Factors affecting chemical‐based purification of DNA from Saccharomyces cerevisiae

Extraction of high molecular weight chromosomal DNA from yeast cells is a procedure that is performed frequently for experiments involving polymerase chain reaction (PCR), Southern blotting and other DNA analysis techniques. We have investigated several parameters affecting DNA yield and quality, using a simple chemical‐based purification procedure that was modelled on alkaline lysis methods developed for bacterial cells. The three major steps of the procedure, cell lysis, protein removal and DNA precipitation, were optimized by testing the impacts of several chemicals, including sodium dodecyl sulphate (SDS), sodium hydroxide, Tris buffer, sodium acetate and potassium acetate. Other parameters, such as the effect of elevated temperatures on cell lysis, were also investigated. A rapid, optimized protocol was derived for the purification of DNA from small cell cultures that can be readily digested with restriction enzymes and used as a template for PCR. Average yield was calculated to be approximately 1.7 µg DNA/10⁸ cells, which is similar to the theoretical maximum amount obtainable from haploid yeast cells. Copyright © 2011 John Wiley & Sons, Ltd.     

Amplification Facilitators and Pre‐Processing Methods for PCR Detection of Strictly Anaerobic Beer‐Spoilage Bacteria of the Class Clostridia in Brewery Samples

The aim of this study was to evaluate easy pre‐PCR processing procedures to allow rapid and reliable detection of strictly anaerobic beer‐spoilage bacteria throughout the brewing process by end‐point and real‐time PCR techniques. The efficiencies of the new procedures were evaluated using spiked brewery samples and specific PCRs for the target group bacteria. We found for the first time that the inclusion of 0.25% (w/v) bovine serum albumin (BSA) or 0.5% (w/v) polyvinyl pyrrolidone (PVP) in the end‐point PCR mixture reduces the inhibiting effect of brewery sample extracts (3–10%, v/v) on PCR. Membrane filtration with a PVP or a sodium tri‐polyphosphate‐EDTA wash, and cross‐flow filtration were the most promising new methods to reduce inhibitors from beer samples before cell lysis. Together with BSA, they allowed the analysis of 10% (v/v) of crude extracts instead of <3% (v/v). Moreover, we developed a one‐hour procedure to prepare target DNA from process samples containing brewer's yeast. It involved removal of inhibitors by a two‐step centrifugation followed by physical disruption of cells. The detection limit of the procedure was 10¹‐10³ CFU/mL. The developed procedures help to reduce the risk of partial or complete PCR failure due to inhibition and target DNA losses, with minimal sample handling.     

Rapid protein extraction from Saccharomyces cerevisiae

We have developed and evaluated an easy and rapid method for extraction of proteins from yeast cells for sodium dodecyl sulphate (SDS)–gel electrophoresis and Western blotting. The procedure comprises a centrifugation step to harvest the cells, addition of a sample buffer and heating, then another centrifugation step before applying the extracted proteins found in the supernatant to an SDS gel. It is applicable to the study of large numbers of samples in 1 day. This procedure is easier, quicker, and as efficient as procedures using base and 2-mercaptoethanol, but somewhat less efficient than lysis with glass beads under certain conditions.     

Immunogold labelling to localize polyphenol oxidase (PPO) during wilting of red clover leaf tissue and the effect of removing cellular matrices on PPO protection of glycerol‐based lipid in the rumen

