PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “enzyme engineering”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 991 records · Page 55Linked to original sources
PubMed1999

Crosslinking of enzymes for improved stability and performance.

Stabilization of enzymes without diminishing and diluting their activity is crucial for their use as industrial catalysts. One practically proven approach involves crystallization of the enzyme (size 1-200 microm) followed by subsequent crosslinking. In many cases, the resulting crosslinked enzyme crystals exhibit increased storage stability as well as stability in applications. The technology is complementary to protein engineering methods that aim at boosting the inherent stability of the enzyme.

Biotechnology↗
PubMed2002

Genetically engineered GABA-producing cells demonstrate anticonvulsant effects and long-term transgene expression when transplanted into the central piriform cortex of rats.

Local application of GABA-potentiating agents can prevent or reduce the development and maintenance of behavioral seizures induced by limbic kindling in rats. Microinjection and lesion studies suggest that the transition zone between anterior and posterior piriform cortex (PC), termed here central PC, is a potential target for transplantation of GABA-producing cells. In the present study, we transplanted conditionally immortalized mouse cortical neurons, engineered with the GABA-synthesizing enzyme GAD(65), to the central PC of rats. Suspensions of 1.5 x 10(5) cells in 1 microl were transplanted bilaterally. Control animals received transplantation of beta-galactosidase (beta-gal)-expressing cells. All rats were subsequently kindled through a chronically implanted electrode placed in the basolateral amygdala. The pre- and postkindling threshold currents for eliciting behavioral seizures were determined before and after kindling. We found the prekindling partial seizure threshold to be significantly increased by about 200% in the rats that received the GABA-producing cells compared to rats receiving beta-gal-producing transplants. After kindling, the seizure threshold tended to be higher by 100% in rats that received GABA-producing cells, although the difference from controls was not statistically significant. GABA-producing transplants had no significant effect on the rate of amygdala kindling, but the latency to the first generalized seizure during kindling was significantly increased in animals receiving GABA-producing cells. The transplanted cells showed long-term GAD(65) expression as verified immunohistologically after termination of the experiments. The findings substantiate and extend previous findings that the central PC is part of the anatomical substrate that facilitates propagation from partial to generalized seizures. The data demonstrate that genetically engineered cells have the potential to raise seizure thresholds when transplanted to the central PC.

Amygdala↗
PubMed2006

Modulation of hypotensive effects of kinins by cathepsin K.

Kinins are pro-inflammatory peptides, which participate in the maintenance of cardiovascular homeostasis, and play a key role in numerous diseases, including lung fibrosis and hypertension. Evidence has been provided recently for the presence of alternative mechanisms of bradykinin generation and/or degradation. Here we showed that cathepsin K may act as a potent kinin-degrading enzyme in bloodstream. Contrary to cathepsin L, cathepsin K attenuates kallikrein-induced decrease of rat blood pressure, and reduces the hypotensive effect of bradykinin in a dose-dependent manner. Moreover, we identified, by engineering the S2 subsite of both recombinant enzymes, two critical residues involved respectively in the kininase activity of cathepsin K, i.e. Tyr67/Leu205, versus kininogenase activity of cathepsin L, i.e. Leu67/Ala205. In conclusion, according to its ability to modulate hypotensive effects of kinins, we propose that cathepsin K is a kininase of biological relevance, in complement of well-documented neutral endopeptidase or angiotensin-converting enzyme.

Animals↗
PubMed1994

Engineering surface charges in a subtilisin: the effects on electrophoretic and ion-exchange behaviour.

The introduction or removal of multiple charged amino acid residues in the subtilisin Savinase by genetic engineering allowed us to modify the electrostatic properties of this enzyme in a systematic way. The effects of these charge changes were investigated experimentally using ion-exchange chromatography and electrophoretic mobility in native gels all under identical conditions. The experiments clearly demonstrated that the overall charge of a given protein is not the only factor determining electrophoretic mobility at low or moderate ionic strengths. For a series of variants having identical overall positive charge a linear relation was observed between mobility towards the cathode and the total number of charged residues present. This effect was found to depend on the type of (chloride) salt used: calcium ions give rise to complete screening of all negative charges, whereas only partial screening is found for magnesium and sodium ions. In contrast, in the presence of sodium phosphate the overall charge of the enzyme becomes slightly negative. These data indicate that cations as well as anions may strongly perturb the overall charge of proteins depending on the type of salt and on the number of charged amino acid residues present. The ion-exchange behaviour demonstrated similar results, i.e. showing stronger enzyme adsorption with increasing numbers of surface charges on a cation-exchange column run below the isoelectric point of the proteins. However, the apparent sign reversal noted above for electrophoresis with sodium phosphate did not appear in the ion-exchange experiments. This work provides further insight into the adsorption of proteins to surface and the role played by small ions, particularly when electrostatic forces dominate the adsorption process.

