Thermostable invertases from Paecylomyces variotii produced under submerged and solid-state fermentation using agroindustrial residues

The filamentous fungus Paecylomices variotii was able to produce high levels of cell extract and extracellular invertases when grown under submerged fermentation (SbmF) and solid-state fermentation, using agroindustrial products or residues as substrates, mainly soy bran and wheat bran, at 40A degrees C for 72 h and 96 h, respectively. Addition of glucose or fructose (a parts per thousand yen1%; w/v) in SbmF inhibited enzyme production, while the addition of 1% (w/v) peptone as organic nitrogen source enhanced the production by 3.7-fold. However, 1% (w/v) (NH4)(2)HPO4 inhibited enzyme production around 80%. The extracellular form was purified until electrophoretic homogeneity (10.5-fold with 33% recovery) by DEAE-Fractogel and Sephacryl S-200 chromatography. The enzyme is a monomer with molecular mass of 102 kDa estimated by SDS-PAGE with carbohydrate content of 53.6%. Optima of temperature and pH for both, extracellular and cell extract invertases, were 60A degrees C and 4.0-4.5, respectively. Both invertases were stable for 1 h at 60A degrees C with half-lives of 10 min at 70A degrees C. Mg2+, Ba2+ and Mn2+ activated both extracellular and cell extract invertases from P. variotii. The kinetic parameters K-m and V-max for the purified extracellular enzyme corresponded to 2.5 mM and 481 U/mg prot(-1), respectively.

Bone healing pattern in surgically created circumferential defects around submerged implants: an experimental study in dog

Objective: To describe the healing of marginal defects below or above 1 mm of dimension around submerged implants in a dog model. Material and methods: In 12 Labrador dogs, all mandibular premolars and first molars were extracted bilaterally. After 3 months of healing, full-thickness flaps were elevated in the edentulous region of the right side of the mandible. Two recipient sites were prepared and the marginal 5mm were widened to such an extent to obtain, after implant installation, a marginal gap of 0.5mm at the mesial site (small defect) and of 1.25mm at the distal site (large defect). Titanium healing caps were affixed to the implants and the flaps were sutured allowing a fully submerged healing. The experimental procedures were subsequently performed in the left side of the mandible. The timing of the experiments and sacrifices were planned in such a way to obtain biopsies representing the healing after 5, 10, 20 and 30 days. Ground sections were prepared and histomorphometrically analyzed. Results: The filling of the defect with newly formed bone was incomplete after 1 month of healing in all specimens. Bone formation occurred from the base and the lateral walls of the defects. A larger volume of new bone was formed in the large compared with the small defects. Most of the new bone at the large defect was formed between the 10- and the 20-day period of healing. After 1 month of healing, the outline of the newly formed bone was, however, located at a similar distance from the implant surface (about 0.4mm) at both defect types. Only minor newly formed bone in contact with the implant, starting from the base of the defects, was seen at the large defects (about 0.8mm) while a larger amount was detected at the small defects (about 2.2 mm). Conclusion: Marginal defects around titanium implants appeared to regenerate in 20-30 days by means of a distance osteogenesis. The bone fill of the defects was, however, incomplete after 1 month.

Characterization of a thermostable extracellular tannase produced under submerged fermentation by Aspergillus ochraceus

Background: Tannases are enzymes that may be used in different industrial sectors as, for example, food and pharmaceutical. They are obtained mainly from microorganisms, as filamentous fungi. However, the diversity of fungi stays poorly explored for tannase production. In this article, Aspergillus ochraceus is presented as a new source of tannase with interesting features for biotechnological applications. Results: Extracellular tannase production was induced when the fungus was cultured in Khanna medium with tannic acid as carbon source. The extracellular tannase was purified 9-fold with 2% recovery and a single band corresponding to 85 kDa was observed in SDS-PAGE. The native apparent molecular mass was estimated as 112 kDa. Optima of temperature and pH were 40 degrees C and 5.0, respectively. The enzyme was fully stable from 40 degrees C to 60 degrees C during 1 hr. The activity was enhanced by Mn2+ (33-39%) and NH4+ (15%). The purified tannase hydrolyzed tannic acid and methyl gallate with Km of 0.76 mM and 0.72 mM, respectively, and Vmax of 0.92 U/mg protein and 0.68 U/mg protein, respectively. The analysis of a partial sequence of the tannase encoding gene showed an open read frame of 567 bp and a sequence of 199 amino acids were predicted. TLC analysis revealed the presence of gallic acid as a tannic acid hydrolysis product. Conclusion: The extracellular tannase produced by A. ochraceus showed distinctive characteristics such as monomeric structure and activation by Mn2+, suggesting a new kind of fungal tannases with biotechnological potential. Further, it was the first time that a partial gene sequence for A. ochraceus tannase was described.

'Synthetic lipase' production from a newly isolated Sporidiobolus pararoseus strain by submerged fermentation

The lipase produced by a newly isolate Sporidiobolus pararoseus strain has potential catalysis ability for esterification reactions. In order to improve its synthetic activity, this work aimed at optimizing 'synthetic lipase' production by submerged fermentation of a conventional media based on peptone, yeast extract, NaCl and olive oil using experimental design technique. According to the results obtained in the first experimental design (2(4-1)), yeast extract and NaCl concentrations were tested to further optimization by response surface methodology. The maximum 'synthetic lipase' activity obtained was 26.9 U/mL in the optimized media (5.0, 6.8, 7.0 and 1.0% (wt/v) of peptone, yeast extract, NaCl and olive oil, respectively), representing a 6.36-fold increase compared to the initial medium. The time course of 'synthetic lipase' production in the optimized condition was evaluated in terms of synthetic activity, protease activity, biomass and total carbon and the maximum synthetic activity was observed during the stationary phase of growth.