Penicillin acylase purification with the aid of hydrophobic charge induction chromatography

The aim of this work was to test a chromatographic support, 4-mercaptoethyl pyridine (4-MEP) Hypercel, for penicillin acylase purification by using pure penicillin acylase and crude extract. Two equilibration buffers with various salt concentrations and different flow rates were tested. The relationships between electrostatic and hydrophobic interactions and proteins are demonstrated. (NH4)2SO4 proved preferable because no salting-in occurred, contrary to NaCl. The recovery and purification fold were similar to those obtained in pseudo-affinity chromatography with a three-fold reduction of the (NH4)2SO4 concentration.     

Expression of the colH Gene Encoding Clostridium histolyticum Collagenase in Bacillus subtilis and Its Application to Enzyme Purification

The colH gene encoding 116-kDa collagenase of Clostridium histolyticum (cColH) was cloned into an Escherichia coli-Bacillus subtilis shuttle vector to develop a method for purification of recombinant collagenase (rColH). When plasmid pJCM310 containing the colH gene was introduced into B. subtilis DB104 and the transformant was grown in LB broth at 37 C, stability of the plasmid was not maintained. However, stability was partly improved by growing the transformant in a modified LB broth containing 0.5 M sodium succinate with gentle shaking at 35 C. When the transformant was grown to an optical density of 0.4 at 600 nm in this medium, pJCM310 was stable and rColH was produced in sufficient amounts. rColH was purified to homogeneity by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The yield of rColH from an 800-ml culture was 0.53 mg and its specific activity was estimated to be 1,210 U per mg of protein. The purified rColH was capable of degrading native type-I collagen fibril from bovine achilles tendon, as was demonstrated by zymography. A comparison of the N-terminal amino acid sequence between cColH and rColH revealed that rColH has 10 extra N-terminal amino acid residues. However, the peptide mapping of rColH with V8 protease was virtually identical to that of cColH. Furthermore, the molecular mass of rColH was estimated to be 112,999 Da by mass spectrometry, coinciding with the value of 112,977 Da, which was predicted from the nucleotide sequence of the colH gene. Therefore, the recombinant B. subtilis culture is capable of serving as a useful source for enzyme purification.     

Collagenase digestion of bone fragments in microgravity

The effect of a quiescent microgravity fluid environment on the activity of collagenase directed at demineralized bone fragments was investigated over a period of 10 days. Enzyme treatment resulted in greater mass loss in microgravity, with nearly three times the loss of mass during Space Shuttle mission STS-62 compared to the stationary ground control. Clinorotation enhanced the loss of mass relative to a stationary control, but this increase was still significantly less than the increase with exposure to microgravity. This suggests the detrimental influence of turbulence on the enzyme function and the benefit of using microgravity to provide both low turbulence and uniformity of unequally dense materials within the reaction chamber. The results are considered for their general applicability to a variety of bioprocessing applications that may be enhanced in microgravity. (c) 1996 John Wiley & Sons, Inc.     

Collagenase production in an Antarctic strain of Arthrobotrys tortor Jarowaja

This paper describes the results of a comparative screening between the nematophagous Antarctic fungus Arthrobotrys tortor and other species of that genus for the production of extracellular collagenases. The nematode species used in this study was Caenorhabditis elegans, feeding on Escherichia coli cultures. Determination of collagenase activity was made using insoluble collagen from bovine Achilles tendon and determining the amount of solubilized hydroxyproline produced. The results show that the total amount of collagenase produced by the Antarctic strain of A. tortor was about threefold higher than that observed for the other species. In the Antarctic strain, collagenase was shown to be a constitutive enzyme. This revised version was published online in June 2006 with corrections to the Cover Date.     

纯化超氧化物歧化酶的新工艺

为探索简便实用纯化ＳＯＤ的工艺路线,以人或猪血红细胞溶血上清液,经铜胺中空纤维透析器（分子量截留值为１５ｋＤ）透析和超滤,收集分子量大于１５．０ｋＤ的物质,再加热６０℃１０ｍｉｎ,离心取上清即得。Ｃｕ、ＺｎＳＯＤ和ＭｎＳＯＤ分子量分别为３２．０ｋＤ和８０．０ｋＤ。人血和猪血纯化的ＳＯＤ总收率分别为８８．２％和８９．２％,比活性分别为１７４２９Ｕ／ｍｇ和１８２２８Ｕ／ｍｇ。工艺简便实用,适于工业纯化生产。     

A new rapid and simple method for large-scale purification of mycobacterial lipoarabinomannan

Lipoarabinomannan (LAM) is a major and structurally important outer cell wall component of all mycobacteria. LAM is also generally regarded as an important immunomodulating substance affecting several immunologic networks and hence important in the pathogenesis of mycobacterial infections. We here describe a new method for large-scale purification of mycobacterial LAM. A crude cell wall preparation was prepared from batch-grown Mycobacterium tuberculosis H37Rv. From this cell wall preparation LAM was purified by sequential extractions and chromatographic steps. From 20 g dry weight cell wall preparation 313 mg of highly purified (> 98%) LAM was obtained in only 3 days. The LAM content of the final purification step was quantified by ELISA using reference LAM as standard. The identity and purity of the LAM preparation was further confirmed by comparison with reference LAM preparation from M. tuberculosis strain Erdman in polyacrylamide gel electrophoresis and Western blots, using reference anti-LAM monoclonals CS-35 and CS-40.     

