Enzymatic cross-linking to improve the handling properties of dough prepared within a normal and reduced NaCl environment.

This research examines the use of three enzymes [glucose oxidase (GOX), hexose oxidase (HOX), and xylanase (XYL)] and their combinations [GOX-XYL and HOX-XYL] on the dough handling properties of CDC Plentiful and Stettler wheat cultivars prepared at reduced (1.0% wt. by flour) and normal (2.0% wt. by flour) NaCl levels. Properties investigated include dough rheology, stickiness, and ratio of resistance to extension and extensibility. The inclusion of XYL and its combinations with GOX and HOX increased the stickiness, yielded lower dough strength indicated by rheology, and reduced the ratio of resistance to extension and extensibility. The inclusion of oxidative enzymes yielded a stronger dough, where HOX addition to dough had the lowest stickiness values and highest |G*| values, whereas GOX addition led to the highest ratio of resistance to extension-extensibility. NaCl only had minor effects overall on dough strength and stickiness for the cultivars studied. Overall, superior dough handling properties were observed with oxidative enzyme addition (GOX and HOX) suggesting that the increased crosslinking that occurs could aid in improving low sodium bread dough properties.

A halotolerant bifunctional ß-xylosidase/α-l-arabinofuranosidase from Colletotrichum graminicola: Purification and biochemical characterization.

A ß-xylosidase from Colletotrichum graminicola (Bxcg) was purified. The enzyme showed high halotolerance, retaining about 63% of the control activity in the presence of 2.5molL-1 NaCl. The presence of NaCl has not affected the optimum reaction temperature (65°C), but the optimum pH was slightly altered (from 4.5 to 5.0) at high salt concentrations. Bxcg was fully stable at 50°C for 24h and over a wide pH range even in the presence of NaCl. In the absence of salt Bxcg hydrolyzed p-nitrophenyl-ß-d-xylopyranoside with maximum velocity of 348.8±11.5Umg-1 and high catalytic efficiency (1432.7±47.3Lmmol-1s-1). Bxcg revealed to be a bifunctional enzyme with both ß-xylosidase and α-l-arabinofuranosidase activities, and hydrolyzed xylooligosaccharides containing up to six pentose residues. The enzyme showed high synergistic effect (3.1-fold) with an endo-xylanase for the hydrolysis of beechwood xylan, either in the absence or presence of 0.5molL-1 NaCl, and was tolerant to different organic solvents and surfactants. This is the first report of a halotolerant bifunctional ß-xylosidase/α-l-arabinofuranosidase from C. graminicola, and the enzyme showed attractive properties for application in lignocellulose hydrolysis, particularly under high salinity and/or in the presence of residues of pretreatment steps.

Characteristics of thermostable endoxylanase and ß-xylosidase of the extremely thermophilic bacterium Geobacillus thermodenitrificans TSAA1 and its applicability in generating xylooligosaccharides and xylose from agro-residues.

An extremely thermophilic bacterial isolate that produces a high titer of thermostable endoxylanase and ß-xylosidase extracellularly in an inducible manner was identified as Geobacillus thermodenitrificans TSAA1. The distinctive features of this strain are alkalitolerance and halotolerance. The endoxylanase is active over a broad range of pH (5.0-10.0) and temperatures (30-100 °C) with optima at pH 7.5 and 70 °C, while ß-xylosidase is optimally active at pH 7.0 and 60 °C. The T 1/2 values of the endoxylanase and ß-xylosidase are 30 min at 80 °C, and 180 min at 70 °C, respectively. The endoxylanase activity is stimulated by dithiothreitol, but inhibited strongly by EDAC and Woodward's reagent K. N-BS and DEPC strongly inhibited ß-xylosidase. MALDI-ToF (MS/MS) analysis of tryptic digest of ß-xylosidase revealed similarity with that of G. thermodenitrificans NG 80-2, and suggested that this belongs to the GH 52 glycosyl hydrolase super family. The action of endoxylanase on birch wood xylan and agro-residues such as wheat bran and wheat straw liberated xylooligosaccharides similar to endoxylanases of the family 10 glycoside hydrolases, while the enzyme preparation having both endoxylanase and ß-xylosidase liberated xylose as main hydrolysis product.

Properties and application of a partially purified alkaline xylanase from an alkalophilic fungus Aspergillus nidulans KK-99.

