acylases and peptidases are very important industrial enzymes. they are used for production of peptide and semisynthetic antibiotics. They play a very significant role in peptide chemistry.

1. Acylases and Peptidases

2. Content
Introduction
What is Acylases and Peptidases
Classification of Penicillin G acylases
Characteristic features of PGA gene expression
Reaction catalysed by PGA
Peptidase
Characteristics features
Advantages of using protease in peptide sysnthesis
Applications of Acylases and Peptidases

3. Penicillin acylase (EC 3.5.1.11) is a serine type of esterase which possesses both esterase and amidase activity, selectively
hydrolyzing the phenyl acetyl moiety from both esters and amides.
discovered 60 years ago as a catalyst of the hydrolysis of the amide bond in penicillin antibiotics
class of hydrolases, a subclass of aminohydrolases, and represents a group of so-called N-terminal nucleophilic
hydrolases.
Ubiquitous in nature.
The physiological role of the enzyme remains poorly understood.
It seems possible that its main function is in utilizing heterocyclic compounds as a source of carbon. PA has been
extensively studied for more than 50 years. In practice, this enzyme is commonly used to produce 6-aminopenicillanic
acid, which is the main synthon in the synthesis of penicillin antibiotics.
PA is also used for the synthesis of various semi-synthetic β-lactam antibiotics.
Broad substrate specificity and high regio-, chemo- and stereoselectivity of the enzyme are used for the production of
chiral compounds (which are more and more in demand in modern pharmaceutics), as well as for the protection of
hydroxy and amino groups in peptide and fine organic synthesis.
Currently, the most commonly used PA is that from Escherichia coli (EcPA).
This enzyme has been better studied and characterized in comparison with the other PAs; however, the efficiency of the
acyl transfer into β-lactam cores, catalysed by EcPA, is not high enough to make the enzyme competitive as compared
with the out-of-date methods of antibiotic synthesis.

4. Penicillin acylases represent a group
of β-lactam acylases and can be
classified according to the type of
the hydrolysed substrate. Therefore,
enzymes can be grouped as those
that hydrolyse penicillin G, penicillin
V, or ampicillin. In 1963 it was
suggested to divide penicillin
acylases into classes I and II . Class I
enzymes basically hydolyse penicillin
V (phenoximethylpenicillin), while
class II enzymes use penicillin G
(benzylpenicillin) as a substrate.
Later, the class III, including the
enzymes which hydrolyse ampicillin,
was added
Classification of Penicilllin G acylase

5. Sources and Localization of Penicillin Acylases
Penicillin acylase activity was also detected in bacteria, yeast, and fungi .
At the present time, PAs from more than 40 different microorganisms have been
described. Many genes of penicillin acylases were found in annotated genomes of
microorganisms.
Depending on the species of the microorganism, the enzyme can dwell either outside
or inside the cell.
Localization in periplasma is chrachteristic for active forms of G-class penicillin acylases
(class II). Extracellular expression is also typical for some strains producing penicillin
acylases V (class I) and penicillin acylases G (class II). The physiological role of PAs
remains unclear despite a 60-year-long history of studying them. It is highly probable
that PAs are needed for the utilization of aromatic amides as carbon source

6. Characteristic features of Penicillin G Acylase Gene expression
A-G gene encodes a precursor polypeptide which consists of 4 structural elements: a signal peptide, αand β-
subunits, and an inter-subunit spacer. The mature PA-G molecule is a heterodimer with a molecular weight of
86 kDa. It consists of two subunits, α- and β-, with molecular masses of 23 and 63 kDa, respectively. In addition,
the molecule contains a bound Ca2+ ion, which, according to data, is important for enzyme processing .
Posttranslational modification of PA-G is a multistage process, which has been well studied for the enzyme from
E.coli. The first step includes transport of the inactive precursor from the cytoplasm to the periplasmic
compartment, a process drived by the signal peptide, which is then removed after the transport is completed.
Afterwards, the inter-subunit spacer undergoes two-step proteolysis, which results in the formation of an active
heterodimer

8. Peptidases
Enzyme Commission nomenclature distinguishes between hydrolases acting on peptidic bonds (EC
3.4) and other amide bonds (EC 3.5). In 1984 all of the sub-subclasses EC 3.4.1-10 were abandoned.
Enzymes cleaving peptide bonds (peptidases, proteases) were divided into two sets of sub-
subclasses.
EC 3.4.11-19 covers peptidases (exopeptidases, carboxy- and aminopeptidases) which cleave single
amino acids or dipeptides from the ends of peptide chains, whereas
EC 3.4.21-24 covers proteinases (endopeptidases, proteolytic enzymes, peptidyl-peptide hydrolases).
Which have no preference for terminal residue cleavage.
Enzymes which cannot be allocated to a specific sub-subclass are assigned as an interim measure to
3.4.99 (Anonymous, 1984).
Proteases are divided into four sub-subclasses: serine proteases (EC 3.4.21.X), thioproteases (EC
3.4.22.X), aspartyl proteases (EC 3.4.23.X), and metalloproteases (EC 3.4.24.X).

9. The key mechanistic features of each are as follows:
(1) Serine proteases -contain the catalytic triad Asp, His, Ser.
Amide hydrolysis proceeds via nucleophilic attack of a serine hydroxyl group on the amide carbonyl
to form a covalent acyl-enzyme intermediate with loss of the amine component. The nucleophilicity
of the serine hydroxyl is enhanced bythe adjacent histidine residue, which acts as a general base.
Subsequent reaction of this intermediate with a water molecule yields the product acid.
The serine proteases are divided by sequence homology into the chymotrypsin family (e.g., trypsin),
the subtilisin family, and an undefined group which shows no sequence homology.
(2)Thioproteases - sometimes called cysteine proteases. These proteases follow a similar pathway
to the serine proteases except that the nucleophile is a thiolate anion from the cysteine residue of
the active site. Thus the acyl-enzyme is now a thioester.
Common thioproteases are papain (from papaya latex), ficin (from figs), bromelain (from
pineapple), cathepsin (from mammals), and bacterial peptidases such as clostripain.

10. (3) Aspartyl proteases - so-called because a pair of aspartic acid residues are involved
in the cleavage step.
These act as a general base/general acid to activate a bound water molecule which
attacks the amide carbonyl. Pepsin is an example used in synthesis.
(4) Metalloproteases - these require a divalent metal cation, frequently zinc, which is
bound to specific amino acid residues and the amide carbonyl oxygen.
The attacking water molecule is again activated by a carboxylate anion. No acyl-
enzyme intermediate is formed in this case.

11. Advantages of using proteases in peptide synthesis
mild conditions, freedom from racemization, minimal protection of reacting fragments, and a very
high degree of regio- and enantioselectivity. Synthesis can be carried out either under
thermodynamic or kinetic control, as depicted in Fig. 6.

12. In the thermodynamically controlled process, which is the reverse of
hydrolysis, the equilibrium has to be moved to the right by modifying
the reaction conditions to favor product formation.
For example, use of organic solvents with low water content, biphasic
systems, and product precipitation by careful selection of protecting
groups have all been used in this way.
In contrast, the kinetic aminolysis reaction proceeds via a covalent
acyl-enzyme intermediate which can either be hydrolyzed to the acid
by water or amidated by an added nucleophile such as an amine or
second amino acid fragment.

13. Applications of Penicillin G acylases and peptidases
Synthesis of 6-APA by free enzyme
production of pure chiral compounds
Enantioselective hydrolysis
Protection and deprotection of reactive amino groups
Synthesis of dipeptides
Degradation of organophosphorus compounds
Bioactive peptide synthesis

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