Amino Acid Sequence↗
PubMed2006

Engineering of cyclodextrin glucanotransferase on the cell surface of Saccharomyces cerevisiae for improved cyclodextrin production.

The cyclodextrin glucanotransferase (CGTase) gene (cgt) from Bacillus circulans 251 was cloned into plasmid pYD1, which allowed regulated expression, secretion, and detection. The expression of CGTase with a-agglutinin at the N-terminal end on the extracellular surface of Saccharomyces cerevisiae was confirmed by immunofluorescence microscopy. This surface-anchored CGTase gave the yeast the ability to directly utilize starch as a sole carbon source and the ability to produce the anticipated products, cyclodextrins, as well as glucose and maltose. The resulting glucose and maltose, which are efficient acceptors in the CGTase coupling reaction, could be consumed by yeast fermentation and thus facilitated cyclodextrin production. On the other hand, ethanol produced by the yeast may form a complex with cyclodextrin and shift the equilibrium in favor of cyclodextrin production. The yeast with immobilized CGTase produced 24.07 mg/ml cyclodextrins when it was incubated in yeast medium supplemented with 4% starch.

Biotechnology↗
PubMed1989

Genetically engineered V79 Chinese hamster cell expression of purified cytochrome P-450IIB1 monooxygenase activity.

Chinese hamster V79 fibroblasts, frequently used as target cells in short-term tests for mutagenicity, do not possess measurable monooxygenase activity; in particular, enzymatic oxidation of testosterone (T) cannot be demonstrated. If these V79 cells, however, had been transfected with the cDNA-encoding rat liver cytochrome P-450IIB1 under control of the SV40 early promoter, they stably expressed monooxygenase activity. These so-called SD1 cells then oxidatively metabolized T at a rate of 27 pmol/mg protein/min, converting it to 16 alpha- and 16 beta-hydroxy-T as well as 4-androsten-3,17-dione as sole metabolites in a ratio of 1.1:1.0:1.6. The regio- and stereoselective conversion of T by SD1 cells, as well as the quantitative distribution of the metabolites, corresponds well with the results reported for pure cytochrome P-450IIB1 in a reconstituted system.

Animals↗
PubMed1999

Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP.

RNA transcripts containing the hammerhead ribozyme have been engineered to self-destruct in the presence of specific nucleoside 3',5'-cyclic monophosphate compounds. These RNA molecular switches were created by a new combinatorial strategy termed 'allosteric selection,' which favors the emergence of ribozymes that rapidly self-cleave only when incubated with their corresponding effector compounds. Representative RNAs exhibit 5,000-fold activation upon cGMP or cAMP addition, display precise molecular recognition characteristics, and operate with catalytic rates that match those exhibited by unaltered ribozymes. These findings demonstrate that a vast number of ligand-responsive ribozymes with dynamic structural characteristics can be generated in a massively parallel fashion. Moreover, optimized allosteric ribozymes could serve as highly selective sensors of chemical agents or as unique genetic control elements for the programmed destruction of cellular RNAs.

Acids↗
PubMed1996

Constructing an efficient trans-acting genomic HDV ribozyme.

We have engineered a genomic HDV ribozyme to construct several trans-acting ribozymes for use in trans to cleave target RNAs. Among the 10 different combinations attempted, only HDV88-Trans had cleavage activity on the 13-nucleotide substrate, R13, in vitro. To improve the cleavage efficiency, at least in vitro, of the HDV88-Trans ribozyme (kclv = 0.022 min(-1)), we have constructed several variants that differ in forming stem II (length) in the pseudoknot secondary structure model. When cleavage rate constants were analyzed and compared among variants of HDV88-Trans, HDV88-Trans-4 yielded kclv = 1.7 min(-1). HDV88-Trans-4 thus represents the highest active genomic HDV ribozyme that functions in trans thus far constructed, and has activity under physiological conditions (pH 7.1 at 37 degrees C with 1 mM of MgCl2).

