Document Type : Research Paper

Author

Anbar University - College of Education

Abstract

The aim of the research was to produce Cellulase via the use of two isolates
of Cytophaga bacteria. The first isolate, given the local code CR1, was taken from
AL-gazeera region soil in Ramadi grown with okra. The second, given the local
code CR2, was taken from animal waste.
Wheat barn and corn wastes (grimes) were used as only carbon source in
the culture media where the two isolates have been cultivated. However these
wastes were available in big amounts in this environment.
CR2 showed a great ability to produce Cellulase in the liquid medium
culture .The best production was at pH 7.5 and 35c temperature using wheat barn
of 1.5% cellulose concentration. When incubated in an incubator shaker of 125
R.p.m and a bacterial density of 1ml/100ml medium. After 5 days of incubation,
the enzymatic activity was 5.976 unit/ml.
The results have shown that adding calcium and magnesium ions as sulfates
of 0.5g/100ml,concentration gave higher production of the enzyme. The enzyme
activity was 6.121unit/ml and 6.212unit/ml,respectively.
The enzyme produced from the local isolate CR2 was purified following
purification procedure that included precipitation using ammonium sulfates of
90% saturation and ion exchange chromatography via replacing the column
DEAE-Sepharose.This was followed by gel filtration column of Superdex-200.It
was possible 64.11% of the enzyme with 4.109 times of purification. The results of
enzyme description showed that its molecular weight was about 32000 Dalton
using gel filtration chromatography of a Superdex-200 column, and that the
optimum pH of the enzyme activity was 6.0 .The optimum temperature of the
enzyme activity was 50c .The results indicated, also, that the enzyme gave the
highest enzymatic activity after 60 minutes of incubation at a pH6.0 and 50c
temperature.

Keywords

Main Subjects

1- Ojumu, Tunde Vicetor; Solomon Bamidele; Betiku, Layokun; Stephen Kolawole and Amigum, Bauikolele. (2003). Cellulase production by Aspergilluse flavus Linn isolate NSPR 101 fermented in sawdust,bagasse and corncop,African J of Biote.2(6):150-152.                      
2- Heiner, A.P and Teleman, O.(1997)."On the structural crystalline cellulose" in (Abstract Tricel of carbohydrates from Trichoderma reesei and other microorganisms). P: 204; Royal Society of chemistry: Cambridge; UK. 
3- Mandels, M. Andreotti, R and Roch, C. (1976). Measurement of sacchrifying cellulose. Biotechnol. Bioeng Symp.6:21-23.
4-   Ohmiya, K; Sakka, K.Karita, S and Kimura; T. (1997)."Cellulase "In (Biotechnology and Engineering Reviews.14: 365).
5-   Christensen, P.J. (1977).The history, biology, and taxonomy of the Cytophaga group. Can. J. Microbiol.23:1599-1653.
6- Chang, W.T. and Thayer, D.W. (1977).The Cellulase system of a Cytophaga species. Can. J. Microbiol.23:1285-1292.
7-    Hunter, J.B.and J.A.Asenjo. (1987). Kinetics of enzymatic lysis and disruption of yeast cells: Evaluation of two lytic systems with different Properties. Biotechnology and Bioengineering. 30 (4): 471-480.
8- Li.X, Z.Zhou, S.Zhang, F.Jin and P.Gao. (1997). Production and properties of a cellobiose-oxidizing enzyme from a newly isolated celluloltic bacterium Cytophaga sp.LX-7.World Journal of Microbiology and Biotechnology . 13 :683-688.
9- Dahot Umar.M and M.Hanif Noomrio. (1996). Microbial production of Cellulase by Aspergillus fumigates using wheat straw as carbon source. Journal of Islamic Academy of Sciences.9.(4).
10- Takuya Koseki;Shinji Furues; Kinino Iwano; Hiroshi Sakai and Hiroshi Matszawa.(1997).An Aspergillus awamor acetylesterase:Purification of the enzyme ,and cloning and sequencing of the gene. Biochem.J.326: 485-490.
11- Dong Won Kim, Young Kyn Jeong; Young Hun Jung; Jae Kuk Lee;Ki Sung Kim and Haill Ryu.(1995).Kinetic mechanism of cellulose hydrolysis by endo glucanase 1 and Exo glucanase 11 purified from Trichoderma viride. Bull. Korean. Cham. Soc.16: 742-746.
12- Robson, L.M and Chambliss, G.H. (1984). Characterization of the celluloltic activity of a Bacillus isolate. Appl.Environ.Microbial.47: 1039-1046.
13-Fred, J, Stutzenberger. (1970). Cellulase production by Thermomonospora currata isolated from municipal solid waste compost .Appl.Microbial.22 :(2):147-152.
14- Tawfiq, Aziz, Asal. (2000). Isolation and identification of Celluloltic Setreptomyces species from the local soil .A thesis submitted to the College of Science of Saddam University.
15-Kotchoni, O.S; Shonukan; O.O and Gachomo, W.E. (2003). Bacillus pumilus BPCRL 6, a promising candate for Cellulase production under conditions of catabolite repression .African Journal of Biotechnology .2(6):140-146.
16- Micheal, EHimmel. (1999). Improved Cellulase enzyme at NREL.U.S.Department of Energy, Office of Fuels Development. 
17- Mandels Mary. (1974). Production and application of Cellulase laboratory procedures. Handbook, U.S.Army materials laboratories.
18- Miller, G.L.(1959).Use of the dinitrosalicylic acid reagent for the determination of reducing sugars .Anallyt.Chem.31:426-428.
19-Laue, T.M. and Rhodes, D.G. (1990). Gelfilitration In Methods of Enzymology (ed. Deutsher, M.P.) .182:556-587.Academic press.
20- Chen, Po-Jui, Tao-Chunwei, Yao-Tsung Chang, and Liag-Ping Lin. (2004) Purification and Characterization of Carboxymethyl cellulose from Sinorhizobiun fredii. Bot. Bull. Acad. 45: 111-118.
 21-McCarthy, A. J. (1987). Lignocelluloses - degradation by Actinomycetes. FEMS. Microbiol. Rev. 46:145-163.
22-Davies, R. (1963). Microbial extra cellular enzymes their uses and some factors affecting their formation in" biochemistry of industrial microorganism "(Eds Rainbow. and Rose,A.H.,) Academic press, New York.
23-Purohit, S. S, and Mathur, S. K. (1996). Enzymes bioacceleractors in: Biotechnology, Fundamentals, and application publisher. India.
24-Englard, E. M., and Sifters. (1990). Precipitation in: Methods in Enzymology (ed. Murray, E.D. and Dentscher ,P.). 182: 425-441.
25-Chauthaiwale, J.V. and Rao, M.B. (1994).Production and purification of extra cellular D-xylose isomerase from alkaliphilic, thermophillic Bacillus sp. Appl Environ. Microbial .60:4495-4499.
26-Brown, S. H., Sjoholm, C.and Kelly, R.M.(1993). Purification and characterization of a highly thermostable glucose isomerase produced by the extremely thermophillic Eubactrium thermotoga maritime. Biotechnol. Bioeng.41:878-886.