Affinity chromatography of bacterial malate dehydrogenases

A rapid method for the quantitative purification of bacterial malate dehydrogenases (EC 1.1.1.37) has been developed. These enzymes adsorb weakly at low ionic strength to either 5′-AMP or Cibacron blue F3GA agarose derivatives. Sequential elution from these columns first with KC1 then NAD results in complete purification of enzymes fromEscherichia coli andSalmonella typhimurium and nearly complete purification from three other bacteria tried. All the enzymes with exception of aCitrobacter enzyme were immunologically cross-reactive.     

High-level expression and one-step purification of cyclic amidohydrolase family enzymes

The cyclic amidohydrolase family enzymes, including hydantoinase, dihydropyrimidinase, allantoinase and dihydroorotase, are metal-dependent hydrolases and play a crucial role in the metabolism of purine and pyrimidine in prokaryotic and eukaryotic cells. With the increasing demand for the elucidation of enzyme structures and functions, along with industrial applications, the research on the family enzymes has recently been proliferating, but the related enzymes had been purified conventionally by multistep purification procedures. Here, we reported the expression in Escherichia coli cells of maltose-binding protein-fused family enzymes and their one-step purification. The expression levels of the fusion proteins account for 20-35% of the total protein in E. coli, allowing approximately 2-3 mg of the purified proteins by affinity chromatography to be obtained per 0.3 L of bacterial culture. As more promising results, their nascent biochemical properties, after the cleavage of the fusion proteins with Factor Xa, in terms of oligomeric structure, optimal pH, specific activity, and kinetic property, were also conserved as those from the native enzymes. The availability of the family enzymes to fusion strategy shows potential as a convenient procedure to recombinant protein purification and accelerates the structure-function study of the related family enzymes.     

Simultaneous preparation from human placenta of several enzymes of glucose metabolism

A procedure for the simultaneous purification to homogeneity of hexokinase, phosphoglucomutase 1 and 2, aldolase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase from human origin has been developed. Human placenta homogenate was first chromatographed on DE-52 column which retains hexokinase and glucose-6-phosphate dehydrogenase while the other enzymes are recovered in the unabsorbed protein fraction. The other steps in the purification involve Matrex gel and specific affinity chromatography for the DE-52 retained enzymes and phosphocellulose and Matrex gel chromatography for the other enzymes. All the enzymes mentioned were obtained in one week, with recoveries from 14 percent for glucose-6-phosphate dehydrogenase to 75 percent for hexokinase. Thus, the procedures utilized seem to be useful in obtaining large amounts of enzymes in a a homogeneous form from an easily available human tissue.     

Specifically increased solubility of enzymes in polyethyleneglycol solutions using polymer-bound triazine dyes

The enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 3-phosphoglycerate kinase (EC 2.7.2.3), present in an extract of Bakers' yeast, are largely kept in solution by minor amounts of polyethylene glycol-bound triazine dyes (Procion yellow HE-3G and Procion olive MX-3G) even when the solution contains such concentrations of polyethylene glycol (12.5% w/w) which normally precipitate the enzymes. The specific prevention from precipitation can be used for purification of enzyme, preferentially in dealing with crude extracts, which has been demonstrated in this work. A 3.4-fold purification of glucose-6-phosphate dehydrogenase has been achieved with good recovery (93%). Further purification has been possible by combining the recovered (enzyme-containing) supernatant liquid with a solution of dextran which generates an aqueous two-phase system. The lower, dextran-containing phase extracts part of the remaining bulk proteins leaving the target enzyme in the upper phase. The advantages of this method for enzyme purification in large scale are discussed.     

果胶酶分离纯化及分析方法的研究进展

果胶酶能降解果胶质,在果汁制造、果酒酿造等方面有着广泛应用。果胶酶分子生物学的迅速发展极大地促进了分离纯化与分析方法的研究。由于不同菌种产生的果胶酶成分复杂程度不同,分离纯化手段和分析方法也不相同。本文对果胶酶分离纯化手段及其分析方法进行了综述。     

An improved method for single step purification of metagenomic DNA

An improved method for purification of intact metagenomic DNA from soil has been developed using Q-Sepharose, which purified the DNA from phenolic and humic acid contaminants in a single step. The entire procedure for purification took only 45 min. A total of 81% of DNA was recovered after purification and there was 84% reduction in humic acid contents. The purified DNA was readily digested with restriction enzymes and can be further used for molecular applications.     

