CHAPTER IV

RECOMBINANT OF PENICILLIN ACYLASE

4.1

INTRODUCTION

Penicillin Acylase (PGA, EC 3.5.1.11), as a subclass of B-lactam antibiotics acylase superfamily, is enzyme that catalyse the selective hydrolysis of relatively stable amide bond in penicillin and some cephalosporins while leaving the labile Bring intact. Penicillin acylase are of great importance in pharmaceutical industry to apply to the production of semi synthetic B-lactam antibiotics via the key intermediates 6-aminopenicillanic acid (6-APA) and 7-amino-3-

deacetoxycephalosporanic acid (7-ADCA). Penicillin acylase is useful as a biocatalyst in many potentially valuable reactions such as protection of amino and hydroxyl groups in peptide synthesis and resolution of racemic mixtures of chiral compounds. (Wen-Jer et al, 2000)

4.2 PENICILLIN ACYLASE PRODUCING BACTERIA

Bacteria such as Bacillus megaterium, Arthrobacter viscosus, Kluyvera cryocrescens and Providencia rettgeri produce penicillin acylase. These penicillin acylase share the same heterodimer protein structure, and their amino acid structure reveal high homology, suggesting that they might originate from the same ancestral gene. The enzyme are synthesized as a single cytoplasmic precursor and their maturation occurs

by removal of signal peptides at the inner membrane, processing of the spacer peptide in the periplasm followed by formation of a biochemically active PGA composed of two different subunits( , approximately 23kDa, and , approximately 65kDa.) (Yong Wen and co, 2005)

4.3 Escherichia coli AS HOST CELL Gene-expression systems is used to produce desired proteins in large amount. In the case of Penicillin acylase production, Escherichia coli is chosen as the host recombinant. This is because the well-known genetic information of E.coli makes it possible for a variety of attempts using genetic engineering, metabolic engineering and protein engineering techniques. Besides that, the growth rate and the ease of cultivation for E.coli make it suitable for industrial application.

4.4 GENETIC MANIPULATION TECHNIQUES Various strategies have been developed for the heterologous overproduction of penicillin acylase (PAC), which is an important industrial enzyme for the production of many b-lactam antibiotics (Shewale et al., 1990). The mature PAC is located in the periplasm and the formation of PAC requires a series of post-translational steps (Sizmann et al., 1990), including translocation and periplasmic processing/folding steps, which are unusual for prokaryotic proteins. The expression of the wild type E. coli acylase gene (pac) is induced by PAA and repressed by glucose, these effects being exerted at the transcriptional level (Merino et al., 1992). It had been suggested that a pac-specific repressor protein, encoded by a putative pacR gene transcribed in the opposite direction to pac from within the pac coding sequence, is the mediator of the induction (Jiang et al., 1997). However, lacZ fusion studies have shown that an intact pac gene is not required for PAA induction (Rao and Garcia, 1999). PAC is normally produced at temperatures between 20 0C and 30 0C. It is not produced at 37
0

C. It is not thought that lack of activity is due to thermal inactivation of the protein

since the optimal enzyme activity is 50 0C (Savidge & Cole, 1975). It is more likely that temperaturesensitive proteolytic processing is involved in the maturation of the

active enzyme (Oh et al., 1987). In addition, improper folding at the higher temperature results in enzyme inactivity (Lindsay and Pain, 1991)

4.4.1 Methods In Genetic Manipulation Techniques.

Genomic DNA from E.coli ATCC11105 and plasmic DNA isolation is carried out according to standard procedures (Sambrook et al. 1989). Purification of PCR products and extraction of DNA fragments from agarose gels is performed using appropriate commercial kits (Bio Rad Co., USA). Electroporation of DNA was done using BTX Electroporation system (BTX Co., USA). Amplification of pac gene was carried out by PCR using a pair of primers (primer 1 and primer 2) and E. coli ATCC11105 genomic DNA as a template under following conditions which is 30 cycles of temperature profile 94 oC for 1 minute, 48 oC for 1 minute and 72 oC for 2 minutes.(Rakesh et al, 2001)

