Rubber 1

Isolation and Purification of Rubber – Degrading Enzymes From a bacterial culture .

Assistant Prof. Kareema Z. Khalef

Introduction : Spence and Van Niel observed in 1963 the formation of clear zones surrounding actinomycetes colonies on a latex-agar plate , an indication of extracellular enzymatic decomposition of natural rubber latex [1] . Since then , there have been a number of reports concerring microbial degradation of natural rubber [2]. A ctinomycetes has been considered the most important organisms with degradation of natural rubber products [3]. Natural rubber in the latex state was degraded by the crude enzyme. There is two Fraction observed from reaction products. One is natural rubber and synthetic isoprene rubber poly (cis – 1,4 – isoprene )[4]. Natural rubber is necessary as a basic material for several items such as tires , gloves , condom , seals. Poly (cis – 1,4 – isoprene ) is subjected to biological mineralization cycle [5] . Xanthomonas Sp. Has the ability to utilize poly (cis - 1 , 4 - isoprene ) is initiated by the oxidative cleavage of the isoprene double bond. Pseudomonas aeruginosa was able to considerably disintegrate natural rubber. The degredative of natural rubber and synthetic poly (cis – 1,4 – isoprene ) by gram – positive and gram – negative bacteria was analyzed by weight loss, gel permeation chromatography and determination of protein content [6] .

There is a Novel type of Heme – dependent oxygenas catalyzes oxidative cleavage of rubber . Enzymatic cleavage of rubber by purified protein was Strictly dependent on the presence of oxygen. Analysis of the amino acid sequence deduced from the cloned gene revealed the presence of two heme –binding motifs covalent attachment of heme to the protein. It must be do Genetic analysis of bacterial strains degrading rubber in order to get specific gene responsible for degradation.

Motivation: Rubber – Degrading by Bacteria is a big problem in rubber industries . The natural rubber is necessary as a basic material for several items such as tires , glovers , condom … etc. So this project will try to solve this problem , to a cheave this goal we have to do many experiment in the biological basis , biotechnology and biochemistry ways.

Aims and Objectives : The project will undertake with the following aims and objectives : 1- The preliminary study of molecular analysis of rubber – Degrading bacteria. 2- Genetic analysis of rubber degrading bacteria.

Expected outcomes: I want to modify the gene which responsible for degradation and I expect that a bacterium Xanthomonas be a potent producer of rubber – Degrading enzyme.

Materials : 1- Gas – liquid chromatography . 2- Gel – permeation chromatography.

3- Scanning electron microscopy . 4- Schiff's reagent staining. 5- Erlenmeyer flasks. 6- Yeast extract. 7- Pentane solution. 8- Luria –Bertani medium.

Proposed Project Planning

Bacteriological Study

Micro organism and Culture

Isolation of Bacterial Species

Identification

Molecular Basis PCR

Preparation of the Gude enzyme

DNA extraction from isolates Purification (gel chromatography) Amplification of Gene Electrophoresis (Agars gel) Sequencing

Electrophoresis (SDS-PAGE)

References: [1] Spence ,D. and C. B. van Niel 1936. Bactrial decomposition of rubber in Hevea latex , Ind . Eng. Chem. 28: 847-850. [2] zuska , B. J. 1981 Rubber , P. 323 – 385 . In A. H. Rose (ed), Economic microbiology , Vol. 6. Microbial biodeterioration . Academic press , Inc. NewYork. [3] Rook, J.J. 1955 Microbiological deterioration of vulcanized rubber . Appl. Microbial . 3:302-309. [4] Tsuchii, A. and Takeda, K. (1990) Rubber- Degrading enzyme from a bacterial culture. Appl. Environ , Microbial 56(1) : 269-274. [5] Bode, H. B. , Zeeck , A. , Pluckhahn , K. and Jendrossek , D. (2000) . Physiological and chemical investigations into microbial degradation of synthetic poly(sic-1 , 4 – Isoprene). Appl. Environ . Microbial . 66 (9) : 3680-3685. [6] Lions A, Reichelt R, Keller A, Stein Buchel A. A gram – negative bacterium identified as pseudomon as aeruginosa AL 98 , is a potent degrader of natural rubber and synthetic (sic-1 , 4 – Isoprene). , 2000 . FEMS microbial lett. 182:155161.