Single pot sequential crystallization-distillation as a new purification procedure

A new purification procedure exploiting the simultaneous presence of a solid, liquid, and gas phase in a low surface area system is proposed and discussed. The assumptions of vanishingly low diffusion coefficients in the solid phase and that of the presence of a single “effective impurity” allow to plan the sequence of operations starting from the knowledge of just the melting and boiling points of the substance to be purified and of those of the “effective impurity”. Examples and results are presented.     

A new streptomycete and a new polyene antibiotic,acmycin

A new antifungal antibiotic named acmycin was isolated from a soil streptomycete. Detailed comparative taxonomic studies showed that the organism differed from three related species of streptomycetes. The organism was referred to asStreptomyces sp. AC₂. The isolated antibiotic appears to be of polyene nature.     

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen ¹.Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77±10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.     

Cloning and expression of a β-1,4-endoglucanase gene from Cellulomonas sp. CelB7 in Escherichia coli; purification and characterization of the recombinant enzyme

Abstract A gene library of a newly isolated Cellulomonas sp. strain was constructed in Escherichia coli and clones were screened for endoglucanase activity using dye-labelled carboxymethylcellulose. Seventeen clones were isolated that carried DNA inserts coding for endoglucanase enzymes. Of the 17 clones, one carrying the gene cegA , was further characterized. The recombinant endoglucanase was purified by FPLC. The endoglucanase was active against carboxymethylcellulose, lichenin and also degraded crystalline cellulose and birchwood xylan. The molecular mass of the enzyme (36 kDa), and its pH (7.4) and temperature (35 °C) optima were determined.     

Trypanosoma congolense: isolation and purification.

Yields of Trypanosoma congolense grown in rats may be increased by placing the rats in a 37 °C environment for 1 hr prior to sacrifice. A further increase in the number of parasites recovered per rat may be achieved by replacement of blood removed by a lactated Ringer's solution with 5% glucose as the rat is being bled from the abdominal aorta. The Ringer's solution serves to maintain intravascular volume during the bleeding procedure and thereby prevents premature cardiac arrest. Erythrocytes in infected blood may be then lysed by raising and rapidly lowering the osmolarity of the blood. This permits separation of the trypanosomes from 95% of the erythrocytes by differential centrifugation. The remaining blood cell contamination may then be removed on a small DEAE-cellulose column. The purified trypanosomes are motile, infective, and intact as judged by electron microscopy. More than 10¹⁰ purified T. congolense can be obtained from three adult rats by these methods.     

Characterization and purification of a mycoplasma membrane-derived macrophage-activating factor

A highly hydrophobic component derived from the membrane ofMycoplasma capricolum has been characterized, purified and assessed for its ability to activate macrophages to tumor cytotoxicity. Initially, crude membranes were evaluated for their solubility in a wide range of solvents. Despite differential solubility in the various solvents, the mycoplasma membranes retained their ability to potentiate macrophage tumor cytotoxicity. Mycoplasma membranes were further characterized by appraising their macrophage-activating ability subsequent to various chemical treatments: cleavage of ester and thioester bonds, oxidation of vicinal hydroxyl groups, and exposure to a broad range of pH. Only strong alkaline treatment (pH>12) caused a reduction in mycoplasma membrane activity: all other chemical treatments were inconsequential. With potential therapeutic applications in mind, mycoplasma membranes were subjected to various physical treatments including heating, freezing/thawing, sonication, lyophilization and storage. The ability of the membranes to induce macrophage activation was stably maintained following all these treatments. Purification of membranes was initiated by a chloroform/methanol lipid extraction. Macrophage-activating ability was found predominantly in the interphase. Proteolytic cleavage with trypsin increased specific activity at least sixfold. Trypsinized fractions were solubilized in 2-chloroethanol and gel filtration was performed on a hydroxylated Sephadex LH-60 column. The active fraction from this column had a further tenfold increase in specific activity. Subsequent rounds of reverse-phase HPLC on this fraction yielded three to four peaks absorbing at 280 nm, of which only one had macrophage-activating ability.     

