Pseudomonas cellulase

Figure 8. Ultracentrifugal patterns of Pseudomonas cellulases Pictures were taken at 45 minutes after rotation had reached a maximum speed of 55,430 r.p.m. at 17°C. Protein concentration was 0.45% (Cellulase A) and 0.7% (Cellulases B and C) in 1/15M phosphate buffer of pH 7.0. The synthetic boundary cell was employed for Cellulases B and C...

Table II. Constituent Sugars Detected on Paper Chromatograms of Acid Hydrolyzate of Pseudomonas Cellulases A, B, and C...

Table VI. Changes in CMC-saccharifying Activities of Pseudomonas Cellulase Components During Incubation with Broken Spheroplast Preparation...

Ps. fluorescens var. cellulosa synthesized a large amount of cellulase in 0.5% cellulose or sophorose medium, and more than 90% of the enzyme formed was released in surrounding medium (exo-type synthesis). In contrast, smaller amounts of cellulase were formed upon cultures on 0.5% cello-oligosaccharides, and more than 90% of the enzyme remained within the cells (endo-type synthesis). Since the major soluble end-products of cellulolysis by Pseudomonas cellulases in vitro were cellobiose and cellotriose (28), the direct C-source utilized by the bacterium should be these sugars even if either cellulose or cello-oligosaccharides were used as C-source. The only difference between these cultures, therefore, may be in the concentration of the end-products in each medium. 

Clostridium thermocellum Cellulase D (27) Pseudomonas fluorescens CMCase (22)... 

Cellulolytic bacteria can be found which produce only cell-bound cellulase such as Cytophaga (12), only cell-free cellulase, such as CelMbrio vulgaris (21), Bacillus sp. (22), Clostridium sp. (23), Acetivibrio cellulofyticus (24), and Thermoactinomyces (25,26), and both cell-bound and cell-free cellulase such as Pseudomonas (27), Bacteroides succinogenes (28), and CelMbrio futvus (29). However, the location of cellulase in bacteria is also dependent upon the environments in which the bacteria are grown and the age of the culture (29,27). 

Figure 1. The organization of catalytic and non-catalytic domains in cellulases from C. fimi and other bacteria. CfCenA, B and C, and CfCex are the endo- and exo-p- 1, 4-glucanases of C. fimi, ClfX is a translated open reading frame from Cellulomonas flavigena (29), CtEGD and PfEndA are endo-p-1, 4-glucanases from Clostridium thermocellum and Pseudomonas fluorescens, respectively (30,31), The primary structures are drawn approximately to scale and are numbered from the amino terminus of the mature protein ClfX is numbered from the start of the open reading frame. Unshaded areas represent catalytic domains, cross-hatched areas indicate cellulose-binding domains, repeated blocks of amino acids are stippled, and black areas represent linker regions.

Pseudomonas fluorescens produced two extracellular (A and B) and one cell-bound (C) cellulase components, the latter being released by treatment with EDTA-lysozyme in isotonic sucrose. Culture with 0.5% glucose formed little cellulase. Cellobiose stimulated only the synthesis of C. The formation of A and B was strikingly enhanced in cultures with cellulose, sophorose, or continuous low concentration of cellobiose. The absence of extracellular cellulase synthesis in 0.5% cellobiose culture may be caused by catabolite repression. The three cellulases were purified and characterized. None of them split cellobiose, but all hydrolyzed various cellodextrins and celluloses. C easily attacked cellotriose and cellotriosyl sorbitol, but A and B had no effect. When pure B was incubated with broken spheroplasts of sophorose-grown cells, a cellulase component indistinguishable from A was formed. 

For the last few years, we have investigated the cellulase components of Pseudomonas fluorescens var. cellulosa in special regard to the physiological relation of their synthesis and localization to the cultural conditions. This pseudomonad is an aerobic mesophilic cellulose-decomposing bacterium isolated earlier by Ueda et al. (39) from field soil. Some of the enzymatic properties of cellulases obtained from it have previously been reported (28). In the present review, the results of our recent studies (42) are described and discussed together with the related works of other authors. [In these studies, activities of cellulase and aryl / -glucosidase were assayed by the same methods as described in a recent paper (31), and that of amylase by the blue value method modified by Fuwa (10)]. 

Cellulase Formation during Bacterial Growth. The cellulolytic Pseudomonas is, like Clostridium and Cellulomonas (12), known to produce cellulases constitutively, though they are very small in amount (0.5-5 CMC-liquefying units/ml. culture) when it was grown on individual various lower alcohols, organic acids, or amino acids as C-source (41). 

A sesquiterpene phytoalexin, C17H26O4, Mr 294.39, oil, [ain -35.9° (CHCI3) formed, together with rish-itin and many other components (including vetispi-ranes), by potatoes Solarium tuberosum) after infection by the putrefactive bacterium Erwinia carotovora. Deacetylation furnishes phytuberol, C,5H240j, Mr 252.35, oil. P. can also be isolated from the culture medium of potato cell cultures after elicitation with Phy-tophthora infestans. Some sorts of tobacco Nicotiana tabacum) produce P. after infection with various Pseudomonas spp. or the tobacco mosaic virus pretreatment with a cellulase shows the same effect. 

Cellulase-producing bacteria include CelMomonas, Clostridium, Pseudomonas, Streptomyces, Thermonospora, and Ruminococcus, but bacterial cellulases digest cotton fibers to lower levels compared to amorphous celluloses (Ray et al., 2006). Cellulases have two functionally distinct domains in their structure, namely catalytic domain and cellulose substrate binding domain, linked by an interdomain, glycosylated linker peptide at either the N or the C terminal of the protein cellulose binding domains exhibit different affinities, specificities, some binding to crystalline cellulose, while others restrict themselves to the disordered regions. Catalytic domain has an active site in the shape of a tunnel or cleft where hydrolytic reactions take place (Cavedon et al., 1990 Lee et al., 1996). 

The fungus Papulaspora thermophila has been shown to degrade filter paper and soluble carboxymethylcellulose. Synthesis of the cellulolytic enzymes could be induced by cellulosic material added to the growth medium. A cytosolic P-D-glucosidase from Pseudomonas fluorescens possesses some exo-cellulase activity. " " ... 

Gray GL, Power SD, Poulouse AJ (1995) Lipase from Pseudomonas Mendocina having cutinase activity. US Patent 5,389,536 Griffiths AD, Tawfik DS (2003) Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. EMBO J 22 24-35 Gusakov AV, Sinitsyn AP, Berlin AG, Markov AV, Ankudimova NV (2000) Surface hydrophobic amino acid residues in cellulase molecules as a structural factor responsible for their high denim-washing performance. Enzyme Microb Technol 27 664-671... 

---