Chromatography of hydroxysteroid dehydrogenases

The structure-function study of hydroxysteroid dehydrogenases has stimulated the development of their chromatography, which in turn reveals more mechanisms of these enzumes. Due to the various membrane associations and mild hydrophobic nature of most of the enzymes studied up to now, hydrophobic interaction chromatography has played a crucial role in their purification, using media such as phenyl-Superose or Sepharose-PEG. At the same time, affinity chromatography, especially the dye-containing columns, proves very efficient for these dehydrogenases, as the latter utilizes adenylyl-containing cofactors. Elution by their specific ligand facilitates their purification. In this paper, the use of detergents in the purification of these enzymes is also reviewed. Hydroxysteroid dehydrogenase preparation is further improved by rapid purification which facilitates the elimination of protein microheterogeneity, caused in vitro by oxidation, reduction or partial proteolysis. This process was shown to increase the crystallizability of the enzymes [Lin et al., J. Cryst. Growth, 122 (1992) 242–245; Zhu et al., J. Mol. Biol., 234 (1993) 242–244]. The fast purification permitted a simpler procedure and better combination of various columns than conventional chromatography. This leads to even higher efficiency, yielding homogeneous and highly active preparations.     

A new look at xylanases: an overview of purification strategies

Interest in xylanases from different sources has increased markedly in the past decade, in part because of the application of these enzymes in the pulp and paper industry. Purity and purification costs are becoming important issues in modern biotechnology as the industry matures and competitive products reach the marketplace. Thus, new paths for successful and efficient xylanase recovery have to be followed. This article reviews the isolation and purification methods used for the recovery of microbial xylanases. Origins and applications of xylanases are described, highlighting the special features of this class of enzymes, such as the carbohydrate-binding domains (CBDs) and their importance in the development of affinity methodologies to increase and facilitate xylanase purification. Implications of recombinant DNA technology for the isolation and purification of xylanases are evaluated. Several purification procedures are analyzed, taking into consideration the sequence of the methods used in each and the number of times each method is used. New directions to improve xylanase separation and purification from fermentation media are described.     

Preparative affinity electrophoresis: application to human erythrocyte carbonic anhydrase

A modification of affinity electrophoresis for preparative purposes is described. This method has been applied to the purification of human erythrocyte carbonic anhydrases B and C. During conventional affinity chromatography some hemoglobin contamination occurs. By introduction of an electrophoretic purification step after the immobilization of carbonic anhydrase to the affinity gel, the hemoglobin impurity is reduced about eight and two times in the preparations of the B and C enzymes, respectively, compared to the enzymes purified by affinity chromatography.     

Biotin-ubiquitin tagging of mammalian proteins in Escherichia coli

Ubiquitin has been used in protein expression for enhancing yields and biological activities of recombinant proteins. Biotin binds tightly and specifically to avidin and has been widely utilized as a tag for protein purification and monitoring. Here, we report a versatile system that takes the advantages of both biotin and ubiquitin for protein expression, purification, and monitoring. The tripartite system contained coding sequences for a leader biotinylation peptide, ubiquitin, and biotin holoenzyme synthetase in two reading frames under the control of T7 promoter. The expression and purification of several large mammalian enzymes as biotin-ubiquitin fusions were accomplished including human ubiquitin activating enzyme, SUMO activating enzymes, and aspartyl-tRNA synthetase. Expressed proteins were purified by one-step affinity column chromatography on monomeric avidin columns and purified proteins exhibited active function. Additionally, the ubiquitin protein hydrolase UBP41, expressed and purified as biotin-UBP41, efficiently and specifically cleaved off the biotin-ubiquitin tag from biotin-ubiquitin fusions to produce unmodified proteins. The present expression system should be useful for the expression, purification, and functional characterization of mammalian proteins and the construction of protein microarrays.     

