Glu-537 of beta-galactosidase (EC 3.2.1.23) was replaced by Asp, Gln and Val using synthetic oligonucleotides. The kcat values of the purified enzyme mixtures were reduced by about 100-fold for the Asp mutant, 30,000-60,000-fold for the Val mutant and 160,000-300,000-fold for the Gln mutant. The greatest differences in properties from the wild-type enzyme were found for the Asp-substituted enzyme: the Km values increased (from 0.12 to 0.42 mM for o-nitrophenyl beta-D-galactopyranoside), and from 0.04 to 0.37 mM for p-nitrophenyl beta-D-galactopyranoside), the Ki value for isopropyl beta-D-galactopyranoside increased (from 0.11 to 0.30 mM), the stability to heat decreased and methanol did not act as an acceptor. The enzymes with the other two substitutions had properties similar to those of the wild-type. For all three substituted enzymes, the inhibitory effects of the transition-state analogues (2-deoxy-2-amino-D-galactose and L-ribose) and the Mg2+ effects were similar to those of the normal enzyme. As all of the properties (except the kcat values) of the Gln- and Val-substituted enzyme preparations were similar to those of the wild-type enzyme, the activities in those preparations were probably due to the presence of a few wild-type enzyme molecules (formed from misreads) among the substituted enzymes. The enzymes with Gln and Val substitutions appear to be totally inactive. The results obtained support a recent suggestion that Glu-537 is an important catalytic residue of beta-galactosidase.

No abstract

No abstract

Aims: The objective of this research was to study the ability of the basidiomycete Ganoderma lucidum to degrade starch and upgrade nutritional value of cornmeal during solid-state fermentation (SSF). Methods and Results: On the basal medium that consisted of cornmeal and salt solution, a-amylase activity of G. lucidum reached its maximum value of 267 U g)1 of culture on day 20 after inoculation. Prolongation of fermentation time from 10 to 25 days increased significantly the degradation rate of starch and ergosterol yield (a kind of physiologically active substances of G. lucidum, also as an indicator of mycelial biomass) (P < 0AE01). Supplementation of glucose, sucrose or maltose to the basal medium also caused a significant increase in either the degradation rate of starch or the ergosterol yield as compared with control (P < 0AE01). Among five kinds of nitrogen sources supplemented, yeast extract, casamino acid and peptone were more effective than (NH 4 ) 2 SO 4 and NH 4 NO 3 , and yeast extract gave the highest degradation rate of starch and ergosterol yield, followed by peptone. Through orthogonal experiments, the theoretical optimum culture medium for SSF of this fungus was the following: 100 g cornmeal, ground to 30-mesh powder, moistened with 67 ml of nutrient salt solution supplemented with 3 g yeast extract and 7AE5 g glucose per litre. Conclusions: Under the optimum culture condition, the degradation rate of starch reached its maximum values of 70AE4%; the starch content of the fermented product decreased from 64AE5 to 25AE3%, while the reducing sugar content increased from 4AE2 to 20AE6%. SSF also produced a significant increase (P < 0AE01) from 11AE0 to 16AE5% in protein content. Significance and Impact of the Study: After SSF by G. lucidum, the digesting and absorbing ratio of cornmeal was strikingly increased and some active substances originated from G. lucidum remained in the fermented product. This implied that cornmeal could be processed into many kinds of special functional foods by SSF of G. lucidum.

Two variants of D-hydantoinase (HYD), created by deletion of one amino acid residue of at either the N-or C-terminus, were expressed in Escherichia coli and purified by twostep chromatography. Compared with HYD, HYDc1 with the C-terminal Arg deletion retained 43% activity, while HYDn1 with the N-terminal Ser deletion had no activity using DLHydantoin as substrate. Based on HYD dimer with a molecular weight of 103 kDa, HYDc1 is a monomer of 52 kDa and HYDn1 is a mixture of dimer and monomer. Moreover, HYDc1 displayed higher pH stability and lower thermal stability compared to HYD. In addition, the secondary and tertiary structures of HYDc1 were not significantly changed in contrast to the ones of HYDn1. All data imply that the C-terminal Arg of the HYD is crucial for homodimeric architecture of the enzyme, but non-essential for catalysis, while the N-terminal Ser is required for both conformation and catalysis of the enzyme.

Aims:To determine the effect of oxidative stress and exogenous b-carotene on sclerotial differentiation and carotenoid yield of Penicillium sp. PT95. Methods and Results: In this experiment, high oxidative stress was applied by inclusion of FeCl 3 (10 lmol l )1 ) in the growth medium and by light exposure. Low oxidative stress was applied by omitting iron from the growth medium and by incubation in the dark. Supplementation of exogenous b-carotene (as antioxidant) to the basal medium caused a concentration-dependent delay of sclerotial differentiation (up to 72 h), decrease of sclerotial biomass (up to 43%) and reduction of carotenoid yield (up to 92%). On the contrary, the exogenous b-carotene also caused a concentration-dependent decrease of lipid peroxidation in colonies of this fungus.Conclusions: Under high oxidative stress growth condition, the sclerotial biomass and carotenoid yield of PT95 strain in each plate culture reached 141 mg and 30AE03 lg, which were 1AE53 and 3AE51 times higher respectively, than that at low oxidative stress growth condition. Significance and Impact of the Study: These data prompted us to consider that in order to attain higher sclerotial biomass and pigment yield, the strain PT95 should be grown under high oxidative stress and in the absence of antioxidants.

Various inocula and grains were evaluated for carotenoid production by solid-state fermentation using Penicillium sp. PT95. Millet medium was more effective in both sclerotia growth and carotenoid production than other grain media. An inoculum in the form of sclerotia yielded higher sclerotia biomass compared to either a spore inoculum or a mycelial pellet inoculum. Adding wheat bran to grain medium favored the formation of sclerotia. However, neither the inoculum type nor addition of wheat bran resulted in a significant change in the carotenoid content of sclerotia. Among grain media supplemented with wheat bran (wheat bran:grain =1:4 w/w, dry basis), a medium consisting of rice and wheat bran gave the highest sclerotia biomass (15.10 g/100 g grain), a medium consisting of buckwheat and wheat bran gave the highest content of carotenoid in sclerotia (0.826 mg/g dry sclerotia), and a medium consisting of millet and wheat bran gave the highest carotenoid yield (11.457 mg/100 g grain).

No abstract