Figure 4.1 The pac gene inserted into plasmid of E.coli

4.4.2 Growth Curve

Two ml of overnight culture of pacC recombinant E. coli strain was added to 1-l flasks containing 200 ml LB medium. The flasks were incubated at 28 oC, 150 rpm for 48 h. Samples were withdrawn at intervals and analyzed for PAC activity, pH value and cell growth (measured turbidometrically at 600 nm ). (Rakesh et al, 2001)

4.4.3 Assay For Penicillin Acylase Activity PAC activity of cells was analyzed. The bacterial colonies grown on LA plates were analyzed for qualitative PAC production by screen plate technique (Meevootisom et al. 1983), whereas quantitative assay of PAC was done by colorimetric method (Balasingham et al. 1972) and HPLC analysis. For HPLC, a C-18 column (150 x 4.6 mm) was used. The mobile phase consisted of water/acetonitrite/0.05 M KH2PO4 buffer, pH 6.5. The flow rate of mobile phase was 1.2 ml/min. The product (6-APA) was detected at 254 nm. One unit of enzyme activity is defined as the amount of enzyme required to produce 1 mol 6-APA per min from benzyl penicillin at 37 oC in 0.05M phosphate buffer, pH 7.5. (Rakesh et al, 2001)

Figure 4.2 Pac formation pathway based on the expression of wild type in E.coli. The structural wild type pac gene enclodes a popypeptide precursor (preproPAC) composed of starting at the N terminus a single peptide (S), and a subunit (), a connecting peptide (C ) and a subunit (). The signal peptide directs the export of preproPAC into the periplasm and is cleaved after translocation.

4.4.4 Plasmid Stability

To determine the stability of recombinant plasmids in E. coli 6212 host, the recombinant E. coli was incubated in 250 ml flasks containing 25 ml LB (28 0C, 150 rpm, 24 h). Daily, 1 ml culture was inoculated into fresh LB medium and incubated under same conditions .The cultures were analyzed for the presence of recombinant asdC pacC plasmid. The cultures were also analyzed for PAC activity by colorimetric method and HPLC analysis.

Figure 4.3 - Schematic diagram of plasmid Prt4. Expression of pac gene is regulated by ptrc promoter and arrow indicates direction of transcription.

4.5 PROBLEMS IN RECOMBINANT TECHNIQUES

The instability of the plasmid carrying active penicillin acylase gene can affect the production of penicillin acylase. The plasmid injected into the E.coli may not be stable hence not producing the desired product. In the absence of any selective pressure and under fully induced production of penicillin acylase the number of cells lacking plasmids increased during cultivation. Cells still harboring plasmids fully maintained their ability to synthesize the enzyme. However, when a selection for Tetr was applied,

the

number

of

selected

cells

containing

the

actively synthesizing

gene

decreased. Changes occurring in plasmid DNA revealed high frequency of insertions affecting expression of penicillin acylase gene.

