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 (SDS-PAGE)
Electrophoresis (Agars gel)

Sequencing
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. 182:155-
161.