Three-phase partitioning of trypsin inhibitor from legume seeds

Three-phase partitioning (TPP) was used to isolate trypsin inhibitors from navy bean (NB), red kidney bean (RK) and adzuki bean (AZ) from the Royal Project Foundation in Thailand. The method was to mix the crude extract with solid ammonium sulfate (30% saturation, w/v) and tert-butanol (t-butanol) in order to obtain the three phases. The trypsin inhibitor was purified to 5-, 14- and 7-fold with 315%, 441% and 228% recovery for NB, RK and AZ, respectively. The SDS-PAGE showed the major inhibitor band with the molecular weights (MWs) of 132, 118 and 13 kDa for NB, RK and AZ, respectively. The fractions from NB and AZ showed higher pH stability compared to that of the RK, and they had the optimum pH ranges of 7–9. The highest relative inhibitory activity of the fractions of NB and RK were found at 50 °C, and all fractions were quite stable at 90 °C for 60 min of incubation. Increasing the concentration of salt (up to 3%, w/v) did not significantly decrease the inhibitory activity of all fractions (p > 0.05). The trypsin inhibitors from the three legumes were unable to inhibit the autolysis of Pacific whiting and arrowtooth flounder muscles.     

Influence of preculture treatment and types of explants on shoot growth and in vitro flowering of feathered amaranth (Celosia argentea var. plumose)

The shoots developed from both the shoot tip and nodal explants of feathered amaranth (Celosia argentea var. plumosa—feathered cockscomb or plumed cockscomb) after 8 weeks of culture in the presence of either paclobutrazol or benzyladenine (BA) were shorter than those developed on basal Murashige and Skoog (MS) medium (Physiol Plant, 15:473–497, 1962) alone. However, this retarding effect was more pronounced in the nodal explant culture. Shoot tip explants from 2-week-old seedlings were more adversely affected by 0.85 or 1.7 μM paclobutrazol than those from older seedlings. In contrast, regardless of preculture duration investigated nodal explants did not exhibit different response to three different concentrations of paclobutazol. The response to 2.2 or 4.4 μM BA appeared to be largely independent of the age of the shoot tip explants or preculture treatment of nodal explants. Shoots developed from nodal explants produced a higher number of terminal inflorescence than those from shoot tip explants. Moreover, only lateral shoots from nodal explant culture formed inflorescence. Increased preculture duration on basal MS medium could generally lessen the inhibitory effect of lower concentrations of paclobutazol or BA on terminal or lateral inflorescence formation in nodal explant culture.     

Activity of immobilized papain dehydrated by n-propanol in low-water media

A propanol-rinsed enzyme preparations (PREP) of papain showed an activity of 59 nmol min(-1) (mg powder)(-1) in tert-butanol at the optimal water activity of 0.2. The immobilized papain was stable in aqueous media for 3 d at 4 degrees C. Solid-state buffers (bases and their HCl salts) suspended in the organic medium decreased the initial rate in all cases tested. The operational stability during Z-Gly-Phe-NH2 synthesis was improved when solid cysteine was added, doubling the yield after 24 h.     

Effect of solid-state buffers on the catalytic activity of papain in low water media

The catalytic activity of papain in the synthesis of Z-Gly-Phe-NH₂ in tert-butanol has been studied in the presence of solid-state acid–base buffers (acids and their sodium salts). All buffer pairs tested reduced the reaction rate compared with the control, particularly the most acidic and basic (assessed by either aqueous pK_a or the response of an organic phase indicator). The highest rates, close to the control, were found with glutamic acid/glutamate-Na, PIPES/PIPES-Na and NaH₂PO₄/Na₂HPO₄. However, these pairs were unable to erase the pH memory phenomenon, or to overcome the effect of spiking with acetic acid. Hence, at least these buffers do not seem to be able to affect the protonation state and catalytic activity of papain. In the last aqueous solution before drying, the presence of activating agents (cysteine plus EDTA) was more important than buffer ions.     

The Immunoreactive Exo-1,3-β-Glucanase from the Pathogenic Oomycete Pythium insidiosum Is Temperature Regulated and Exhibits Glycoside Hydrolase Activity

The oomycete organism, Pythium insidiosum, is the etiologic agent of the life-threatening infectious disease called “pythiosis”. Diagnosis and treatment of pythiosis is difficult and challenging. Novel methods for early diagnosis and effective treatment are urgently needed. Recently, we reported a 74-kDa immunodominant protein of P. insidiosum, which could be a diagnostic target, vaccine candidate, and virulence factor. The protein was identified as a putative exo-1,3-ß-glucanase (Exo1). This study reports on genetic, immunological, and biochemical characteristics of Exo1. The full-length exo1 coding sequence (2,229 bases) was cloned. Phylogenetic analysis showed that exo1 is grouped with glucanase-encoding genes of other oomycetes, and is far different from glucanase-encoding genes of fungi. exo1 was up-regulated upon exposure to body temperature, and its gene product is predicted to contain BglC and X8 domains, which are involved in carbohydrate transport, binding, and metabolism. Based on its sequence, Exo1 belongs to the Glycoside Hydrolase family 5 (GH5). Exo1, expressed in E. coli, exhibited ß-glucanase and cellulase activities. Exo1 is a major intracellular immunoreactive protein that can trigger host immune responses during infection. Since GH5 enzyme-encoding genes are not present in human genomes, Exo1 could be a useful target for drug and vaccine development against this pathogen.