Pullulan specificity

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

Biely et al (59) first reported the presence of acetyl xylan esterases in fungal cellulolytic and hemicellulolytic systems Trichoderma reesei, Asper llus niger, Schizophyllum commune and Aureobasidium pullulans. As compared with plant and animal esterases, these fungal esterases exhibited high specific activities... 

Abstract Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review, we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran, and schizophyllan each have unique properties and potential for modification, and these results are discussed with the focus on facile synthetic routes and favorable performance in biological systems. Many of these carbohydrates have been used to develop alternative types of biomaterials for nucleic acid delivery to typical polyplexes, and these novel materials are discussed. Also presented are polymeric vehicles that incorporate copolymerized carbohydrates into polymer backbones based on polyethylenimine and polylysine and their effect on transfection and biocompatibility. Unique scaffolds, such as clusters and polymers based on cyclodextrin (CD), are also discussed, with the focus on recent successes in vivo and in the clinic. These results are presented with the emphasis on the role of carbohydrate and charge on transfection. Use of carbohydrates as molecular recognition ligands for cell-type specific dehvery is also briefly... 

The research performed by several groups on pullulans has demonstrated their potential as nucleic acid delivery vehicles. Although most of the pullulan-based delivery systems yielded low toxicity, some modifications of the backbone or introduction of substituents resulted in higher toxicity. Such modifications are unavoidable because the parent structure is incapable of efficient delivery and lacks target specificity. 

The plant and bacterial enzymes capable of hydrolyzing pullulan do not have identical specificities. In particular, the plant enzymes have little or no action on glycogen and phytoglycogen under conditions in which they readily hydrolyze amylopectin and its /3-dextrin. To stress this difference (the bacterial enzymes are capable of degrading both glycogen and phytoglycogen), Manners (1997) recommended different nomenclature for bacterial enzymes, to be called pullulanase, and the plant enzymes, to be called limit dextrinases. 

Pullulanase type I hydrolyzes a-1,6 linkages in amylopectin, pullulan or limit dextrins with high specificity. Pullulan is completely degraded in a random fashion... 

Information on the hydrolytic activity in marine sediments has been obtained from the use of model substrates labeled with fluorescent dyes such as methylumbelliferone (MUF) or fluorescein. These substrates may be small dimeric molecules, the hydrolytic cleavage of which releases the fluorescence signal, which is then indicative of the activity of specific enzymes such as glucosidase, chitobiase, lipase, ami-nopeptidase or esterase (Chrost 1991). Also large fluorescently labeled polymers such as the polysaccharides laminarin or pullulan have been used in experiments to demonstrate the mechanism and kinetics of bacterial degradation (Amosti 1996). 

Ethyl-2-(/J)-hydroxy-2-(T,2, 3. 4 -tetrahydro-T,T.4, 4 -tetramethyl-6 -naphthalenyl) acetate 53 (Figure 16.14) and the corresponding acid 54 were prepared as intermediates in the synthesis of the retinoic acid receptor gamma-specific agonist [86]. Enantioselective microbial reduction of ethyl 2-oxo-2-(T,2, 3. 4 -tetrahydro-T,T,4, 4 -tetramethyl-6-naphthalenyl) acetate 55 to alcohol 53 was carried out using Aureobasidiumpullulans SC 13849 at a 98% yield and with an EE of 96%. At the end of the reaction, hydroxyester 53 was adsorbed onto XAD-16 resin and, after filtration, recovered in 94% yield from the resin with acetonitrile extraction. The recovered (/ )-hydroxyester 53 was treated with Chirazyme L-2 or pig liver esterase to convert it to the corresponding (/ )-hydroxyacid 54 in quantitative yield. The enantioselective microbial reduction of ketoamide 55 to the corresponding (/ )-hydroxyamide 52 by A. pullulans SC 13849 has also been demonstrated [86]. 

The FTIR characterization of dextrin, miCTocrystalline cellulose, hydroxypropyl methylcellulose, pullulan, alginate, carrageenan, chitosan, gnar gum, and gelatin biomaterials was successfully performed. Also specific markers for each tested biomaterials were identified. 

Iwamoto et al. suggested that the tissue distribution of an O/W emulsion can be controlled by coating their surfaces with a cell-specific cholesterol-bearing polysaccharide, such as pullulan, amylopectin, and mannan (10,24). 

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