Effect of substitution at C-6 on the susceptibility of pullulan to pullulanases. Enzymatic degradation of modified pullulans.

Pullulan, with all of the primary hydroxyl groups modified, is an excellent substrate for defining the effect of degree of substitution on biodegradability because of the uniform distribution of substituents on the polysaccharide. 6-Chloro-6-deoxypullulan and 3,6-anhydropullulan are highly resistant to hydrolysis by the four different types of pullulanase. 6-Azido-6-deoxypullulan is resistant to three types but susceptible to hydrolysis by the fourth, isopullulanase. Neopullulanase is strongly inhibited by 6-chloro-6-deoxypullulan and 6-azido-6-deoxypullulan, the other pullulanases much less so.

A novel acid phosphatase excreted by Penicillium funiculosum that hydrolyzes both phosphodiesters and phosphomonoesters with aryl leaving groups.

An acid phosphatase has been purified from the culture broth of Penicillium funiculosum by procedures including SP-Sephadex column chromatography and Sephacryl S-200 gel filtration. The phosphatase appears to be a 76 kDa heterodimer composed of 51 and 26 kDa subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme hydrolyzes both phosphodiesters and phosphomonoesters, but only those with aryl leaving groups. At the early phase of degradation of bis-p-nitrophenyl phosphate by the enzyme, inorganic phosphate and the intermediary product, p-nitrophenyl phosphate, are liberated at approximately the same rate. This indicates that the intermediary phosphomonoester produced on the enzyme is further hydrolyzed in situ or dissociates into the medium at approximately the same probability.

Catalytic versatility of trehalase: synthesis of alpha-D-glucopyranosyl alpha-D-xylopyranoside from beta-D-glucosyl fluoride and alpha-D-xylose.

Trehalase was previously shown (see ref. 5) to hydrolyze alpha-D-glucosyl fluoride, forming beta-D-glucose, and to synthesize alpha, alpha-trehalose from beta-D-glucosyl fluoride plus alpha-D-glucose. Present observations further define the enzyme's separate cosubstrate requirements in utilizing these nonglycosidic substrates. alpha-D-Glucopyranose and alpha-D-xylopyranose were found to be uniquely effective in enabling Trichoderma reesei trehalase to catalyze reactions with beta-D-glucosyl fluoride. As little as 0.2mM added alpha-D-glucose (0.4mM alpha-D-xylose) substantially increased the rate of enzymically catalyzed release of fluoride from 25mM beta-D-glucosyl fluoride at 0 degrees. Digests of beta-D-glucosyl fluoride plus alpha-D-xylose yielded the alpha, alpha-trehalose analog, alpha-D-glucopyranosyl alpha-D-xylopyranoside, as a transient (i.e., subsequently hydrolyzed) transfer-product. The need for an aldopyranose acceptor having an axial 1-OH group when beta-D-glucosyl fluoride is the donor, and for water when alpha-D-glucosyl fluoride is the substrate, indicates that the catalytic groups of trehalose have the flexibility to catalyze different stereochemical reactions.

Stability of the cellulase of Trichoderma reesei under use conditions.

Enzyme stability studies have been reinvestigated under the conditions used for cellulose hydrolysis (pH 4.8, 50 degrees C, 24 hr). The cellobiohydrolase (CBH) component as measured on Avicel is less stable than other enzymes of the cellulase complex, and is 60% inactivated by merthiolate (and other Hg compounds) under the above conditions. Endo-beta-1,4-glucanase is much more stable, and more resistant to merthiolate and other compounds. Under unshaken conditions the Avicelase of the Rutgers strain C 30 shows greater stability to heat than that of other available strains. Biocides must be selected not only for their ability to prevent contamination, but also for their compatibility with cellulases. Tetracycline and chlortetracycline are inexpensive, effective in very low concentrations, have no harmful effect on the enzymes, and are compatible with the yeasts that subsequently grow on the sugar solutions to produce alcohol. Attempts have been made to stabilize the enzymes by chemical modification in such a way as to maintain their solubility. Glutaraldehyde treatment greatly increased the enzyme size, lowered the pI values, and gave a slight shift in the pH activity curve. There was, unfortunately, no increase in enzyme stability, and the activity of enzymes on solid celluloses was adversely affected. Shaking greatly reduced the hydrolysis of Avicel by Trichoderma reesei C 30 enzyme. The adverse effect was accompanied by a decrease in recoverable enzyme and protein.

alpha,alpha-Trehalase of Trichoderma reesei.

A simple adsorption and elution of the trehalase of Trichoderma reesei on bentonite increased the specific activity 70-80 times, with a recovery of 90%. This alpha,alpha-trehalase has an optimum pH of 4.4, a pl of 5.7, a Km of 3.1 X 10(-3) M, and a specific activity of 50 mumol/mg. min-1.

beta-Glucosidase: microbial production and effect on enzymatic hydrolysis of cellulose.

The enzymatic conversion of cellulose is catalyzed by a multiple enzyme system. The Trichoderma enzyme system has been studied extensively and has insufficient beta-glucosidase (EC 3.2.1.21) activity for the practical saccharification of celluose. The black aspergilli (A. niger and A. phoenicis) were superior producers of beta-glucosidase and a method for production of this enzyme in liquid culture is presented. When Trichoderma cellulase preparations are supplemented with beta-glucosidase from Aspergillus during practical saccharifications, glucose is the predominant product and the rate of saccharification is significantly increased. The stimulatory effect of beta-glucosidase appears to be due to the removal of inhibitory levels of cellobiose.

Modified substrates and modified products as inducers of carbohydrases.

Cellobiose and isomaltose are both inducers and repressors of cellulase and dextranase, respectively. The repression can be avoided by supplying the disaccharide slowly. This has been done by use of palmitate and acetate esters which are hydrolyzed by esterases of the growing organism to yield the inducer. Sucrase yields, also, are greatly increased (to 80 times) by substituting sucrose monopalmitate for sucrose in the culture medium.

Surfactants as stimulants of enzyme production by microorganisms.

The addition of Tween 80 and sucrose monopalmitate, nonionic surfactants, to fungal cultures resulted in marked increases in yields of the enzymes cellulase, amylase, sucrase, beta-1 --> 3 glucanase, xylanase, purine nucleosidase, and benzoyl esterase. The action appears to be an effect of the surfactant on cell permeability.

Purine ribonucleosidase g from Aspergillus foetidus.

Nucleosidase g was prepared by growing Aspergillus foetidus on bran, and was purified by passage through a diethylaminoethyl-Sephadex column. The enzyme acted on the purine ribosides (except xanthosine) and on their 5'-phosphates. Action on the latter was a good means for preparing ribose-5-phosphate.