Purification and characterization of two different xylanases from the thermophilic actinomycete Microtetraspora flexuosa SIIX.

Two endoxylanases were isolated from the xylanolytic enzyme system of the thermophilic actinomycete Microtetraspora flexuosa SIIX, and purified by ammonium sulfate fractionation, DEAE-Sepharose chromatography, gel filtration on Sephacryl S 200 and fast protein liquid chromatography on Q-Sepharose. The molecular masses of xylanase I and II were 26.3 and 16.8 kDa, and isoelectric points were 8.4 and 9.45, respectively. Optimal enzyme activities were obtained at 80 degrees C and pH 6.0. The thermostability of both xylanases was greatly diminished during purification but could be restored by preincubation of the purified enzymes in the presence of xylan. The half-lives at 80 degrees C were approximately 25 min. The kinetic constants of xylanases I and II determined with Remazol-brilliant-blue xylan were Vmax of 1537 and 353 mumol.min-1.mg protein-1 and K(m) values of 2.44 and 1.07 mg.ml-1, respectively. Purified xylanases utilized xylan as well as small oligosaccharides such as xylotriose as substrate. They did not exhibit xylobiase or debranching activities. The predominant products of arabinoxylan hydrolysis were xylobiose and xylotriose, the latter being hydrolysed to xylobiose and xylose upon further incubation. In addition, fragments containing arabinose side chains accumulated. The xylanases did not act on crystalline or amorphous cellulose indicating a possible application in biobleaching processes.

Production and properties of xylanases from thermophilic actinomycetes.

30 strains of xylanolytic thermophilic actinomycetes were isolated from composted grass and cattle manure and identified as members of the genera Thermomonospora, Saccharomonospora, Microbispora, Streptomyces and Actinomadura. Screening of these strains for extracellular xylanase indicated that strains of Saccharomonospora and Microbispora generally were poor xylanase producers (0.5-1.5 U/ml) whereas relatively high activities were observed in cultures of Streptomyces and Actinomadura (4-12 U/ml). A preliminary characterization of the enzymes of strains of the latter genera suggested that xylanases of all the strains of Actinomadura exhibited higher thermostabilities than those of Streptomyces. To evaluate the potential of thermophilic Actinomadura for industrial applications, xylanases of three strains were studied in more detail. The highest activity levels for xylanases were observed in cultures grown on xylan and wheat bran. The optimal pH and temperature for xylanase activities ranged from 6.0 to 7.0 and 70 to 80 degrees C. The enzymes exhibited considerable thermostability at their optimum temperature. The half-lives at 75 degrees C were in the range from 6.5 to 17 h. Hydrolysis of xylan by extracellular xylanases yielded xylobiose, xylose and arabinose as principal products. Estimated by the amount of reducing sugars liberated the degree of hydrolysis was 55 to 65%. Complete utilization of xylan is presumably achieved by beta-xylosidase activities which could be shown to be largely cell-associated in the 3 Actinomadura strains.

Correlation of pancreatic enzyme levels with the patient's recovery from acute edematous pancreatitis.

Levels of total amylase, pancreatic isoamylase, lipase and immunoreactive trypsin were measured in 27 consecutive patients with noncomplicated (methemalbumin negative, no pseudocysts on ultrasound) acute pancreatitis during the acute, subacute, and recovery phases. At the beginning of the recovery phase (no pain, start of oral food intake), amylase and pancreatic isoamylase were below normal, whereas lipase (12% of the patients) and especially trypsin (52% of the patients) were still three times above the upper limit of normal. None of the patients showed a relapse of pain at the time of oral food intake. The conclusion is that amylase and pancreatic isoamylase reflect more accurately the patient's recovery pattern in acute pancreatitis than the other enzymes. Trypsin, however, may be significant in cases of delayed diagnosis. Thus, the decision to start oral food intake should be based on the patient's clinical state rather than on pancreatic enzyme levels.