Importance of spore mutants for fed-batch and continuous fermentation of Bacillus subtilis

To alleviate plasmid instability and to prolong the production phase of subtilisin, integrable plasmid and spore mutants are used. Compared with batch-type shake flask cultures, spore mutants' ability to produce subtilisin can be well pronounced in fed-batch and continuous cultures. Hence, the two culture methods make it possible to identify the peculiar characteristics of the spore mutants unobtainable in batch culture. Spore mutants can enhance subtilisin productivity and prolong subtilisin production time in fed-batch culture as well as enable us to use very low dilution rates (<0.1 h(-1)) without losing productivity in continuous culture, thereby improving the conversion yield of the nitrogen source. At 0.05 h(-1) the spollG mutant of Bacillus subtilis DB104 (Deltanpr Deltaapr) (Em(r)) spollG (Bim(r)):: pMK101 (Cm(r)) showed a subtilisin yield about ten times higher than that from wild-type DB104 (Deltanpr Deltaapr)::pMK101 (Cm(r)). (c) 1995 John Wiley & Sons, Inc.     

Changes in Esterification of the Uronic Acid Groups of Cell Wall Polysaccharides during Elongation of Maize Coleoptiles

Cell walls of grasses have two major polysaccharides that contain uronic acids, the hemicellulosic glucuronoarabinoxylans and the galactosyluronic acid-rich pectins. A technique whereby esterified uronic acid carboxyl groups are reduced selectively to yield their respective 6,6-dideuterio neutral sugars was used to determine the extent of esterification and changes in esterification of these two uronic acids during elongation of maize (Zea mays L.) coleoptiles. The glucosyluronic acids of glucuronoarabinoxylans did not appear to be esterified at any time during coleoptile elongation. The galactosyluronic acids of embryonal coleoptiles were about 65% esterified, but this proportion increased to nearly 80% during the rapid elongation phase before returning to about 60% at the end of elongation. Methyl esters accounted for about two-thirds of the total esterified galacturonic acid in cell walls of unexpanded coleoptiles. The proportion of methyl esters decreased throughout elongation and did not account for the increase in the proportion of esterified galactosyluronic acid units during growth. The results indicate that the galactosyluronic acid units of grass pectic polysaccharides may be converted to other kinds of esters or form ester-like chemical interactions during expansion of the cell wall. Accumulation of novel esters or ester-like interactions is coincident with covalent attachment of polymers containing galactosyluronic acid units to the cell wall.     

Inhibition of Sucrose Enhancer Effect of the Potato Proteinase Inhibitor II Promoter by Salicylic Acid

Effect of salicylic acid (SA) on the expression of the potato proteinase inhibitor (PI) II promoter was studied with transgenic tobacco plants (Nicotiana tabacum) carrying a gene fusion between the PI-II promoter and the chloramphenicol acetyltransferase (cat) reporter. As previously observed, the PI-II promoter was inducible by wounding and the promoter activity was further enhanced by sucrose. Addition of SA did not influence the wound induction of the PI-II promoter but significantly inhibited the sucrose response. The 5′-deletion mutant −573 was unable to respond to wounding but did respond to sucrose and SA. The 3′-deletion analysis indicated the presence of a sucrose-responsive element between −574 and −520. A study of the insertion mutants revealed the function of another sucrose-responsive element between −522 and −500. Enhancer effects of these sucrose-responsive elements were inhibited by SA. These studies suggest that SA inhibits PI-II promoter activity by decreasing the sucrose response. Analysis of SA-related chemicals revealed that only acetyl-SA showed a similar inhibitory effect, and other hydroxybenzoic acids had little or no effect on the sucrose enhancer activity. Therefore, it seems that the interaction between SA and the receptor molecule is specific.     

Current concepts of cell-cycle regulation and its relationship to oocyte maturation, fertilization and embryo development

Genetic and biochemical approaches have contributed to an explosion of literature on cell-cycle control. Regulation of the cell-cycle is controlled by a series of kinases and phosphatases. Key control points are during the G(1)-S and G(2)-M transitions. During both transitions, cyclins interact with a specific kinase to allow a cell to pass through that phase. The meiotic maturation of oocytes, fertilization and embryo development are all events influenced by cell-cycle regulation. Understanding cell-cycle control should provide new ways for gamete and embryo biologists to approach culture and development problems.     

A new biosensor for specific determination of sucrose using an oxidoreductase of Zymomonas mobilis and invertase

A new biosensor for specific determination of sucrose was developed using an oxidoreductase of Zymomonas mobilis and invertase. Cells of Z. mobilis were permeabilized with toluene in order to utilize the enzymes of glucose-fructose oxidoreductase and gluconolactonase inside the intact cells. Permeabilized cells and invertase were coimmobilized in a gelatin membrane, and a whole cell enzyme electrode was constructed by fixing the membrane on a pH electrode. The production of hydrogen ion was detected using the biosensor-connected microcomputer, and the concentration of sucrose was determined by using both the initial rate and the steady-state methods. Optimum conditions for biosensor response were pH 6.2 and temperature 35 degrees C. The effect of interfering compounds on the electrode response was investigated, and the interference by various sugars was eliminated by determining sucrose concentration using the steady-state method. The biosensor developed is simple and reproducible, and the calibration curve for sucrose is linear up to 70 g/L.     

