3rd International Conferences and Workshops on Basic and Applied Sciences 2010 ISBN: 978-979-19096-1-7

B013

Active Side of Lactonase, a Barrier to Bacterial Communication, an Anti-Infective Alternative of

Shrimp Disease.

Fadjar, M1, Tripuspaningsih, N.N2., Soegianto, A.A.2, And Aulanni'am3

1 Department of Aquaculture Fac. of Fisheries and Marine Science

Brawijaya University, Malang, Indonesia e-mail: f4dj4r_02@yahoo.com

2 Fac. of Science and Technology

Airlangga University, Surabaya, Indonesia

3Fac. of Mathematics and Science Brawijaya University, Malang, Indonesia

Abstract

Lactonase is an enzyme produced by several species of bacteria with the aim to hydrolyzes the acylated homoserine lactones (AHL) ring, resulting a barrier of bacterial communication and increasing of bacterial density can be prevented , so the virulence of the bacteria can not be happen. Lactonase recombinant protein was derived from cloning of Lactonase gene of Pseudomonas aeruginosa PAO1, which produced a Lactonase recombinant protein with a molecular mass of 30 kDa, Lactonase recombinant protein showed 100% similarity with the putative lactonase. This protein has an active ingredient of Beta lactamase. It has an active side in the Cys (cysteine) , located in the 124th of amino acids.

Keywords: active side, Lactonase, bacterial communication, anti-infective

1 Introduction

In the implementation of best aquaculture in Indonesia should be introduced to the technology most likely to keep production costs for both small and large scale businesses.. Currently, the destruction of bacterial quorum sensing has been designed as an anti-infective strategies are new and some techniques that can be used to interfere with quorum sensing has been investigated. This technique emphasizes: (1)

inhibition of signal molecule biosynthesis, (2) application of quorum sensing antagonists (including those that occur naturally as the synthesis of furanone, quorum sensing antagonists (including those that occur naturally as the synthesis of furanone, quorum sensing antagonist molecules and active ingredients from plants and algae), (3) inactivation of a chemical quorum sensing by oxidized halogen antimicrobials, (4) biodegradation by a bacterial signal molecule lactonase and acylase eukaryotic bacteria, and (5) application of quorum sensing agonists [1]. Through the introduction of bacterial communication means, it is expected to develop a way of controlling bacteria that are not always based on antibiotics, but in a "family" by preventing the occurrence of bacterial mass gathering or when it is already happening mass gathering, used the way which can damage bacterial communication. In this approach no attempt is made to eradicate the bacteria, but allowing the bacteria to live together as long as his behavior is not destructive, among others, by inhibiting its quorum sensing. The phenomenon of bacterial quorum sensing in Vibrio not just happen, but it turns out almost all types of Gram-negative bacterial quorum sensing displays as one regulator of behavior. Gram-positive bacteria using a peptide or a specific protein called paraoxonase (PON) for a similar purpose as the function of acylhomoserine lactone (AHL) in Gram-negative bacteria, which degrade the AHL. Quorum sensing is an important target to prevent disease by making small molecule antagonists are able to weaken the virulence through

Fadjar, Active Side of Lactonase, a Barrier to Bacterial Communication, an Anti-Infective Alternative of Shrimp Disease.

B013

the blockade of bacterial cell communication [2]. Two groups of enzymes, namely acyl-homoserine lactone lactonase (AHL-lactonase) and acyl-homoserine lactone acylase (AHL-acylase), can degrade AHL by lactone and amide bonds hydrolyze [3,4,5, 6, 7, 8., 9, 10].

The use of AHL-Lactonase enzymes based on the fact that the AHL-Lactonase so far is the special enzymes that degrade the AHL to be seen by looking for the active site

2 Methodology

Recombinant protein, Lactonase, was got from cloning of Lactonase encoding gene from P. aeruginosa PAO1, and transformed to E. coli M15 Purification of the Lactonase protein. was done from the resulting supernatant by column chromatography using Ni-TED protocol, and finally by a polishing step on Q-Sepharose columns (from Amersham Biosciences). Fractions were analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot, then pooled based on

the presence of the appropriate bands. Tracing the active side residues was done using www.ncbi.nlm.nih.gov web site.