Sucrose metabolism in green algae. I. The presence of sucrose synthetase and sucrose phosphate synthetase.

The presence of sucrose synthetase and sucrose phosphate synthetase has been demonstrated in two species of green algae: Chlorella vulgaris and Scenedesmus obliquus. Partial purification from crude extracts allowed the determination of the kinetic constants of algae enzymes. They are very similar to the ones reported for enzymes from higher plants.     

The binding of aminoacyl-tRNA synthetases to triazine dye conjugates.

The binding of thirteen aminoacyl-tRNA synthetases to thirty two immobilised procion dyes has been investigated. Most dyes bind one or more enzymes. The amino acid substrates are not normally potent eluants, with the notable exception of tryptophan eluting tryptophanyl-tRNA synthetase from Brown MX-5BR. Phosphate is frequently extremely effective, much more than expected by simple considerations of ionic strength, indicating that many of the dyes are able to mimic the phosphate groups of the phosphodiester backbone of the nucleic acid. Procedures for the purification of methionyl-, tryptophanyl- and tyrosyl-tRNA synthetases are presented and compared to the conventional purifications of these enzymes. The results indicate the general applicability of these dye columns to the purification of most enzymes of of nucleic acid metabolism and the necessity of investigating as many different dyes as possible for any individual enzyme.     

Immunoaffinity chromatography of enzymes.

Immunoaffinity chromatography of enzymes represents an attractive purification technique suitable for one-step and large-scale purification of enzymes to homogeneity. Monoclonal and polyclonal antibodies can be used equally well. The broad use of the technique is restricted by the harsh elution conditions which are often required. The efforts to overcome these limitations and to optimize the method are reviewed, viz. proenzyme purification, purification of enzymes as part of multienzyme complexes carried out by a mild dissociation step, specific elution by substrates and effectors, enzyme stabilization, electrophoretical desorption and negative elution by adsorbing impurities from the crude extract, and hypotonic elution. The current practice is discussed considering antibody and enzyme selection, optimization of elution conditions, and washing steps using different media. Representative examples are given for various approaches.     

Isolation and some properties of homogenous nuclease S1 from alpha-amyloryzin

A purification method for isolating homogeneous single-strand specific nuclease S1 from alpha-amyloryzin has been developed. The yield was about 16% and purification factor--9000. Nuclease S1 thus obtained was proved to be free of contaminations of any others nucleolytic enzymes. It is shown for the first time that ribo- and deoxy-dinucleosidemonophosphates are hydrolyzed by nuclease S1 to form 5'-nucleotides with pH optimum for ApA equal to 4.6.     

Separation and characterization of four enzyme forms of beta-galactosidase from Saccharomyces lactis

beta-Galactosidase from Saccharomyces lactis has been purified to serve as a model for the kinetic behavior of human lactase in adult lactase deficiency. Enzymes from both species are neutral and follow Michaelis-Menten kinetics. beta-Galactosidase of S. lactis is more readily available than the human lactase. An enzyme preparation from S. lactis (Maxilact 40,000), which is used commercially to hydrolyze lactose in milk, has been found to contain four isozymes of beta-galactosidase. Methods have been developed for the separation and purification of each of the four enzymes. The enzymes were found to differ in molecular mass, kinetic behavior, isoelectric point, response to pH, specific volume and sensitivity to metal ions. The four enzymes had apparent molecular masses of 630 kDa, 550 kDa, 41 kDa and 19 kDa. Their specificity constants (kcat/Km) were found to be 42.0, 355.2, 0.38 and 0.48 mM-1 s-1, respectively. The techniques of reiterated ultrafiltration used for the isolation of these isozymes may be applicable to other purification processes.     

Group-specific adsorbents linked with dyes - structural analogs of coenzymes used for isolation and purification of enzymes: a review

The paper reviews the data in the literature related to the use of group-specific adsorbents linked with dyes-Cibacron blue F3GA, blue dextran and procion red HE-3B-for the study, isolation and purification of coenzyme-dependent enzymes. Particular attention is given to the nature and specificity of interactions between these dyes and sites of coenzyme binding in dehydrogenases, kinases and enzymes involved in nucleic acid metabolism. Recent findings on the practical application of such adsorbents for the isolation and purification of coenzyme-dependent enzymes are presented.     

