Comparative characterization of complete and truncated forms of Lactobacillus amylovorus alpha-amylase and role of the C-terminal direct repeats in raw-starch binding. (65/1178)

Two constructs derived from the alpha-amylase gene (amyA) of Lactobacillus amylovorus were expressed in Lactobacillus plantarum, and their expression products were purified, characterized, and compared. These products correspond to the complete (AmyA) and truncated (AmyADelta) forms of alpha-amylase; AmyADelta lacks the 66-kDa carboxyl-terminal direct-repeating-unit region. AmyA and AmyADelta exhibit similar amylase activities towards a range of soluble substrates (amylose, amylopectin and alpha-cyclodextrin, and soluble starch). The specific activities of the enzymes towards soluble starch are similar, but the K(M) and V(max) values of AmyADelta were slightly higher than those of AmyA, whereas the thermal stability of AmyADelta was lower than that of AmyA. In contrast to AmyA, AmyADelta is unable to bind to beta-cyclodextrin and is only weakly active towards glycogen. More striking is the fact that AmyADelta cannot bind or hydrolyze raw starch, demonstrating that the carboxyl-terminal repeating-unit domain of AmyA is required for raw-starch binding activity.  (+info)

The influence of secretory-protein charge on late stages of secretion from the Gram-positive bacterium Bacillus subtilis. (66/1178)

Following their secretion across the cytoplasmic membrane, processed secretory proteins of Bacillus subtilis must fold into their native conformation prior to translocation through the cell wall and release into the culture medium. The rate and efficiency of folding are critical in determining the yields of intact secretory proteins. The B. subtilis membrane is surrounded by a thick cell wall comprising a heteropolymeric matrix of peptidoglycan and anionic polymers. The latter confer a high density of negative charge on the wall, endowing it with ion-exchange properties, and secretory proteins destined for the culture medium must traverse the wall as the last stage in the export process. To determine the influence of charge on late stages in the secretion of proteins from this bacterium, we have used sequence data from two related alpha-amylases, to engineer the net charge of AmyL, an alpha-amylase from Bacillus licheniformis that is normally secreted efficiently from B. subtilis. While AmyL has a pI of 7.0, chimaeric enzymes with pI values of 5.0 and 10.0 were produced and characterized. Despite the engineered changes to their physico-chemical properties, the chimaeric enzymes retained many of the enzymic characteristics of AmyL. We show that the positively charged protein interacts with the cell wall in a manner that influences its secretion.  (+info)

A complex containing both trypsin inhibitor and dehydroascorbate reductase activities isolated from mitochondria of etiolated mung bean (Vigna radiata L. (Wilczek) cv. Tainan no. 5) seedlings. (67/1178)

A complex containing trypsin inhibitor (TI) activity was extracted with 0.1 M TRIS buffer (pH 7.9) from trypsin-treated mitochondria of etiolated mung bean seedlings, and further purified with a Superdex 200 FPLC column. This partially purified complex with an M(r) about 820 kDa exhibited additional dehydroascorbate (DHA) reductase activity with specific activities of 0.21, 1.53 and 1.54 mumol ascorbate formed min-1 mg-1 protein at pH 6.0, 6.5 and 7.0, respectively, when glutathione was added. Much lower DHA reductase activity (0.013 and 0.026 mumol ascorbate formed min-1 mg-1 protein at pH 6.5 and 7.0, respectively) was found when glutathione was omitted. The isolated complex gave positive results when it was tested by TI activity staining after SDS-PAGE, and could be recognized by a polyclonal antibody which was raised against 38 kDa sweet potato Kunitz-type TI, one of the root storage proteins of sweet potato. The possible physiological functions of this complex with both TI and DHA reductase activities were discussed.  (+info)

The significance of alpha-amylase under anoxia stress in tolerant rhizomes (Acorus calamus L.) and non-tolerant tubers (Solanum tuberosum l., var. Desiree). (68/1178)

Rhizomes of Acorus calamus L. were able to maintain a functional alpha-amylase under anoxia, whereas a steep decrease in the enzyme protein content and activity took place in potato tubers. The stress-induced control in tubers occurred on the translational level. It is suggested that this decrease is one of the key factors with regard to anoxia intolerance.  (+info)

New type of starch-binding domain: the direct repeat motif in the C-terminal region of Bacillus sp. no. 195 alpha-amylase contributes to starch binding and raw starch degrading. (69/1178)

