Specificity of amino acid acylases.
نویسندگان
چکیده
The observation that chloroacetyl-m-alanine is asymmetrically hydrolyzed by crude kidney preparations at pH 7 (1) led to the development of a general and simple procedure for the resolution of racemic amino acids (2-12). This procedure was based upon the action of a concentrated enzyme preparation obtained from hog kidney which asymmetrically hydrolyzed most N-acylated racemic amino acids, and which in consequence was designated “acylase.” The enzyme concentrate was obtained by an alcohol fractionation of the homogenate at low temperature, and in most cases represented a 4to 6-fold concentration in activity against chloroacetyl-m-alanine which was employed as the test substrate. It was further assumed that this concentration in activity held toward all other susceptible acylated amino acids. Despite this modest enrichment in acylase activity, the concentrates were readily applied to the resolution of several amino acids (2-ll), and led to the preparation of enantiomorphs which possessed an optical purity greater than 99.9 per cent (13).* We report in this paper the preparation of a much more active acylase from hog kidney by a simplified procedure involving fractionation with ammonium sulfate and acetone. When this new acylase preparation was tested with a large number of N-acylated amino acids, two new observations were made. The first was that the concentrate hydrolyzed all acylated amino acids studied at a rate roughly 30 times greater than that observed with the crude homogenate, with the exception of acylated aspartic acid. This substance was hydrolyzed at a much slower rate than by the crude homogenate. This suggested that N-acylated aspartic acid was hydrolyzed
منابع مشابه
Mutational analysis of a key residue in the substrate specificity of a cephalosporin acylase.
beta-Lactam acylases are crucial for the synthesis of semisynthetic cephalosporins and penicillins. Unfortunately, there are no cephalosporin acylases known that can efficiently hydrolyse the amino-adipic side chain of Cephalosporin C. In a previous directed evolution experiment, residue Asn266 of the glutaryl acylase from Pseudomonas SY-77 was identified as being important for substrate specif...
متن کاملModeling and Experimental Analysis of Cephalosporin C Acylase and Its Mutant
7-amino cephalosporanic acid (7-ACA) is the crucial intermediate for the synthesis of semi-synthetic antibiotics, which is currently prepared by two-step biocatalysis using D-amino acid oxidase and glutaryl-7-amino cephalosporanic acid acylase (GL-7-ACA acylase) starting from cephalosporin C (CPC). Compared with the two-step enzymatic method, one-step method is more efficient and economical. Bu...
متن کاملNucleotide sequences of the genes for two distinct cephalosporin acylases from a Pseudomonas strain.
Two genes, acyI and acyII, for distinct cephalosporin acylases from Pseudomonas sp. strain SE83 (A. Matsuda, K. Matsuyama, K. Yamamoto, S. Ichikawa, and K.I. Komatsu, J. Bacteriol. 169:5815-5820, 1987) were sequenced. Each sequence contained a single open reading frame for two nonidentical subunits, predicting a common precursor. Some homologies at the amino acid level were found between the ac...
متن کاملThe use of chromogenic reference substrates for the kinetic analysis of penicillin acylases.
Determination of kinetic parameters of penicillin acylases for phenylacetylated compounds is complicated due to the low K(m) values for these substrates, the lack of a spectroscopic signal, and the strong product inhibition by phenylacetic acid. To overcome these difficulties, a spectrophotometric method was developed, with which kinetic parameters could be determined by measuring the effects o...
متن کاملCloning, sequence analysis, and expression in Escherichia coli of the gene encoding an alpha-amino acid ester hydrolase from Acetobacter turbidans.
The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing beta-lactam antibiotics, such as cephalexin and ampicillin. N-terminal amino acid sequencing of the purified alpha-amino acid ester hydrolase allowed cloning and genetic characterization of the corresponding gene from an A. turbidans genomic library. The gene, designated aehA, en...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- The Journal of biological chemistry
دوره 194 1 شماره
صفحات -
تاریخ انتشار 1952