An alkalophilic Aspergillus nidulans KK-99 produced an alkaline, thermostable xylanase (40 IU/ml) in a basal medium supplemented with wheat bran (2% w/v) and KNO3 (at 0.15% N) pH 10.0 and 37 degrees C. The partially purified xylanase was optimally active at pH 8.0 and 55 degrees C. The xylanase was stable in a broad pH range of 4.0-9.5 for 1 h at 55 degrees C, retaining more than 80% of its activity. The enzyme exhibited greater binding affinity for xylan from hardwood than from softwood. The xylanase activity was stimulated (+25%) by Na+ and Fe2+ and was strongly inhibited (maximum by 70%) by Tween-20, 40, 60, SDS, acetic anhydride, phenylmethane sulphonyl fluoride, Triton-X-100. The xylanase dose of 1.0 IU/g dry weight pulp gave optimum bleach boosting of Kraft pulp at pH 8.0 and temperature 55 degrees C for 3 h reaction time.

Purification and characterization of an intracellular alpha-D-xylosidase II from Aspergillus flavus MO-5.

Alpha-D-Xylosidase II activity from Aspergillus flavus MO-5 was increased roughly 5- to 10-fold by use of xylose instead of methyl alpha-D-xylopyranoside (alpha-MX) as a carbon source. The enzyme was purified to an electrophoretically pure state by successive chromatography on Q-Sepharose, Phenyl Superose, PL-SAX, and TSK-gel G3000SWXL. The purified enzyme hydrolyzed isoprimeverose [alpha-D-xylopyranosyl-(1-->6)-D-glucopyranose] and p-nitrophenyl alpha-D-xylopyranoside (alpha-p-NPX), but not alpha-MX or xyloglucan oligosaccharide. The apparent Km and Vmax of the enzyme for alpha-p-NPX and isoprimeverose were 0.97 mM and 28.0 mumol/min/mg protein, and 47.62 mM and 2.0 mumol/min/mg protein, respectively. This enzyme had an apparent molecular weight of 67,000 by SDS-polyacrylamide gel electrophoresis and 180,000 by gel filtration chromatography (TSK-gel G3000SWXL). The enzyme showed the highest activity at pH 6.0 and 40 degrees C, and was stable in the pH range from 6.0 to 7.0 and at the temperatures up to 40 degrees C. The activity was inhibited by Cu2+, Zn2+, Hg2+, p-CMB, SDS, Fe3+, and N-ethylmaleimide. This enzyme had nothing in common with alpha-D-xylosidase I and four alpha-D-xylosidases reported already.

Positional isomers of thioxylobiose, their synthesis and inducing ability for D-xylan-degrading enzymes in the yeast Cryptococcus albidus.

Isomeric S-linked 2-thioxylobiose 10, 3-thioxylobiose 17, and 4-thioxylobiose 19 were conveniently prepared by SN2 displacement of suitable triflylglycoses with the sodium salt of 2,3,4-tri-O-acetyl-1-thio-beta-D-glucopyranose, either in N,N-dimethylformamide, or in oxolan in the presence of a sodium complexing agent. Allyl 3,5-O-isopropylidene-2-O-trifluoromethanesulfonyl-beta-D-lyxofu ranoside was a convenient electrophilic precursor for 10, which was smoothly obtained after a short sequence of deprotection involving conversion to the 1-propenyl glycoside. 1,2:5,6-Di-O-isopropylidene-3-O-trifluoromethylsulfonyl-alpha-D-++ +allofuranose and 1,2,3-tri-O-benzoyl-4-O-trifluoromethylsulfonyl-beta-L-arabinop yranose were the respective precursors for 17 and 19. 4-Thioxylobiose has a highly stimulatory effect on the synthesis of enzymes of the xylanolytic system in the yeast Cryptococcus albidus when applied to the cells in the presence of the natural disaccharide inducer (1----4)-beta-D-xylobiose.

Purification and characterization of beta-xylosidase activities from the yeast Arxula adeninivorans.

beta-Xylosidase activity has been detected in cell-free extracts, in culture fluids and as cell wall-bound enzyme of Arxula adeninivorans. With chromatographic procedures used to purify the activity two different forms of beta-xylosidase (denoted beta X-1 and beta X-2) from the cell-free extract and from the culture medium could be separated, but only one form (beta X-2) was present in the cell wall. Both forms are glycoproteins and were deglycosylated by endoglycosidase H treatment. The molecular masses of the enzymes under native and denaturing conditions suggest that beta X-2 is the dimer of beta X-1. M(r) of the native beta X-1 was 60 kDa and after deglycosylation 39 kDa determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Some enzymatic properties of beta X-1 and beta X-2 and of their deglycosylated products were studied and showed, with one exception, wide similarities. The maximum activity was reached at pH 5.0 and 60 degrees C. The enzymes hydrolyze only beta-glycosidic bound beta-xylopyranosides and the Km values for p-nitrophenyl-beta-xylopyranoside were determined to be 0.23-0.33 mM. The beta-xylosidase activity was inhibited competitively by xylose. The deglycosylated enzymes were, however, stronger inhibited (Ki = 2.1 mM) as their glycosylated ones (Ki = 5.8 mM).