Surface Modification of Silica Gels as Carrier for AminoacylaseImmobilization

The surface of silica gels was modified by an diethylamino group and an hydroxygroup, simultaneously. The activity of the aminoacylase immobilized on such amino-hydroxy-silica gels was 316.8 U/g. By its catalysis, the conversions of tested N-acetyl-D,L-amino acids toL-amino acids were 85%.

Kinetics of Inactivation of Aminoacylase I During Modification of Its Thiol Groups by DPDS and PCMB

The kinetics theory of the substrate reaction during modification of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of aminoacylase I by DPDS and PCMB.From the results obtained we have found that the inactivation reaction of aminoacylase I by DPDS is noncomplexing inhibition,and PCMB reaction is complexing inhibition.The microscopic constants for the reaction of the inactivator with free enzyme and the enzyme-substrate complex were determined.

Crystallization and preliminary X-ray studies of porcine kidney aminoacylase Ⅰ

AMINOACYLASE Ⅰ（ACY-1） （E. C. 3. 5. 1. 14 ） participated in amino acid metabolism existingin mammalian kidney and microorganisms, and catalyzes reversible hydrolysis of acylaminoacids. ACY-1 consists of 772 amino acids, and the molecular weight is 85 500 daltons. It is adimeric metalloprotein having two Zn2+ in the molecule. The nucleotide and the amino acid se-quence of porcine and human ACY-1 have been determined. The nucleotide sequence and

THE IMMOBILIZATION OF AMINOACYLASE AND THE RESOLUTION OF D,L-PHENYLALANINE

A polymer with suitable physical characters and matched functional groups was synthesized,and used as a supporter to immobilize aminoacylase. The results showed that this supporterhad high immobilizing capacity and high selectivity for aminoacylase. Immobilized amino-acylase had high specific activity. In this paper, we determine the physical and chemicalcharacters of aminoacylase for resolution of D,L--phenylalanine, including its optimum tem-perature, pH, ion concentration, activated ion, substrate concentration, thermal stability anddenaturation. The immobilized aminoacylase was used to resolve the D,L-phenylalaninecontinuously. Thc resolved products was separated and purified with ion-exchange resins. L-phenylalanine solution was concentrated by vacuum evaporation and its hydrochloride wasformed. Checking of final product with polarimeter showed high yield and purity.

Enhancement of Aminoacylase Activity by Sodium Citrate

Kidney and other tissues of animals and humans have a high concentration of citrate which is an important intermediate substance in the citrate cycle. Citrate may play an important physiological role in metabolism. In this paper, we studied the interaction of the sodium salt of citrate with aminoacylase which is an important enzyme in metabolism and found sodium citrate can enhance the activity of aminoacylase.The maximum enzyme activity induced by sodium citrate increased approximately 3 folds over the enzyme activity without sodium citrate. The initial reaction rates (V) for different concentrations of sodium citrate were obtained, showing that sodium citrate is a non competitive activator. The result of the ANS binding fluorescence measurements for aminoacylase indicated that increasing sodium citrate concentrations markedly increased the ANS binding fluorescence with a blue shift of the emission spectra peak. This suggests the formation of more hydrophobic regions. Aggregates formed quickly when aminoacylase was incubated with sodium citrate (0.3 mol/L) and guanidinium chloride (03.5 mol/L). Aminoacylase lost enzyme activity in the guanidinium chloride more quickly in the presence of sodium citrate than in the absence of sodium citrate. The intrinsic fluorescence emission intensity decreased more quickly and the red shift of the emission spectra peak was larger than that without sodium citrate.

Unfolding and Inactivation During Thermal Denaturation of Aminoacylase

Many researchers have compared inactivation with conformational changes of a number of enzymes during denaturation by guanidine hydrochloride and urea. The results obtained show that inactivation occurs before noticeable conformational change of the enzyme molecule as a whole can be detected. The inactivation rate constants

Inactivation of aminoacylase at low urea concentrations not due to dissociation of dimeric enzyme

Aminoacylase （EC 3. 5. 1. 14） is a dimeric enzyme containing one Zn2+ ion per subunit with a molecular weight of 43000 u. Zn2+ ion is located at the active site, and it is essential for enzyme activity. It is well known that the presence of Zn2+ ion helps to keep the conformation of the active site in a strained state required for the catalysis of

Aminoacylase from pig kidney contains no disulfide bonds

Both non-reduced/reduced(NR/R)two-dimensional diagonal SDS-PAGE and NR/Rone-dimensional SDS-PAGE showed no disulfide bonds in aminoacylase from pig kidney.Eight andfour thiol groups were modified in the native enzyme by 2-chloromercuri-4-nitrophenol(MNP)andEllman’s reagent,5,5’-dithiobis(2-nitrobenzoic add)(DTNB),and another two and six thiol groupscould be exposed and modified in 7mol/L guanidine hydrochloride,respectively.The enzyme denaturedwith guanidine or urea was found to contain a total of ten thiol groups.This is in good agreement with therecently deduced amino acid sequence from cloned cDNA.It is therefore clear that no disulfide bridges existin aminoacylase from pig kidney.

Cause of Irreversible Inactivation of Aminoacylase in Urea Solution

Some have suggested that the refolding of denatured aminoacylase is difficult. The present study reports that aminoacylase denatured in urea concentrations more than 4?mol/L was markedly but partially unfolded and could be reactivated and refolded very rapidly. However, the extent of reactivation decreased with increasing time suggesting an irreversible inactivation occurred in a biphasic course. The fast phase of the irreversible inactivation is suggested to accompany the unfolding. Oxidation of the thiol groups exposed by unfolding may be one of the main causes of the second phase of the irreversible inactivation.

Comparison between conformational change and inactivation rates of aminoacylase during denaturation in urea solutions

The kinetic method of the substrate reaction in the presence of mactivator previously described by Tsou has been applied to the determination of inactivation rates of aminoacylase during denaturation in urea solutions. The protective effect of substrate on the inactivation of aminoacylase by urea has been investigated. Simultaneously, the comparison between conformational change and inactivation rates of enzyme in the urea solutions of different concentrations has been studied. Results obtained show that the inactivation rate constants of the enzyme are larger than the rate constants of conformational changes. The present results show that the active site of metal enzyme-aminoacylase is also located in a limited and flexible region of the molecule that is more sensitive to denaturants than the enzyme as a whole.

Secondary Structure of Holo-Enzyme and Apo-Enzyme of Aminoacylase Using CD and FTIR Spectroscopy

Aminoacylase is a dimeric metal enzyme containing one Zn2+-ion per subunit of active site.It is essential for the activity of enzyme.Fourier transform-infrared spectroscopy has been used for the studyon the secondary structure of holo-enzyme and ago-enzyme of aminoaeylase from pig kidney.Resolution en-hancement of the amide I secondary structure-sensitive overlapped component bands has been achieved bymeans of the Fourier self-deconvolution and the Fourier derivation.The effect of Zn2+-ion on the secondarystructure of aminoacylase was observed clearly.After the removal of Zn2+in aminoacylase,the extent of theordered structure was decreased markedly.It suggests that the conformation st or near the active site ofaminoacylase contains more ordered structures,and the presence of Zn2+helps to keep the conformation ofthe active site required for the catalysis of the enzyme.