Expression, purification and characterization of cloning-grade zinc finger nuclease

The limited number of naturally occurring rare-cutting restriction enzymes and the slow and tedious engineering of existing restriction enzymes for novel specificities have prompted the design of new strategies for the development of restriction enzymes with specificities for long DNA sequences. One possibility is using zinc finger nucleases (ZFNs)—synthetic restriction enzymes that are custom-designed to target and cleave long DNA sequences and which have been recently shown useful for DNA cloning. Here we report on the purification and biochemical analysis of ZFN-10, a custom-made ZFN. We show that Ni-affinity and gel-filtration purification methods are sufficient to produce a cloning-grade enzyme. We show that ZFN-10 can function as an accurate and reliable ZFN using the same reagents and protocols used for naturally occurring and commercially available recombinant restriction enzymes. We also show that ZFN-10 tolerates a set of target-site substitutions which can be predicted from the specificities of recognition helices incorporated into the structure of its DNA-binding domain. The relative simplicity of ZFN-10 design, expression, purification and analysis suggests that novel ZFNs can potentially be designed and applied for various recombinant DNA applications.     

Affinity purification of bacterial luciferase and NAD(P)H:FMN oxidoreductases by FMN-sepharose for analytical applications

A modified purification method for bacterial luciferases and NAD(P)H:FMN oxidoreductases is described which uses FMN-Sepharose alone or coupled to DEAE ion exchange chromatography for the simultaneous purification of luciferase and the various oxidoreductases from Vibrio harveyi, a bright mutant of Vibrio harveyi, Vibrio fischeri, and Photobacterium phosphoreum. This purification method is compared with DEAE-Sepharose CI 6B fractionations from these organisms. Both methods allow the separation of oxidoreductases specific for either NADH or NADPH. The use of FMN-Sepharose coupled to DEAE-Sepharose fractionation allows the isolation of highly purified enzymes. Lacking interfering factors, these are very suitable for various analytical applications based on bacterial bioluminescence enzymes. The partially purified enzymes from the affinity column have higher specific activities than those obtained using DEAE-Sepharose.     

Purification and comparative studies of alcohol dehydrogenases

Alcohol dehydrogenases from various animal and plant sources were purified by a common procedure which employed DEAE, Sephadex-G100 and affinity chromatographies. The procedure achieves an 80-130 fold purification for animal enzymes. However, only a 5-15 fold purification for plant enzymes was attained because of the instability of these enzymes. Purified alcohol dehydrogenases from animal and plant sources differ in coenzyme and substrate specificities. The enzymes from mammalian, avian and fish livers display aldehyde oxidizing and esterolytic activities in addition to alcohol oxidizing activity. However, the enzymes from plants and yeast show only the oxidative activity toward alcohols. Chemical modifications have been performed to identify amino acid residues which are essential to the oxidative and esterolytic activities of alcohol dehydrogenases.     

Expanded bed affinity purification of bacterial α-amylase and cellulase on composite substrate analogue–cellulose matrices

Expanded bed purification of α-amylase and cellulase directly from unclarified fermentation broth was carried out on specially prepared composite affinity matrices. The concept used was incorporation of polymeric substrates/substrate analogue during cross-linking of cellulose to prepare rigid, porous, cross-linked composite affinity matrices for target enzymes. Of the several polymeric substrates/substrate-analogue used, alginic acid (AA) and microcrystalline cellulose (MCC) when used to prepare cross-linked composite matrices with cellulose, resulted in best affinity purification matrices for α-amylase and cellulase, respectively. These matrices were suitable for purification of the enzymes by batch, packed bed as well as expanded bed purification protocols. The optimized expanded bed protocol for α-amylase from Bacillus spp. B3 gave 51-fold purification on AA-CELBEADS with 69% recovery, whereas, cellulase from Bacillus spp. B21 was purified on MCC-CELBEADS to 18-fold purification with 97% recovery. The SDS-PAGE of both purified preparations showed single bands indicating significant purification on composite affinity adsorbents in a single step strategy.