BACKGROUND: The enzyme polyphenol oxidase (PPO) reduces the extent of proteolysis and lipolysis within red clover fed to ruminants. PPO catalyses the conversion of phenols to quinones, which can react with nucleophilic cellular constituents (e.g. proteins) forming protein–phenol complexes that may reduce protein solubility, bioavailability to rumen microbes and deactivate plant enzymes. In this study, we localized PPO in red clover leaf tissue by immunogold labelling and investigated whether red clover lipid was protected in the absence of PPO‐induced protein–phenol complexes and plant enzymes (lipases). RESULTS: PPO protein was detected to a greater extent (P < 0.001) within the chloroplasts of mesophyll cells in stressed (cut/crushed and wilted for 1 h) than freshly cut leaves for both palisade (61.6 and 25.6 Au label per chloroplast, respectively) and spongy mesophyll cells (94.5 and 40.6 Au label per chloroplast, respectively). Hydrolysis of lipid and C18 polyunsaturated fatty acid biohydrogenation during in vitro batch culture was lower (P < 0.05) for wild‐type red clover than for red clover with PPO expression reduced to undetectable levels but only when cellular matrices containing protein–phenol complexes were present. CONCLUSION: Damaging of the leaves resulted in over a doubling of PPO detected within mesophyll cells, potentially as a consequence of conversion of the enzyme from latent to active form. PPO reduction of microbial lipolysis was apparent in macerated red clover tissue but not in the absence of the proteinaceous cellular matrix, suggesting that the PPO mechanism for reducing lipolysis may be primarily through the entrapment of lipid within protein–phenol complexes. Copyright © 2009 Society of Chemical Industry     

Chlorella viruses as a source of novel enzymes

A special advantage has been conferred upon Chlorella cells as tools in biotechnology when viruses (Phycodnaviridae) infecting Chlorella cells were discovered and isolated. The viruses are large icosahedral particles (150-200 nm in diameter), containing a giant, 330-380 kbp long, linear dsDNA genome. Recently, the nucleotide sequence of the 330,740-bp genome of PBCV-1, the prototype virus of Phycodnaviridae, was determined, and up to 702 open reading frames (ORFs) were identified along the genome. The possible genes present include those encoding a variety of enzymes involved in the modification of DNA, RNA, protein and polysaccharides as well as those involved in the metabolism of sugars, amino acids, lipids, nucleotides and nucleosides. Many of these genes are actually expressed during viral infection, with functional enzymes detected in the host cytoplasm or incorporated into the virion. The successful utilization of these viral enzymes as various DNA restriction and modification enzymes (Cvi enzymes) that are now commercially available is well documented. Also noteworthy are virion-associated chitinase and chitosanase activities that have potentially important applications in the recycling of natural resources. The virions of Chlorella viruses contain more than 50 different structural proteins, ranging in size from 10 to 200 kDa. Some of these proteins may be replaced with useful foreign proteins using recombinant DNA technology. The proteins of interest can be recovered easily from the viral particles, and collected by centrifugation after complete lysis of the host Chlorella cells.     

The cytotoxic effect of Bowman–Birk isoinhibitors,IBB1 and IBBD2, from soybean (Glycine max) on HT29 human colorectal cancer cells is related to their intrinsic ability to inhibit serine proteases

Bowman–Birk inhibitors (BBI) from soybean and related proteins are naturally occurring protease inhibitors with potential health‐promoting properties within the gastrointestinal tract. In this work, we have investigated the effects of soybean BBI proteins on HT29 colon adenocarcinoma cells, compared with non‐malignant colonic fibroblast CCD‐18Co cells. Two major soybean isoinhibitors, IBB1 and IBBD2, showing considerable amino acid sequence divergence within their inhibitory domains, were purified in order to examine their functional properties, including their individual effects on the proliferation of HT29 colon cancer cells. IBB1 inhibited both trypsin and chymotrypsin whereas IBBD2 inhibited trypsin only. Despite showing significant differences in their enzyme inhibitory properties, the median inhibitory concentration values determined for IBB1 and IBBD2 on HT29 cell growth were not significantly different (39.9±2.3 and 48.3±3.5 μM, respectively). The cell cycle distribution pattern of HT29 colon cancer cells was affected by BBI treatment in a dose‐dependent manner, with cells becoming blocked in the G0–G1 phase. Chemically inactive soybean BBI had a weak but non‐significant effect on the proliferation of HT29 cells. The anti‐proliferative properties of BBI isoinhibitors from soybean reveal that both trypsin‐ and chymotrypsin‐like proteases involved in carcinogenesis should be considered as potential targets of BBI‐like proteins.     

