Regulation of tyrosine aminotransferase in rat liver. XI. Studies on the relationship of enzyme stability to enzyme turnover in cultured hepatoma cells.
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Amino Acids↗
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Results for “Enzyme Stability”
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Lipoprotein lipase activity in F1 heart cell cultures. Effect of dialyzable serum factors on enzyme stability and enzyme synthesis.
F1 heat cell cultures were grown in F-10 medium containing 20% fetal calf and horse serum and after 6-7 days showed high activity of lipoprotein lipase. When the culture medium contained 20% serum which had been dialyzed against F-10 medium, a 75% decline in lipoprotein lipase activity occurred after 3 h of incubation. Cultures incubated with 20% dialyzed serum and dialysate, obtained after 24 h dialysis of serum against F-10 medium, retained full enzyme activity. Restoration of enzyme activity, lost upon incubation with dialyzed serum, became apparent only 2 h after incubation of the cells with dialysate and dialyzed serum and was complete after 24 h. The effectiveness of the dialysate was not affected by trichloroacetic acid precipitation, ether extraction, exposure to pronase or to 80 degrees C for 10 min; it was retained after chromatography of DEAE cellulose but was lost after elution from CM cellulose colums. Addition of spermidine and spermine to culture media containing dialyzed serum did prevent partially the decline in lipoprotein lipase activity of the heart cell cultures. These polyamines were also able to stabilize lipoprotein lipase activity of heart homogenates incubated at 37 degrees C. However, these compounds were not effective in resoration of enzyme activity of cultured cells lost after exposure to dialyzed serum. It appears that positively charged low molecular weight molecules present in sera of various species are required for the stabilization and synthesis of lipoprotein lipase in heart cell cultures.
Animals↗
Directed evolution of enzyme stability.
Modern enzyme development relies to an increasing extent on strategies based on diversity generation followed by screening for variants with optimised properties. In principle, these directed evolution strategies might be used for optimising any enzyme property, which can be screened for in an economically feasible way, even if the molecular basis of that property is not known. Stability is an interesting property of enzymes because (1) it is of great industrial importance, (2) it is relatively easy to screen for, and (3) the molecular basis of stability relates closely to contemporary issues in protein science such as the protein folding problem and protein folding diseases. Thus, engineering enzyme stability is of both commercial and scientific interest. Here, we review how directed evolution has contributed to the development of stable enzymes and to new insight into the principles of protein stability. Several recent examples are described. These examples show that directed evolution is an effective strategy to obtain stable enzymes, especially when used in combination with rational or semi-rational engineering strategies. With respect to the principles of protein stability, some important lessons to learn from recent efforts in directed evolution are (1) that there are many structural ways to stabilize a protein, which are not always easy to rationalize, (2) that proteins may very well be stabilized by optimizing their surfaces, and (3) that high thermal stability may be obtained without forfeiture of catalytic performance at low temperatures.
Animals↗
[Chemical modification of lysine epsilon-NH2-groups in horseradish peroxidase. Its effect on enzyme stability. Temperature dependence of thermo-inactivation constants for native and modified peroxidase].
Thermostability of horseradish peroxidase modified by acetic, propionic, butyric, valeric and succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) is studied within the temperature range of 56-80 degrees C. Acylation of 4 amino groups and arylation of 3 amino groups with TNBS are found to stabilize the enzyme, while modification of 6 groups decreases the enzyme stability. Chemical modification of peroxidase does not change its pH-dependence with respect to enzyme thermostability. Thermodynamic activation parameters of irreversible thermoinactivation are determined for native and modified peroxidase. Native peroxidase has deltaH not equal to = 30+/-1 kcal/mole and deltaS not equal to = 14 e. e.; modified by acid anhydrides peroxidase has deltaH not equal to within 64-87 kcal/mole and deltaS not equal to within 110-178 e. e. depending on the nature of a modifying agent. The effect of the structure of a radical introduced into the enzyme molecule, and of a number of modified epsilon-amino groups on thermoinactivation deltaH not equal to and deltaS not equal to values is discussed.
Acylation↗
Principles of enzyme stabilization. V. The possibility of enzyme selfstabilization under the action of potentially reversible intramolecular cross-linkages of different length.
The denaturing action of guanidine . HCl on modified alpha-chymotrypsin (EC 3.4.21.1) preparations has been studied. The consecutive treatment of alpha-chymotrypsin with N-acetyl-homocysteine thiolactone, 5,5'-dithio-bis-(2-nitrobenzoic acid) and dithiols of HS-(CH2)n-SH type, with n ranging from 4 to 10, leads to enzyme stabilization as a result of protein modification. A greater stabilization effect can be achieved by enriching the protein molecule with groups reactive towards dithiols, after first modifying carboxygroups. In this case dithiol with n=5 forms an intramolecular cross-linkage. If an equimolecular mixture of different dithiols is used for enzyme modification, the enzyme gradually 'selects' 1,5-dithiol for the formation of an intramolecular cross-linkage instead of the initial one-point modification. The use of potentially reversible cross-linkages may be generally employed for the preparation of stabilized water-soluble enzymes via the mechanism of selfstabilization.