4.6 INCREASING PRODUCTION BY ENHANCING EXPRESSION OF PAC

Classical mutation and selection procedures have been applied to enhance the production of PAC from many micro-organisms. Recombinant DNA technology has made it possible to clone and express the pac gene from many sources. The gene has been cloned into multicopy vectors to increase gene dosage. This has been achieved from species including E. coli (Oh et al., 1987), Arthrobacter viscosus (Ohashi et al., 1989), P. rettgeri (Daumy et al., 1986), Kluyvera citrophila (Barbero et al., 1986), Bacillus megaterium (Meevootisom and Saunders, 1987), and A. faecalis (Verhaert et al., 1997) . Segregational instability of recombinant plasmids in the absence of selective pressure is widespread. To overcome this effect, the E. coli pac gene has been cloned in pYA292, which is stable in strain 6212 in the absence of challenge. The resultant clone produces higher PAC levels (Vohra et al., 2001). The location of the mature PAC protein can determine the efficiency of production. The penicillin acylases from gram negative bacteria accumulate within the periplasmic space, whereas those from gram positive bacteria tend to be secreted. The transcription of the pac gene in E. coli is inefficient due to the nonoptimal disposition of bases present in the regulatory sequence. The translation of the message is also relatively poor due to there only being four bases between the ATG start codon and the ribosome binding site. PAC expression has been enhanced by placing the pac gene under the control of a strong promoter and optimising the spacing upstream of the translational start (Chou et al., 1999). However, one of the drawbacks to this approach was the subsequent production of inclusion bodies which reduced the level of PAC activity recovered. Since active PAC is formed in the periplasmic space of E. coli, engineering of strains to improve transport and folding would be expected to be beneficial to PAC production. This has led to significant improvement in recombinant PAC production. For the commercial manufacture of PAC, combinations of the aforementioned

approaches have been applied to production strains to improve the efficiency of the process.

4.7 IMMOBILISATION

Penicillin acylase enzymes have proved effective in their role as biocatalysts for both synthesis and hydrolysis, but their industrial application is often limited by the lack of long term operational stability, and of adequate steps for the recovery and re-use of the enzyme. Immobilization of enzymes provides a suitable tool to help tackling with all these requirements. Enzyme immobilization overcomes major problems that are inherent to the use of soluble enzymes. These include product contamination, difculty of separation from the reaction mixture, and limited reuse, while enabling the use of enzymes in continuous mode of operation . A further benet of immobilization, which is often quoted, is enhanced stability, under both storage and operational conditions, since immobilization provides a protective (micro) environment towards denaturation by heat or organic solvents, or by autolysis . However, immobilization brings along some drawbacks, such as: (a) loss of activity following immobilization and diffusion limitations, which reduce the productivity of the immobilized enzyme system as compared to the free form; and (b) increased process costs, inherent to the immobilization step and to the processing of the spent immobilized biocatalyst The major techniques formimmobilization reported in the literature include covalent binding, ionic and hydrophobic adsorption, aggregation and entrapment. There are a few types of immobilization which can be used for penicillin acylase. (Susana Bernardino, 2011)

4.6.1 Cross-linking

The widespread use of this methodology is somehow hampered by the ability of enzymes to resist chemical cross-linking. Furthermore, diffusion limitations are also still prone to occur, due to aggregation The difculty in the handling and in the recovery of biocatalyst particles, due to their low mechanical stability, is also a main drawback of cross-linking immobilization. (Susana Bernardino, 2011)

4.6.2 Sol-Gel Technique

One promising method for the immobilization of enzymes is a solgel technique, where enzymes are con- ned within a chemically inert silica support, which is There are several advantages of the solgel matrixes, in general: (a) they are easily obtained in a variety of forms: monoliths, optically grade polished monoliths, thin lms, bers, powders, etc., that can be miniaturized to micron size, and can be furthermore attached to most other materials (plastic, paper, metals, etc.); (b) the process occurs at near room temperature, which avoids protein denaturation; (c) the easy insertion of substituting groups into the matrix provides the entrapped enzymes with benecial microenvironments; (d) the matrixes have controllable surface area, average pore size and pore size distribution; (e) they are thermally stable, much beyond the range of temperatures which is of relevance for biocatalysts; (f) are not photodegradable and, in the case of SiO2 matrixes, are not involved in light-induced reactions with the matrix; (g) do not degrade electrochemically; (h) can be transparent well into the UV range (*250 nm for SiO2), and are highly suitable for optical applications; (i) allow control of conductivity; (j) have a potential advantage over the use of surfaces in the form of adsorption or covalent bonding, since adsorbed molecules easily leak out, and covalently attached molecules are prone to chemical degradation of the anchoring bond.(Susana Bernardino, 2011)

Figure 4.4 SEM micrograph of silica sol-gel particles of penicillin acylase crushed in a mortar.