The alpha-amylase from Bacillus sp. no. 195 (BAA) consists of two domains: one is the catalytic domain similar to alpha-amylases from animals and Streptomyces in the N-terminal region; the other is the functionally unknown domain composed of an approx. 90-residue direct repeat in the C-terminal region. The gene coding for BAA was expressed in Streptomyces lividans TK24. Three active forms of the gene products were found. The pH and thermal profiles of BAAs, and their catalytic activities for p-nitrophenyl maltopentaoside and soluble starch, showed almost the same behaviours. The largest, 69 kDa, form (BAA-alpha) was of the same molecular mass as that of the mature protein estimated from the nucleotide sequence, and had raw-starch-binding and -degrading abilities. The second largest, 60 kDa, form (BAA-beta), whose molecular mass was the same as that of the natural enzyme from Bacillus sp. no. 195, was generated by proteolytic processing between the two repeat sequences in the C-terminal region, and had lower activities for raw starch binding and degrading than those of BAA-alpha. The smallest, 50 kDa, form (BAA-gamma) contained only the N-terminal catalytic domain as a result of removal of the C-terminal repeat sequence, which led to loss of binding and degradation of insoluble starches. Thus the starch adsorption capacity and raw-starch-degrading activity of BAAs depends on the existence of the repeat sequence in the C-terminal region. BAA-alpha was specifically adsorbed on starch or dextran (alpha-1,4 or alpha-1,6 glucan), and specifically desorbed with maltose or beta-cyclodextrin. These observations indicated that the repeat sequence of the enzyme was functional in the starch-binding domain (SBD). We propose the designation of the homologues to the SBD of glucoamylase from Aspergillus niger as family I SBDs, the homologues to that of glucoamylase from Rhizopus oryzae as family II, and the homologues of this repeat sequence of BAA as family III.  (+info)

Dietary carbohydrate source and energy intake influence the expression of pancreatic alpha-amylase in lambs. (70/1178)

In ruminants, pancreatic alpha-amylase is the primary enzyme responsible for the initial hydrolysis of alpha-linked glucose in the small intestinal lumen. The objective of this experiment was to examine the effects of altered dietary starch and energy supply on the expression of pancreatic alpha-amylase mRNA, protein and activity in lambs. Wether lambs (n = 24; 28 +/- 0.5 kg body weight) were fed low or high starch diets at 1.2 or 1.8 x net energy of maintenance for at least 28 d before tissue collection. Lambs fed the high energy/high starch diet tended to have more pancreatic alpha-amylase protein (54.5 kDa; P: = 0.08) and had greater activity (P: = 0.03), but alpha-amylase mRNA (1.6 kb) tended to be lower (P: = 0.17). Additionally, rumen fluid total short-chain fatty acid concentration was greater (P: = 0.04) and plasma glucose concentration tended to be greater (P: = 0.07) in lambs fed the high energy/high starch diet. However, pancreatic trypsinogen protein (25. 5 kDa) and jejunal maltase activity were not influenced by dietary treatment, suggesting that different regulatory systems are involved in regulating the tissue protein or activity levels of these two enzymes compared with alpha-amylase. These data suggest that dietary regulation of pancreatic alpha-amylase expression in ruminants is complex and probably regulated by transcriptional and post-transcriptional events.  (+info)

Comparison of four methods to assess fungal alpha-amylase in flour dust. (71/1178)

The aim of the study was to compare four different immunological methods to analyse fungal alpha-amylase in flour dust samples. Three European research groups have independently developed four immuno assay based methods to measure alpha-amylase in air samples. Three of the methods use polyclonal antibodies and one method uses monoclonal antibodies. Eighty personal samples have been collected during two to three work-shifts in four bakeries. Sampling was performed with PAS-6 inhalable dust samplers and aliquots from each sample were analysed by the three laboratories. The agreement between the four methods was high compared with agreement between immunological methods to measure other allergens in the air, e.g. for rat allergens. For the three methods with polyclonal antibodies the mean differences for individual samples was less than a factor of two. The arithmetic means (AM) of the estimated alpha-amylase exposure were 12.5, 11.3, 8.6 and 25.9ng/m(3) for the respective methods with values ranging from below the detection limit to 192, 215, 207 and 615 ng/m(3). The AM for all samples analysed by the methods with polyclonal antibodies varied by about a factor of 1.5. About one-third of the values were below or at the detection limit for all methods. In a regression analysis the squared correlation coefficients (R(2)) between the methods varied between 0.91 and 0.95 for the log transformed values. For workplace monitoring, results from the methods using polyclonal antibodies will be relatively comparable. High levels of alpha-amylase might differ in absolute numbers with a factor of two or more between the different methods but will anyway be considered as high and should result in preventive actions. On the other hand, this study also shows that despite the relative agreement between methods, there is a clear need for standardization.  (+info)

Structural equilibrium fluctuations in mesophilic and thermophilic alpha-amylase. (72/1178)

By comparing a mesophilic alpha-amylase with its thermophilic homolog, we investigated the relationship between thermal stability and internal equilibrium fluctuations. Fourier transform infrared spectroscopy monitoring hydrogen/deuterium (H/D) exchange kinetics and incoherent neutron scattering measuring picosecond dynamics were used to study dynamic features of the folded state at room temperature. Fairly similar rates of slowly exchanging amide protons indicate about the same free energy of stabilization DeltaG(stab) for both enzymes at room temperature. With respect to motions on shorter time scales, the thermophilic enzyme is characterized by an unexpected higher structural flexibility as compared to the mesophilic counterpart. In particular, the picosecond dynamics revealed a higher degree of conformational freedom for the thermophilic alpha-amylase. The mechanism proposed for increasing thermal stability in the present case is characterized by entropic stabilization and by flattening of the curvature of DeltaG(stab) as a function of temperature.  (+info)