Chymotrypsin↗
Enzyme stability in downstream processing. Part 1: enzyme inactivation, stability and stabilization.
In biotechnological recovery processes the instability of the product can lead to large losses in the sequence of recovery processes needed to purify the product. As the cost of the final active product is strongly dependent on the recovery yield, this will lead to an increase in product cost. Therefore knowledge of factors that influence stability is important. This Part 1 contains a review on the factors that influence stability. As stability is very important in enzyme purification this review deals about enzymes and their ability to retain catalytic activity. Inactivation mechanisms and agents are discussed. A short review is given of enzyme structure and stability. This is followed by stabilization strategies and methods.
Journal Article↗
Enzyme stabilization: state of the art.
'Enzyme stabilization' is one of the most important fields in basic and applied enzymology. In basic enzymology, it is of particular relevance to understand enzyme stabilization principles first elucidating how and why the enzymes lose their biological activity and then deriving structure-stability relationships existing in enzymatic molecules. In applied enzymology, the most significant goal is to achieve useful compounds by biocatalysis. Enzymes are good catalysts in terms of high catalytic and specific activity with ability to function under mild conditions. However, they are not always ideal catalysts for practical applications because they are generally unstable and they inactivate rapidly through several mechanisms. In order to enhance enzyme stability, many strategies have been pursued in recent years. The present article is an attempt to provide detailed information about these strategies.
Animals↗
Enzyme stability in systems with organic solvents.
This review deals with enzyme stability in organic solvent systems. Based on the current state of the problem, catalytic activity is chosen as the main tool for testing enzyme stability. Various enzyme properties being regarded as their "stability" at present, a classification of the types of enzyme stability most often discussed in the literature is put forward. Aggregation state of the biocatalytic system formed a basis for the analysis of enzyme stability in organic solvent systems. Regularities of enzyme function in homogeneous solutions in water--organic co-solvent mixtures and suspensions in practically anhydrous solvents are discussed, kinetic data being analyzed in parallel to results of structural studies. Based on the elaborated regularities, up-to-date methods for enzyme stabilization in organic solvent systems are considered.
Catalysis↗
Utilization of immobilized archaeal chaperonin for enzyme stabilization.
CpkA and CpkB are thermostable chaperonins from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. CpkA or CpkB was immobilized onto agarose beads by the carbodiimide coupling method. In order to investigate whether immobilized CpkA or CpkB stabilizes a foreign enzyme, their ability to stabilize enzyme was examined using beta-D-galactosidase from Escherichia coli as a model target. In the absence of chaperonin beads, 38.1% of the total soluble protein was precipitated by heat treatment at 53 degrees C for 30 min. The protein structure of the residual soluble fraction was examined by circular dichroism, indicating that beta-D-galactosidase exists as a mixture of the active folded form and the inactive unfolded form. The specific activity of the residual soluble fraction decreased to 85.1% that of the unheated level (from 149.0 U/mg to 127.0 U/mg), indicating that the active folded form in the heated soluble fraction was 85.2%. In the presence of CpkA- or CpkB-beads, 28.6% or 34.3% of beta-d-galactosidase was precipitated by the same heat treatment. However, the specific activity in the soluble fraction was almost maintained (CpkA, from 151.0 U/mg to 140.3 U/mg; CpkB, from 149.0 U/mg to 140.3 U/mg). These results indicated that CpkA- or CpkB-beads are useful for keeping the enzyme active.
Journal Article↗
Stability of firefly luciferase in tricine buffer and in a commercial enzyme stabilizer.
A solution of firefly luciferase in AuthentiZyme Enzyme Stabilizer retains full activity when stored in an ice bath (0.5 degrees C) during one day. These solutions have the advantage that no additional protein (other than the luciferase) is present, which is desirable for proteolytic digestion and protein derivatization experiments. For longer-term experiments, firefly luciferase solutions in 0.05 mol/l Tricine buffer at pH 7.8, 10 mmol/l MgSO4, 1 mmol/l EDTA, and 1 mmol/l DTT which contain 100 micrograms/ml of bovine serum albumin are stable for 6 weeks if frozen and thawed only once.
Animals↗
The principles of enzyme stabilization. I. Increase in thermostability of enzymes covalently bound to a complementary surface of a polymer support in a multipoint fashion.
