excitation source. Furthermore, analysis of the emission spectra permitted the detection of light scattering, binding effects, and chemical modification of the probe. A microspectrofluorometer was constructed to analyze low light level emission spectra from intracellular 1,4-DHPN. The instrument consists of a modified Leitz inverted microscope (E. Leitz, Inc., Rockleigh, NJ) with a Ploem illuminator adapted for broadband excitation and objective focusing capability. The emission spectra were collected by focusing the fluorescence from the cell onto the entrance slit of an imaging monochromator, which was scanned by a SIT camera interfaced with a computer. This permitted the acquisition of fluorescence emission spectra extending from 391-588 nm in approximately 33 ms. pHi measured in the cultured toad kidney epithelial cell line, A6, was 7.49 +/- 0.04 (n = 12) with an external pH of 7.6. A6 cells were found to regulate pHi in response to both acute acid and alkali loads and maintained pHi relatively constant over a wide range of external pH values. The technique described in this report overcomes several of the difficulties encountered with other fluorescent pH probes where excitation spectroscopy is required to monitor pH.     

Membrane potential and cation content of osteoblast-like cells (UMR 106) assessed by fluorescent dyes

A number of cellular functions have recently been associated with alterations of the membrane potential in non-excitable cells. To assess the electrophysiologic regulation of osteoblast function, a method for measuring the membrane potential (Em) of a rat osteogenic sarcoma cell line (UMR 106) by the voltage-sensitive oxonol dye di-BA-C4(3) was developed. The fluorescent signal of di-BA-C4(3) was calibrated through a null point method using the protonophore FCCP. At null point, Em is equivalent to H+ equilibrium potential, and may be calculated by the Nernst equation. Intracellular pH (pHi) changes induced by the protonophore were monitored using BCECF, a pH-sensitive fluorescent probe. In the presence of FCCP, intracellular pH was found to be linearly correlated to extracellular pH (pHo). Therefore, the value of pHi at null point was extrapolated as well. With this technique, we estimated the plasma membrane potential of the "putative" rat osteoblasts (UMR 106) as -28.3 +/- 4.0 mV (n = 10). This method corrected the 16% overestimation of Em derived from the assumption that pHi does not change during the calibration procedure, as described in previous studies employing pH null point techniques. With null point methods, using BCECF and the carboxylic ionophores nigericin and monensin, intracellular concentrations of potassium and sodium were also measured and found to be 125 +/- 0.7 mM (n = 3) and 24 +/- 5.3 mM (n = 3), respectively. Although the Em of UMR 106 cells was dependent on extracellular potassium concentration, these cells did not behave as a potassium electrode. The sodium/potassium permeability ratio, calculated by the Goldman equation, was estimated at 0.317. This high membrane permeability to sodium may contribute to the genesis of the low plasma membrane potential of UMR 106 cells.     

Role of caveolin-1 and cholesterol in transmembrane fatty acid movement

We have created by transfection a series of HEK 293 cell lines that express varying amounts of caveolin-1 to test the possible effect of this protein on the transport and metabolism of long chain fatty acids (FA) in cells with this gain of function. We used an extracellular fluorescent probe (ADIFAB) to monitor binding of exogenous FA to the plasma membrane and an intracellular pH probe to monitor FA equilibration across the plasma membrane. Real-time fluorescence measurements showed rapid binding of oleic acid to the extracellular side of the plasma membrane and a rapid translocation across the lipid bilayer by the flip-flop mechanism (<5 s). Two cell lines expressing levels of caveolin-1 roughly comparable to that of adipocytes, which have a very high level of endogenous expression of caveolin-1, showed a relatively slow change in intracellular pH (t(1/2) < 100 s) in addition to the fast changes in fluorescence. We interpret this additional second phase to represent translocation of additional FA from the outer to inner leaflet of the plasma membrane. The slower kinetics could represent either slower flip-flop of FA across highly organized, rigid regions of the plasma membrane or binding of FA to caveolin-1 in the intracellular leaflet of the plasma membrane. The kinetics of palmitate and elaidate (a trans FA) transmembrane movement were identical to that for oleate. These results were observed in the absence of the putative FA transport protein, CD36, and in the absence of any changes in expression of fatty acid transport proteins (FATP) 2 and 4, and are in direct correlation with increased cellular free cholesterol content. FA metabolism was slow in all cell lines and was not enhanced by caveolin-1 expression. We conclude that transport of FA across the plasma membrane is modulated by caveolin-1 and cholesterol and is not dependent on the putative FA transport proteins CD36 and FATP.     

Cytoplasmic pH in cultured porcine thyroid cells: alkalinization by epidermal growth factor

We studied the effects of epidermal growth factor (EGF), thyroid-stimulating hormone (TSH) and amiloride on cytoplasmic pH (pHi) in cultured porcine thyroid cells. We used 2',7'-bis(2-carboxyethyl)-5- (and 6-)carboxyfluorescein (BCECF), an internalized fluorescent pH indicator, to measure pHi. EGF stimulated thyroid cell alkalinization and proliferation, which were blocked by amiloride. EGF-stimulated thyroid cell alkalinization depended on extracellular Na+ concentrations. EGF stimulation resulted in an activation of Na+/H+ exchange, which alkalinized the cells. The results indicated that Na+/H+ exchange or cell alkalinization might function as a transmembrane signal transducer in the action of EGF. In the present system, TSH did not stimulate alkalinization or proliferation.