A Rapid Method for the Recovery,Quantification and Electrophoretic Analysis of Proteins from Beer

A previously‐developed method for protein recovery from wine has been applied to beer and beer foam samples. The method involves the complexation of proteins with dodecyl sulfate (added as sodium salts) and subsequently the insolubilization of the protein‐detergent complexes by addition of potassium ions (added as KCl). The protocol allows preparation of proteins from a few hundred microliters of beverage in a few minutes. The precipitated proteins are free from interfering materials and are directly utilizable for quantitative and electrophoretic assays.     

In Vitro and Mouse Evaluation of Methods for Protecting Whey Protein and Casein from Ruminal Degradation

A series of in vitro buffer (protein solubility) and rumen fermentation (ammonia production) studies were conducted to evaluate varying formaldehyde (.25, .5, 1, and 3%) and tannic acid (.5, 1, 2, 3, and 6%), and three heating times (1, 2, and 3 h at 104 C) on protection of whey protein concentrate and casein from ruminal degradation. Subsequent trials of mouse growth were conducted to evaluate the effectiveness of protection of treated proteins. Formaldehyde (.25 to 3%) reduced solubility of whey protein and casein to less than 10% of the untreated in pH 6.8 buffer and approximately 30% of the untreated in pH 2.5 buffer plus pepsin. Formaldehyde reduced ammonia production, indicating protection and reduced solubility of whey protein and casein under rumen conditions in vitro. Tannic acid (.5 to 6%) did not greatly reduce protein solubility and ammonia production. Heat treatment reduced protein solubility to less than 10% of the untreated in pH 6.8 buffer and to 50% of the untreated in pH 2.5 buffer and reduced ammonia production to approximately 20% of the untreated. Growth and feed intake of mice were decreased with 1% formaldehyde treated diets (17% protein), indicating overprotection of protein. Growth decreased as tannic acid increased. Mouse growth also decreased as the length of heat treatment increased. Diets with .5% formaldehydecasein depressed gains. Based on these in vitro and mouse-evaluation studies, formaldehyde less than .5% of the dry weight is required to protect whey protein and casein from ruminal degradation and permit solubilization in the lower intestinal tract.     

Prion‐associated proteins in yeast: comparative analysis of isogenic [PSI+] and [psi−] strains

A large group of prion‐associated proteins was identified in yeast cells using a new approach, comparative analysis of pellet proteins of crude cell lysates in isogenic strains of Saccharomyces cerevisiae differing by their prion composition. Two‐dimensional (2D) electrophoresis followed by MALDI analysis of the pellet proteins of [PSI⁺] and [psi^?] strains after prion elimination by GuHCl and prion transmission by cytoduction permitted identification of ca. 40 proteins whose aggregation state correlated with the change of prion(s) content. Approximately half of these proteins belonged to chaperones and to enzymes of glucose metabolism. Chaperones are known to be involved in prion metabolism and are expected to be present in prion‐containing aggregates, but glucose metabolism enzymes are not predicted to be present. Nevertheless, several recent data suggest that their presence is not incidental. We detected six proteins involved in oxidative stress response and eight in translation. Also notable is a protease. Most of the identified proteins seem to be prion‐associated, but we cannot exclude the possibility that several proteins may propagate as prions. Copyright © 2009 John Wiley & Sons, Ltd.     

In vitro Digestibility of Whey Protein/K-Casein Complexes Isolated from Heated Concentrated Milk

The disulfide-linked complex of _K-casein and whey proteins that forms when concentrated milk is heated was isolated by centrifugation and column chromatography on Sephacryl S-1000. The rate of hydrolysis of β-lactoglobulin and _K-casein in the complex and the reduced and carboxymethylated components of the complex were measured by polyacrylamide gel electrophoresis. The rates of hydrolysis at pH 2.0 (pepsin) and pH 8.0 (trypsin and chymotrypsin) were similar for _k-casein in the complex and its reduced form. β-Lactoglobulin hydrolysis was faster for the reduced complex than for the complex which was much faster than for the native protein for all three enzymes. The results suggest that heating milk increases the digestibility of whey proteins, despite the formation of large protein complexes between the whey proteins and _K-casein.     