Base Sequence↗
PubMed1994

High density cultivation of a recombinant CD-1 cell line producing prourokinase using a Biosilon microcarrier culture system.

CD-1, a genetically-engineered CHO cell line, was cultivated with a Biosilon microcarrier culture system. We successfully cultivated CD-1 cells to a very high density (over 1 x 10(7) cells/ml). Prourokinase was stably secreted at about 180 IU/10(6) cells/24 h. Experiments showed that CD-1 cells growing on Biosilon microcarriers were able to spontaneously release from the microcarriers, then reattach and proliferate on fresh microcarriers. This makes it very easy to scale up production. The microcarriers could be reused several times without affecting adhesion, proliferation and prourokinase secretion. With CM-PECC membrane radial flow chromatography and MPG chromatography, the prourokinase in conditioned medium could be purified to a specific activity of 1 x 10(5) IU/mg of protein. The purification factor was about 600 fold, and approximately 90% of the biological activity was recovered.

Animals↗
PubMed2001

Production and characterization of recombinant human CLN2 protein for enzyme-replacement therapy in late infantile neuronal ceroid lipofuscinosis.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal recessive childhood disease caused by mutations in the CLN2 gene, which encodes the lysosomal enzyme tripeptidyl peptidase I. As a step towards understanding the protein and developing therapeutics for the disease, we have produced and characterized recombinant human CLN2 (ceroid lipofuscinosis, neuronal 2) protein from Chinese-hamster ovary cells engineered to secrete high levels of the enzyme. The protein was secreted as an inactive soluble proenzyme of approximately 65 kDa that appears as a monomer by gel filtration. Upon acidification, the protein is processed to mature form and acquires activity. The enzyme is efficiently delivered to the lysosomes of LINCL fibroblasts by mannose 6-phosphate-receptor-mediated endocytosis (EC(50) approximately 2 nM), where it remains active for long periods of time (t(1/2) approximately 12 days). In addition, the enzyme is taken up by rat cerebellar granule neurons by mannose 6-phosphate-dependent and -independent mechanisms. Treatment of LINCL fibroblasts with recombinant CLN2 protein restores normal enzyme activity and ameliorates accumulation of the major storage protein, mitochondrial ATP synthase subunit c.

Aminopeptidases↗
PubMed2002

Restriction enzymes in the analysis of genetic alterations responsible for cancer progression.

BACKGROUND: Molecular approaches are increasingly being employed to dissect the genetic changes accompanying tumour formation. These methods can often be confusing to the non-specialist as they include complex molecular steps. This can reduce the usefulness of such molecular data to clinicians. The authors aim to aid interpretation of molecular studies in general by presenting a comprehensive review of one molecular approach, i.e. the use of restriction enzymes in molecular studies of tumour development. METHOD: A review was made of the molecular studies that have employed restriction enzymes in gastrointestinal cancer research. These studies have used restriction enzymes to analyse point mutation induction, gene methylation status and the deletion of chromosomal loci. In addition, emphasis is placed on some of the important considerations for the molecular analysis of tumours that can affect the molecular data obtained. RESULTS: Restriction enzyme digestion has played, and continues to play, a major role in analysing the genetic changes in cancer. Many adaptations of basic restriction enzyme methodologies have enhanced the application of this approach in cancer genetics. CONCLUSION: The availability of 200 different restriction enzymes, each recognizing different sequences in DNA, has been invaluable in studying cancer genetics. It is hoped that current advances in protein engineering will facilitate the creation of novel restriction enzymes with tailor-made sequence specificities. This will further improve the applicability of restriction enzymes in cancer genetics.

DNA Methylation↗
PubMed1992

Biotransformation of caffeine and theophylline in mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms.