Microcarrier culture of bowes melanoma cells in serum-free medium with Human plasma fraction IV-4+ V

Bowes melanoma cells were cultivated successfully in a serum-free medium which was constructed by the concept of maximum retention of proteins from fractionated human plasma having growth stimulatory activities. The cells could be cultivated in the serum-free medium without any adaptation period. The major serum-free component of the medium was the fraction IV-4 + V of the Cohn fractionation process of human plasma. Approximately six times increase of tissue-type plasminogen activator (t-PA) activity as compared with that in serum-free medium even though the cell growth was much slower. In addition, the growth stimulatory activities of thrombin and fibronectin were investigated during the cultivation of Bowes melanoma cells in this serum-free medium. These proteins contributed significantly to the enhanced growth of cells by reducing doubling time to 25 and 35 h as compared with 55 h in the serum-free medium without them. Especially, fibronectin supported cells to propagate near to the maximum cell density achieved in the medium with 10% FBS.     

A new biosensor for specific determination of glucose or fructose using an oxidoreductase of Zymomonas mobilis

Cells of Zymomonas mobilis were permeabilized with toluene in order to utilize the enzymes, glucose-fructose oxidoreductase and gluconolactonase, inside the intact cells. Permeabilized cells were immobilized in a gelatin membrane, and a whole cell enzyme electrode was constructed by fixing the membrane on pH electrode. The biosensor developed was used for specific determination of glucose or fructose by detecting the production rate of hydrogen ion. Optimum conditions for biosensor response were pH 6.2 and temperature of 39 degrees C. The biosensor was highly specific and reproducible, and calibration curves for glucose and fructose were excellent, being linear up to 5 and 50 g/L, respectively.     

Enhanced shikonin production from Lithospermum erythrorhizon by in situ extraction and calcium alginate immobilization

Plant cell cultures of Lithospermum erythrorhizon were carried out to produce shikonin by in situ extraction and cell immobilization in calcium alginate bead in shake flask cultures. In situ product extraction and cell immobilization enhanced shikonin production and facilitated product recovery. In situ extraction by n-hexadecane and cell immobilization by calcium alginate gave higher specific shikonin productivities of 7.4 and 2.5 times, respectively, than those from the cultures of free cells without extraction. Simultaneous use of both techniques increased specific and volumetric productivities of shikonin 25- and 15-fold, respectively. In calcium alginate immobilized cell cultures, n-hexadecane addition at an early stage (before 15 days) was effective for shikonin production, and solvent addition after 15 days of the culture significantly reduced shikonin production. Higher numbers of plant cell immobilized bead inoculation did not increase shikonin production and sucrose consumption. Most of the produced shikonin was dissolved in the solvent layer.     

Windows操作系统下二代测序数据处理平台的建立及高通量信息分析标准流程在个人计算机上的实现

当前二代测序数据的处理广泛使用基于标准版本的Linux操作系统分析方法。这一系统专业性强,成本较高,操作界面不够友好,严重限制了大多数科研人员对数据的自主分析。本文创建了一个基于微软Windows操作系统的全功能二代测序数据的生物信息学分析系统,利用该系统经优选实现当前多种高通量测序数据的主流标准化分析流程。通过RNA-Seq的代表性案例,演算实测数据与传统Linux系统驱动的数据分析结果相比较,结果显示,本系统的组件和流程在常用的数据分析过程中,可以基本取代目前主流的Linux服务器或云计算平台,在运行效率相近的情况下,其操作极为简便且成本大大降低。本系统与所配附的编译软件及流程脚本,不仅为测序数据的生物信息学分析实操演练提供全面的解决方案,而且可以直接应用于专业的测序数据分析中。     

Dissecting the pretransitional conformational changes in aminoacylase I thermal denaturation

Aminoacylase I (ACYI) catalyzes the stereospecific hydrolysis of L-acylamino acids and is generally assumed to be involved in the final step of the degradation of intracellular N-acetylated proteins. Apart from its crucial functions in intracellular amino acid metabolism, ACYI also has substantial commercial importance for the optical resolution of N-acylated DL-amino acids. As a zinc-dependent enzyme, ACYI is quite stable against heat-induced denaturation and can be regarded as a thermostable enzyme with an optimal temperature for activity of approximately 65 degrees C. In this research, the sequential events in ACYI thermal denaturation were investigated by a combination of spectroscopic methods and related resolution-enhancing techniques. Interestingly, the results from fluorescence and infrared (IR) spectroscopy clearly indicated that a pretransitional stage existed at temperatures from 50 degrees C to 66 degrees C. The thermal unfolding of ACYI might be a three-state process involving an aggregation-prone intermediate appearing at approximately 68 degrees C. The pretransitional structural changes involved the partial unfolding of the solvent-exposed beta-sheet structures and the transformation of about half of the Class I Trp fluorophores to Class II. Our results also suggested that the usage of resolution-enhancing techniques could provide valuable information of the step-wise unfolding of proteins.