A novel thermostable alpha-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b: purification and characterization.

High levels of an extracellular alpha-galactosidase are produced by the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b when grown in submerse culture and induced by sucrose. The enzyme was purified 114-fold from the culture supernatant by (NH(4))(2)SO(4) fractionation, and by chromatographical steps including Sepharose CL-6B gel filtration, DEAE-Sepharose FF anion-exchange, Q-Sepharose FF anion-exchange and Superose 12 gel filtration. The purified enzyme exhibits apparent homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and iso-electric focusing (IEF). The native molecular weight of the monomeric alpha-galactosidase is 93 kDa with an isoelectric point of 3.9. The enzyme displays a pH and temperature optimum of 5-5.5 and 65 degrees C, respectively. The purified enzyme retains more than 90% of its activity at 45 degrees C in a pH range from 5.5 to 9.0. The enzyme proves to be a glycoprotein and its carbohydrate content is 5.3%. Kinetic parameters were determined for the substrates p-nitrophenyl-alpha-galactopyranoside, raffinose and stachyose and very similar K(m) values of 1.13 mM, 1.61 mM and 1.17 mM were found. Mn(++) ions activates enzyme activity, whereas inhibitory effects can be observed with Ca(++), Zn(++) and Hg(++). Five min incubation at 65 degrees with 10 mM Ag(+) results in complete inactivation of the purified alpha-galactosidase. Amino acid sequence alignment of N-terminal sequence data allows the alpha-galactosidase from Thermomyces lanuginosus to be classified in glycosyl hydrolase family 36.

Effect of selection for growth rate on the ageing of myofibrils, meat texture properties and the muscle proteolytic potential of m. longissimus in rabbits.

The effect of selection for growth rate on the degradation of the myofibrillar proteins and on meat texture properties of rabbit longissimus muscle at two ageing times (1 and 7 days) was studied as well as its effect on the proteolytic potential of the muscle. Two groups of contemporary animals (20 rabbits per group), one selected for growth rate (S) for 14 generations and the other unselected control group (C) were compared. The control group was formed from the offspring of the embryos belonging to the 7th generation and was compared with selected animals belonging to 21st generation. Myofibrillar protein degradation was studied by SDS-PAGE electrophoresis (12.5% and 4-15% polyacrylamide gels) followed by densitometric analysis of the pherograms. Texture properties were evaluated by Warner-Bratzler (WB) test and Texture profile analysis (TPA). The activities of proteolytic enzymes calpains and cathepsins and of their inhibitors were determined in the muscle at 24h. Densitometric analysis of the pherograms of samples aged 7 days showed an extra 30kDa band and the disappearance of a band with higher molecular weight than the myosin heavy chain with respect to samples aged 24h in both groups of rabbits. TPA results showed that cohesiveness was significantly lower in meat at 7 days than at 24h (P<0.0001), whereas springiness and chewiness presented a clear tendency to be lower at 7 days than at 24h (P=0.0646 and P=0.0764, respectively). Regarding the genetic type, S animals presented higher hardness and chewiness than C rabbits. Shear firmness (WB test) was significantly (P<0.0001) higher for S group, whereas no significant differences in shear force and area were found. No significant effect (P>0.05) of ageing time was detected using WB test. Selection for growth rate did not affect the activities of proteolytic enzymes or their inhibitors.