Primary steps in the metabolism of caffeine and theophylline are cleavage of methyl groups and/or hydroxylation at position 8, mediated by cytochromes P450. V79 Chinese hamster cells genetically engineered for stable expression of single forms of rat cytochromes P450IA1, P450IA2 and P450IIBI and human P450IA2 and rat liver epithelial cells expressing murine P450IA2 were used to overcome problems arising in the proper allocation of metabolic pathways to specific isoforms by conventional techniques. These cell lines were exposed to caffeine and/or theophylline, and concentrations of metabolites formed in the medium were determined by HPLC. Caffeine was metabolized by human, rat and murine P450IA2, resulting in the formation of four primary demethylated and hydroxylated metabolites. However, there were differences in the relative amounts of the metabolites. The human and the mouse P450IA2 isoforms predominantly mediated 3-demethylation of caffeine. The rat cytochrome P450IA2 mediated both 3-demethylation and 1-demethylation of caffeine to a similar extent. Theophylline was metabolized mainly via 8-hydroxylation. All cell lines tested were able to carry out this reaction, with highest activities in cell lines expressing rat or human P450IA2, or rat P450IA1. These results support the hypothesis that caffeine plasma clearance is a specific in vivo probe for determining human P450IA2 activity.

Animals↗
PubMed2003

A comparison of directed evolution approaches using the beta-glucuronidase model system.

Protein engineers can alter the properties of enzymes by directing their evolution in vitro. Many methods to generate molecular diversity and to identify improved clones have been developed, but experimental evolution remains as much an art as a science. We previously used DNA shuffling (sexual recombination) and a histochemical screen to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with improved beta-galactosidase (BGAL) activity. Here, we employ the same model evolutionary system to test the efficiencies of several other techniques: recursive random mutagenesis (asexual), combinatorial cassette mutagenesis (high-frequency recombination) and a versatile high-throughput microplate screen. GUS variants with altered specificity evolved in each trial, but different combinations of mutagenesis and screening techniques effected the fixation of different beneficial mutations. The new microplate screen identified a broader set of mutations than the previously employed X-gal colony screen. Recursive random mutagenesis produced essentially asexual populations, within which beneficial mutations drove each other into extinction (clonal interference); DNA shuffling and combinatorial cassette mutagenesis led instead to the accumulation of beneficial mutations within a single allele. These results explain why recombinational approaches generally increase the efficiency of laboratory evolution.

Directed Molecular Evolution↗
PubMed2002

Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase.

Metabolic engineering studies have generally focused on manipulating enzyme levels through either the amplification, addition, or deletion of a particular pathway. However, with cofactor-dependent production systems, once the enzyme levels are no longer limiting, cofactor availability and the ratio of the reduced to oxidized form of the cofactor can become limiting. Under these situations, cofactor manipulation may become crucial in order to further increase system productivity. Although it is generally known that cofactors play a major role in the production of different fermentation products, their role has not been thoroughly and systematically studied. However, cofactor manipulations can potentially become a powerful tool for metabolic engineering. Nicotinamide adenine dinucleotide (NAD) functions as a cofactor in over 300 oxidation-reduction reactions and regulates various enzymes and genetic processes. The NADH/NAD+ cofactor pair plays a major role in microbial catabolism, in which a carbon source, such as glucose, is oxidized using NAD+ producing reducing equivalents in the form of NADH. It is crucially important for continued cell growth that NADH be oxidized to NAD+ and a redox balance be achieved. Under aerobic growth, oxygen is used as the final electron acceptor. While under anaerobic growth, and in the absence of an alternate oxidizing agent, the regeneration of NAD+ is achieved through fermentation by using NADH to reduce metabolic intermediates. Therefore, an increase in the availability of NADH is expected to have an effect on the metabolic distribution. This paper investigates a genetic means of manipulating the availability of intracellular NADH in vivo by regenerating NADH through the heterologous expression of an NAD(+)-dependent formate dehydrogenase. More specifically, it explores the effect on the metabolic patterns in Escherichia coli under anaerobic and aerobic conditions of substituting the native cofactor-independent formate dehydrogenase (FDH) by and NAD(+)-dependent FDH from Candida boidinii. The over-expression of the NAD(+)-dependent FDH doubled the maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed, increased the final cell density, and provoked a significant change in the final metabolite concentration pattern both anaerobically and aerobically. Under anaerobic conditions, the production of more reduced metabolites was favored, as evidenced by a dramatic increase in the ethanol-to-acetate ratio. Even more interesting is the observation that during aerobic growth, the increased availability of NADH induced a shift to fermentation even in the presence of oxygen by stimulating pathways that are normally inactive under these conditions.