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Acylation↗
Enzyme stabilization strategies based on electrolytes and polyelectrolytes for biosensor applications.
The achievements in the area of enzyme stabilization based on electrolytes, polyelectrolytes and polyols is reviewed, in the context of biosensor applications. Both the storage and operational stabilities of the biosensors can be improved using these stabilizers. The deactivation of the enzymes used for the development of biosensors from thermal shock, proteolytic degradation, and non-specific metal-catalyzed oxidation can be drastically reduced with the use of one or more of these stabilizers. It is attempted to deconvolute the effect of these additives on (a) the storage stability or shelf life, and (b) the operational stabilities of the biosensors. Even though there are a large number of techniques and reports dealing with enzyme stabilization, their application to biosensor technology is still very limited. It is thus concluded that the use of the existing enzyme stabilization techniques will have a drastic effect on the storage and operational stabilities of biosensors in the near future.
Biosensing Techniques↗
Variation in alpha-L-fucosidase properties among 28 inbred mouse strains: six strains have high enzyme activity and heat-stabile enzyme with a variant pH-activity curve; twenty-two strains have low activity and heat-labile enzyme.
Alpha-L-fucosidase in tissues of 28 inbred mouse strains varied with respect to three properties: high or low heat stability, a pH-activity curve with high or low relative activity at pH 2.8, and high or low activity. Alpha-L-fucosidase from six strains (A/J, BDP/J, LP/J, P/J, SEA/GNJ, and 129/J) had high heat stability, high pH 2.8 relative activity, and high activity, whereas the other 22 strains all had low heat stability, low pH 2.8 relative activity, and low activity. The heat-stability difference was seen in all organs tested (brain, liver, kidney, spleen, heart, skeletal muscle, lung, and testis) for two heat-stabile strains (P/J and 129/J) and four heat-labile strains (C57BL/6J, C3H/HeJ, DBA/2J, and BALB/cJ) studied in detail. The findings suggested that two structural variants of alpha-L-fucosidase, probably genetically determined, exist in these 28 inbred mouse strains, although the presence of linkage disequilibrium between alleles of tightly linked structural and regulatory genes could not be excluded.
Animals↗
Crosslinked enzyme aggregates in hierarchically-ordered mesoporous silica: a simple and effective method for enzyme stabilization.
alpha-chymotrypsin (CT) and lipase (LP) were immobilized in hierarchically-ordered mesocellular mesoporous silica (HMMS) in a simple but effective way for the enzyme stabilization, which was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale crosslinked enzyme aggregates (CLEAs) entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). CLEA of alpha-chymotrypsin (CLEA-CT) in HMMS showed a high enzyme loading capacity and significantly increased enzyme stability. No activity decrease of CLEA-CT was observed for 2 weeks under even rigorously shaking condition, while adsorbed CT in HMMS and free CT showed a rapid inactivation due to the enzyme leaching and presumably autolysis, respectively. With the CLEA-CT in HMMS, however, there was no tryptic digestion observed suggesting that the CLEA-CT is not susceptible to autolysis. Moreover, CLEA of lipase (CLEA-LP) in HMMS retained 30% specific activity of free lipase with greatly enhanced stability. This work demonstrates that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity.
Chymotrypsin↗
Glycosylation of Proteins: a new method of enzyme stabilization.
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Drug Stability↗
Approaches to enzyme stabilization.
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Clostridium↗
Domain-specific N-glycosylation of the membrane glycoprotein dipeptidylpeptidase IV (CD26) influences its subcellular trafficking, biological stability, enzyme activity and protein folding.
Dipeptidyl peptidase IV (DPPIV, CD26) is an N-glycosylated type II plasma membrane protein. The primary structure of rat wild-type DPPIV contains eight potential N-glycosylation sites. To investigate the role of N-glycosylation in the function of DPPIV, three of its asparagine residues were separately converted to glutamine by site-directed mutagenesis. The resulting N-glycosylation mutants of rat DPPIV were studied in stable transfected Chinese hamster ovary cells. All three N-glycosylation mutants of DPPIV showed a reduced half-life, as well as differing degrees of inhibition of the processing of their N-glycans. Mutation of the first (Asn83-->Gln) or eighth (Asn686-->Gln) N-glycosylation site had only a small effect on its enzymatic activity, cell-surface expression and dimer formation, whereas the mutation of the sixth N-glycosylation site (Asn319-->Gln) abolished the enzymatic activity, eliminated cell-surface expression and prevented the dimerization of the DPPIV protein. The mutant [Gln319]DPPIV is retained in the cytoplasm and its degradation was drastically increased. Our data suggest that the N-glycosylation at Asn319 is involved in protein trafficking and correct protein folding.
Animals↗
Does coenzyme binding determine enzyme stability?
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Coenzymes↗