Ornithine carbamoyltransferase of Streptomyces fradiae: purification and properties

Abstract The enzyme ornithine carbamoyltransferase was purified from Streptomyces fradiae . A 1200-fold increase in specific activity was achieved by ammonium sulphate precipitation, DEAE-cellulose and aminohexyl-agarose chromatography and gel filtration. The purified enzyme has a M _r of 87 000. Its isoelectric point is 5.3 as determined by isoelectric focusing. Apparent K _m values at pH 7.7 for ornithine and carbamoyl phosphate are 1.8 and 1.2 mM, respectively.     

Collagenase and proteinase induction in implants of bone matrix

Enzymes capable of digesting collagen and non-collagenous proteins are present in implants of bone matrix. In the early stages of bone morphogenesis, implants produce relatively large amounts of trypsin-labile proteins and have high non-collagenolytic neutral proteinase and low collagenase activities. Enzymatic activity is maximal three weeks after implantation. The results indicate that increased synthesis of non-collagenous proteins and non-collagenolytic proteinases precedes the induction of significant amounts of collagenases. The importance of these findings in bone morphogenesis is discussed.     

Large scale enzymatic synthesis of oligosaccharides and a novel purification process

Herein we report the practical chemo enzymatic synthesis of trisaccharide and derivatives of iGb3 and Gb3, and a novel purification process using immobilized yeast to remove the monosaccharide from the reaction mixture. High purity oligosaccharide compounds were achieved in large scale. This study represents a facile enzymatic synthesis of and novel purification process of oligosaccharide.     

Single-step purification and refolding of recombinant mouse and human myelin oligodendrocyte glycoprotein and induction of EAE in mice

The extracellular domain of human and rat MOG (ED-MOG) induces experimental autoimmune encephalomyelitis (EAE) when injected into susceptible animals. EAE is a T cell-mediated disease of the central nervous system commonly used as an animal model for human multiple sclerosis. Here, we describe a straightforward procedure for the purification and refolding of mouse and human ED-MOG overexpressed in Escherichia coli as inclusion bodies. Following solubilization and purification using Ni-NTA resin chromatography under denaturing conditions, a column-based refolding proceeded in renaturation buffer supplemented with a glutathione redox buffer system. Using this approach up to 33 mg of highly pure soluble proteins was obtained per liter of expression culture. The ability of purified proteins to induce EAE was evaluated in three strains of mice. We believe that the strategy described here would facilitate researchers to carry out encephalitogenic as well as structure-function studies of this autoantigen. Additionally, we show for the first time that mouse ED-MOG induces severe disease in mice.     

Partial purification of extracellular exo-inulinase from Ulocladium atrum

Eight fungal species were cultivated on the Czapek liquid medium and a good starting extracellular and intracellular exo-inulinase were selected. Extracellular inulinase from Ulocladium atrum was prepared in the presence of 1% inulin source and 0.2% sodium nitrate as the best carbon and nitrogen sources. Incubation for the U. atrum was increased till it reached its maximum (36 U/ml) at the sixth day of incubation at 30 °C which was the best temperature for the production of exo-inulinase. Effect of all metal ions inhibited inulase production by U. atrum. Exo-inulinase was purified by using ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose. Three active inulinase forms INI, INII and INIII were resolved, each for DEAE cellulose. The specific activity of INI was 1915 U/mg protein which represented 2.65-fold purification over the crude extract with 42.8% recovery pooling of INI placed on CM cellulose chromatography and INI was resolved into INIa, INIb and INIc. The specific activity of INIa was 2479.2 U/mg protein which represented 3.43-fold purification over the crude extract with 24.2% recovery.     

Polyelectrolyte complexes as a tool for purification of plasmid DNA. Background and development

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Plasmid DNA was selectively precipitated from a clarified alkaline lysate using the polycation poly(N,N'-dimethyldiallylammonium) chloride which formed insoluble polyelectrolyte complex (PEC) with the plasmid DNA. Soluble PECs of DNA with polycations have earlier been used for cell transformation, but now the focus has been on insoluble PECs. Both DNA and RNA form stable PECs with synthetic polycations. However, it was possible to find a range of salt concentration where plasmid DNA was quantitatively precipitated whereas RNA remained in solution. The precipitated plasmid DNA was resolubilised at high salt concentration and the polycation was removed by gel-filtration.     

Lactulose: production, purification and potential applications

Lactulose a “bifidus factor” is composed of galactose and fructose, which can be produced by the isomerization of lactose. It is a prebiotic carbohydrate which stimulates the growth of health-promoting bacteria in the gastrointestinal tract, such as bifidobacteria and lactobacilli and at the same time inhibits growth of pathogenic bacteria such as Salmonella. It can also be used for the treatment of constipation, hepatic encephalopathy, tumour prevention, and to maintain blood glucose and insulin level. This review provides comprehensive information on the different techniques used for the production of lactulose, purification and analysis. Besides this mechanism of action and its potential applications in food and pharmaceutical industries have also been discussed.     

A simple method for the purification of myxobacteria

Pure cultures of many myxobacteria could be quickly obtained by treating fruiting bodies with highly dosed mixtures of antibiotics