3 Result

Blastx results showed that this protein showed 100% similarity with hypothetical proteins on the amino acid from the 2nd to the 242th, also called the putative lactonase.

This protein consists of 242 amino acids with the function of molecular activity hydrolyzes the AHL, with the active ingredient Beta lactamase located in the region of amino acids into the amino acids 28 to 220, including the family of metallo-betalactamase (www.ncbi.nlm.nih.gov). From tracing the active side residues via a web-site www.ncbi.nlm.nih.gov, it is known that Beta lactamase has an active hand in the amino acid Cys (cysteine) for sulfur transferase activity. In Lactonase recombinant protein cysteine (C) lies in the sequence of amino acids to 124 and 127 [11] (Figure 1)

Query 43 RIVSRDRWFETRHFYNGISLIHEPYVRPFYRCNMWHVQGRERDVLVDSGSGLVSLCEQLP 222 RIVSRDRWFETRHFYNGISLIHEPYVRPFYRCNMWHVQGRERDVLVDSGSGLVSLCEQLP Sbjct 2 RIVSRDRWFETRHFYNGISLIHEPYVRPFYRCNMWHVQGRERDVLVDSGSGLVSLCEQLP 61 Query 223 WLTERPLLAVASHTHFDHIAGHHEFAERLAHPAEAEILAAPDGDNTLARAYVGDEMFEAH 402 WLTERPLLAVASHTHFDHIAGHHEFAERLAHPAEAEILAAPDGDNTLARAYVGDEMFEAH Sbjct 62 WLTERPLLAVASHTHFDHIAGHHEFAERLAHPAEAEILAAPDGDNTLARAYVGDEMFEAH 121 Query 403 PECPLCYAEYRVRAAPATRLIDEGDVLDLGDRVLQVLHTPGHSPGGISLWEAATQTLFSG 582 PECPLCYAEYRVRAAPATRLIDEGDVLDLGDRVLQVLHTPGHSPGGISLWEAATQTLFSG Sbjct 122 PECPLCYAEYRVRAAPATRLIDEGDVLDLGDRVLQVLHTPGHSPGGISLWEAATQTLFSG 181 Query 583 DIVYDGPLVEDAYHSNLDDYASSLARLRELPVRTVHGGHFASFSGERLREMIVAWFRNHD 762 DIVYDGPLVEDAYHSNLDDYASSLARLRELPVRTVHGGHFASFSGERLREMIVAWFRNHD Sbjct 182 DIVYDGPLVEDAYHSNLDDYASSLARLRELPVRTVHGGHFASFSGERLREMIVAWFRNHD 241 Query 763 R 765 R Sbjct 242 R 242 Figure 1.

Gambar 1: Sequence of Lactonase recombinant protein

A putative lactonase means that this substance is a protein which is not widely known that its activities still require further testing.

C124 sulfrihidril group forming thiophosphate enzyme intermediate with the substrate. Enzyme with the catalytic cysteine has an important role in

biological processes such as setting cycle cells, apoptosis , and signal transduction [12]. With the active side in the C124, AHL-Lactonase is expected to activated lactone bond hydrolysis process in the AHL by the AHL-Lactonase.

3rd International Conferences and Workshops on Basic and Applied Sciences 2010 ISBN: 978-979-19096-1-7

B013

AHL- Lactonase appears as an important enzyme. This enzyme works very well against AHL signals and hydrolyze fourAHL, namely: C4-HSL, 3-oxo-C6-HSL, 3-oxo-C8-HSL, and 3-oxo-C12-HSL [13].

4 Conclusions

Lactonase recombinant protein with a molecular mass of 30 kDa, has similarity with the putative lactonase. This protein has an active ingredient of Beta lactamase and has an active side in the Cys (cysteine) , located in the 124th of amino acids

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