Aerobiosis↗
PubMed1999

Expression, purification and characterization of recombinant mouse MT5-MMP protein products.

We have recently identified the fifth member of the membrane-type matrix metalloproteinase subfamily, MT5-MMP/MMP24, which is expressed in a brain specific manner (Duanqing Pei (1999) J. Biol. Chem. 274, 8925-8932). To further characterize its enzymic properties, an expression construct was engineered to produce MT5-MMP as a soluble and active form by truncating its transmembrane domain. Stable expression cell lines were subsequently established from MDCK cells transfected with this construct. Unfortunately, purification of MT5-MMP from the culture media in large quantity proves to be difficult initially due to its rapid turnover via a mechanism which can be inhibited by a broad spectrum metalloproteinase inhibitor, BB94. Thus, BB94 was included in the cell culture medium and throughout the purification process except the final step of chromatography to protect MT5-MMP from destruction. Purified to homogeneity and free of the synthetic inhibitor, MT5-MMP can activate progelatinase A efficiently in a TIMP2 sensitive fashion. A preliminary screen for its potential substrates among extracellular matrix components identified the proteoglycans as the preferred substrates for MT5-MMP. Furthermore, it is determined that the stability of purified MT5-MMP is temperature dependent with rapid destruction at 37 degrees C, but being relatively stable at temperatures 4 degrees C or lower. These observations establish MT5-MMP as a proteoglycanase with a short half-life at body temperature, which may be critical for tightly controlled turnover of ECM components such as those in the brain.

Animals↗
PubMed2000

Metabolic activation of N-alkylnitrosamines in genetically engineered Salmonella typhimurium expressing CYP2E1 or CYP2A6 together with human NADPH-cytochrome P450 reductase.

A Salmonella typhimurium tester strain YG7108 2E1/OR co-expressing human CYP2E1 together with human NADPH-cytochrome P450 reductase (OR) was established. The mutagen-activating capacity of human CYP2E1 for N-alkylnitrosamines was compared with that of CYP2A6 using the YG7108 2E1/OR and the YG7108 2A6/OR strains of SALMONELLA: Salmonella YG7108 2A6/OR is a derivative of YG7108 co-expressing CYP2A6 together with OR. Eight N-alkylnitrosamines, including N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosomethylphenylamine (NMPhA), N-nitrosopyrrolidine (NPYR), N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were examined. CYP2E1 expressed in the YG7108 2E1/OR cells showed mutagen-activating capacity, as indicated by induced revertants/min/pmol cytochrome P450, for NDMA, NDEA, NDPA, NDBA, NPYR and NNK, but not NMPhA and NNN. CYP2A6 activated NDMA, NDEA, NDPA, NDBA, NMPhA, NPYR, NNN and NNK. The ratio of the mutagen-activating capacity seen with CYP2A6 to that seen with CYP2E1 was calculated for each N-alkylnitrosamine. In the case of NDMA, NPYR and NDEA, the ratio was under 1.0, while the ratio was over 1.0 with NDPA, NDBA, NNK, NMPhA and NNN. We conclude that human CYP2E1 is mainly responsible for the metabolic activation of N-nitrosamines with a relatively short alkyl chain(s), whereas CYP2A6 was predominantly responsible for the metabolic activation of N-alkylnitrosamines possessing a relatively bulky alkyl chain(s).

Aryl Hydrocarbon Hydroxylases↗
PubMed1982

[Biotechnology. Genetic engineering. Chemico-pharmaceutical area].

We have tried to bring up to date the information on in vitro Genetic Manipulations and the Recombinant DNA considering its relations with new technology and possible applications towards the fine chemicals development and specially drug area. Taking into account the scientific development which takes place in the most advanced industrial societies, those countries which are nowadays developing an adequate technology level must face the scientific challenge among them, we can find two which appear as specially important: the Genetic Engineering knowledge and the Molecular Biology developments.

Cloning, Molecular↗