提高双歧杆菌在离心过程中活菌收得率的研究

菌体细胞的分离技术是制备双歧杆菌冻干制剂重要的中间工艺环节。本文建立了研究离心过程国活菌收得率与损失率、存活率相关性试验方法。选择改良ＭＲＳ培养基为离心基质研究了离心力、离心时间、基质ＰＨ对两歧双歧杆菌（Ｂ．ｂｉｆｉｄｕｍ）离心损失率和存活率的影响,确定了活菌收得率最高的适宜离心条件。结果表明：增大离心力和延长离心时间,离心损失率和存活率均显著降低（Ｐ〈０．０１）;影响离心存活率的阈值为４２９７ｇ     

Possible Mechanism of Co‐Flocculation Between Non‐Flocculent Yeasts†

Co‐flocculation between cells of S. cerevisiae NCYC 234 and NCYC 1109, both of which were non‐flocculent when cultivated in YM medium for 20 h, was investigated by chemical modification. Ca²⁺ promoted co‐flocculation. Protein‐denaturants and several carbohydrates caused reversible inhibition of the co‐flocculation in the presence of Ca²⁺. The effect of treatment with proteolytic enzymes and chemical modification of cell surface protein and carbohydrate components suggest strongly that co‐flocculation between cells of NCYC 234 and cells of NCYC 1109 results from interaction between surface protein component of cells of NCYC 1109 and surface carbohydrate component of cells of NCYC 234.     

An improved method for whole protein extraction from yeast Saccharomyces cerevisiae

A new method for protein extraction from yeast Saccharomyces cerevisiae cells is described. This method involves the use of LiAc and NaOH to enhance the permeability of yeast cell wall prior to protein extraction with SDS-PAGE sample buffer. It was safe and efficient compared to other methods reported so far in the literature. The proteins extracted with this new method retained their immunoreactive properties and are suitable for most applications in molecular biology studies.     

Effects of high hydrostatic pressure on membrane proteins of Salmonella typhimurium

Salmonella typhimurium is a leading cause of foodborne diseases. Today high hydrostatic pressure treatments are considered as alternative methods of preservation. To select optimal conditions of treatment, we have to characterize the cell targets of pressure. In this study the action of pressure on the bacterial membrane proteins is analysed. The total membrane extract is obtained by lysis of cells separated by equilibrium density gradient centrifugation. Protein content is analysed by electrophoresis SDS-PAGE and visualised by silver stain. Electrophoretic profiles reveal the presence of three major outer membrane proteins and 12 minor proteins in control bacteria outer membranes. Outer membrane protein content is drastically modified after treatments. In some cases, except for the major proteins OmpA and LamB, other outer membrane proteins seem to totally disappear. LamB is more resistant to hyperbaric exposure when the pH of the media is acidic. This behaviour could be explained by a different conformation adopted by the LamB protein depending on the extracellular pH. This work allows us to define membrane proteins as a target of high hydrostatic pressure treatments. Knowledge of the behaviour of these bacterial membrane proteins subjected to pressure under different conditions (pH, temperature, a(w)...) could allow an increase in the efficiency of treatments.     

Evaluation of different protein sources for aquafeeds by an optimised pH‐stat system

This paper presents the results of some experiments oriented to optimise and standardise the measurement of protein hydrolysis by fish proteases using the pH‐stat technique. Various factors affecting the degree of hydrolysis (DH) were considered (autohydrolysis of the protein sources, type of enzymes utilised, substrate/enzyme ratio and effect of inhibitors). The crude extracts obtained from fish digestive tissues showed their suitability for the determination of protein hydrolysis, rendering better results than those obtained using mixtures of commercial enzymes. DH values for a given protein were greatly affected by the substrate/enzyme ratio, since small modifications in protein concentration resulted in significant variations in DH. Protease inhibitors present in various plant protein sources produced a reduction in DH values. © 2002 Society of Chemical Industry     

A method for determining beta-galactosidase activity of yogurt cultures in skim milk

A method was developed for determining the specific activity of bacterial beta-galactosidase (EC 3.2.1.23) during growth of Streptococcus thermophilus and Lactobacillus bulgaricus in skim milk. Individual and mixed strain cultures of S. thermophilus (St 3642, St14485) and L. bulgaricus (Lb11842, Lb880) were examined for growth (OD at 600 nm and viable cell counts), acid production, and beta-galactosidase activity (expressed as a function of recoverable TCA-precipitable cellular protein). Cultures were inoculated into 10% skim milk (2% inoculum) and incubated at 40 degrees C for 12 h. Aliquots were removed at 2-h intervals and diluted with ice cold EDTA, pH 12. The EDTA chelates calcium and solubilizes milk protein, allowing separation of the bacteria by centrifugation. Cells were then washed twice with 20 mM phosphate buffer and disrupted by sonication. Cell debris and intact cells were removed by centrifugation and the cell-free extract evaluated for beta-galactosidase activity using o-nitrophenyl-beta-D-galactopyranoside as substrate. Specific activities ranged from 0 to 6 units/mg protein. This simple and reproducible method is applicable for enzyme assays and measurement of cellular components where contamination by milk proteins is a potential problem.     

Differences in properties of myofibrillar proteins from bovine semitendinosus muscle after hydrostatic pressure or heat treatment

Hydrostatic pressure (HP) and heat treatments of myofibrillar proteins have both been shown to induce protein denaturation, but different gel formation properties result from these treatments. To characterise differences in the properties of proteins resulting from HP or heat treatment, Ca‐ and Mg‐ATPase activities (ATP, adenosine triphosphate) and protein solubility in 0.1 and 0.6 mol L^?1 KCl buffers (pH 7) were evaluated in this study. The inactivation rate of Ca‐ATPase of myofibrillar proteins (Mf) induced by HP was slower than that of Mg‐ATPase at each of the tested pressures. However, the inactivation rate of Ca‐ATPase induced by heating was faster than that of Mg‐ATPase at each of the tested temperatures. The level of soluble proteins in Mf suspension induced by HP in 0.1 mol L^?1 KCl buffer increased with increasing pressure up to 400 MPa and then decreased slightly at 500 MPa. However, the level of soluble proteins in Mf suspension induced by heat treatment in 0.1 mol L^?1 KCl buffer increased with increasing temperature up to 55°C. According to the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis, the levels of soluble myosin heavy chain and actin in Mf suspension induced by HP in 0.6 mol L^?1 KCl buffer decreased simultaneously at pressures higher than 300 MPa. The level of soluble MHC in 0.6 mol L^?1 KCl buffer decreased gradually with increasing temperature, but there were no changes in the level of soluble actin in 0.6 mol L^?1 KCl buffer with increasing temperature up to 50°C. These results showed that the mechanism of HP‐induced protein denaturation was different from the mechanism underlying heat‐induced protein denaturation. Copyright © 2006 Society of Chemical Industry     

Effect of Bacillus subtilis spo0A mutation on cell wall lytic enzymes and extracellular proteases, and prevention of cell lysis

The Bacillus subtilis spo0A mutant is an adequate host for extracellular protein production (e.g., alpha-amylase). However the mutant was prone to cell lysis. SDS-PAGE and zymography of cell wall lytic proteins indicated that the spo0A mutant contained high amounts of two major autolysins (LytC [CwlB] and LytD [CwlG]) and two minor cell wall lytic enzymes (LytE [CwlF] and LytF [CwlE]). On the other hand, the expression of eight extracellular protease genes was very poor or absent in the spo0A mutant. An eight-extracellular-protease-deficient mutant (Dpr8 strain) was constructed and the strain also exhibited cell lysis. The autolysins from the spo0A mutant were degraded by the supernatant of the wild type but not degraded by that of the Dpr8 mutant. These results suggest that the extensive cell lysis of the spo0A mutant was partially caused by the stability of autolysins via the decrease of the extracellular proteases. The introduction of a major autolysin and/or SigD mutations into the spo0A mutant was effective for preventing cell lysis.