Improving thermostability of Bacillus amyloliquefaciens alpha-amylase by multipoint mutations.

The medium-temperature alpha-amylase of Bacillus amyloliquefaciens is widely used in the food and washing process. Enhancing the thermostability of alpha-amylases and investigating the mechanism of stability are important for enzyme industry development. The optimal temperature and pH of the wild-type BAA and mutant MuBAA (D28E/V118A/S187D/K370 N) were all 60 °C and 6.0, respectively. The mutant MuBAA showed better thermostability at 50 °C and 60 °C, with a specific activity of 206.61 U/mg, which was 99.1% greater than that of the wild-type. By analyzing predicted structures, the improving thermostability of the mutant MuBAA was mainly related to enhanced stabilization of a loop region in domain B via more calcium-binding sites and intramolecular interactions around Asp187. Furthermore, additional intramolecular interactions around sites 28 and 370 in domain A were also beneficial for improving thermostability. Additionally, the decrease of steric hindrance at the active cavity increased the specific activity of the mutant MuBAA. Improving the thermostability of BAA has theoretical reference values for the modification of alpha-amylases.

Buckwheat Antifungal Protein with Biocontrol Potential To Inhibit Fungal ( Botrytis cinerea) Infection of Cherry Tomato.

A 11 kDa antifungal protein FEAP was purified from buckwheat ( Fagopyrum esculentum) seed extract with a procedure involving (NH4)2SO4 precipitation and chromatography on SP-Sepharose, Affi-gel blue gel, Mono S, and Superdex peptide. Its N-terminal sequence was AQXGAQGGGAT, resembling those of buckwheat peptides Fα-AMP1 and Fα-AMP2. FEAP exhibited thermostability (20-100 °C) and acid resistance (pH 1-5). Its antifungal activity was retained in the presence of 10-150 mmol/L of K+, Mn2+, or Fe3+ ions, 10-50 mmol/L of Ca2+ or Mg2+ ions, and 50% methanol, 50% ethanol, 50% isopropanol, or 50% chloroform. Its half-maximal inhibitory concentrations toward spore germination and mycelial growth in Botrytis cinerea were 79.9 and 236.7 µg/mL, respectively. Its antifungal activity was superior to the fungicide cymoxanil mancozeb (248.1 µg/mL). FEAP prevented B. cinerea from infecting excised leaves, intact leaves, and isolated fruits of cherry tomato. Its mechanism involved induction of an increase in cell membrane permeability and a decrease in mitochondrial membrane potential.

Purification of an Antifungal Peptide from Seeds of Brassica oleracea var. gongylodes and Investigation of Its Antifungal Activity and Mechanism of Action.

In this study, a 8.5-kDa antifungal peptide designated as BGAP was purified from the crude extract of the seeds of Brassica oleracea var. gongylodes by employing a protocol that comprised cation exchange chromatography on SP-Sepharose, cation exchange chromatography on Mono S and gel filtration chromatography on Superdex peptide. BGAP showed the highest amino acid sequence similarity to defensin peptides by mass spectrometric analysis. BGAP showed a broad spectrum of antifungal activity with a half maximal inhibitory concentration at 17.33 µg/mL, 12.37 µg/mL, 16.81 µg/mL, and 5.60 µg/mL toward Colletotrichum higginsianum, Exserohilum turcicum, Magnaporthe oryzae and Mycosphaerella arachidicola, respectively. The antifungal activity of BGAP remained stable (i) after heat treatment at 40-100 °C for 15 min; (ii) after exposure to solutions of pH 1-3 and 11-13 for 15 min; (iii) after incubation with solutions containing K⁺, Ca2+, Mg2+, Mn2+ or Fe3+ ions at the concentrations of 20-150 mmol/L for 2 h; and (iv) following treatment with 10% methyl alcohol, 10% ethanol, 10% isopropanol or 10% chloroform for 2 h. Fluorescence staining experiments showed that BGAP brought about an increase in cell membrane permeability, a rise in reactive oxygen species production, a decrease in mitochondrial membrane potential, and an accumulation of chitin at the hyphal tips of Mycosphaerella arachidicola.

Plant antifungal proteins and their applications in agriculture.

Fungi are far more complex organisms than viruses or bacteria and can develop numerous diseases in plants that cause loss of a substantial portion of the crop every year. Plants have developed various mechanisms to defend themselves against these fungi which include the production of low-molecular-weight secondary metabolites and proteins and peptides with antifungal activity. In this review, families of plant antifungal proteins (AFPs) including defensins, lectins, and several others will be summarized. Moreover, the application of AFPs in agriculture will also be analyzed.

Isolation of a ribonuclease with antiproliferative and HIV-1 reverse transcriptase inhibitory activities from Japanese large brown buckwheat seeds.

A ribonuclease, with a molecular mass of 22.5 kDa and an N-terminal sequence exhibiting resemblance to previously isolated buckwheat storage proteins and allergens, was isolated from Japanese large brown buckwheat seeds. The ribonuclease was purified using a simple protocol that comprised ion exchange chromatography on Q-Sepharose and DEAE-cellulose and gel filtration on Superdex 75. The ribonuclease exhibited low activity toward poly U, lower activity toward poly C, and very low activity toward poly A and poly G. The enzyme was activated upon exposure to 10 mM of Fe(2+) and Zn(2+) ions but was inhibited by Ca(2+), Mg(2+), and Mn(2+) ions at the same concentration. The optimum pH and optimum temperature for the enzyme were pH 9 and 60 °C, respectively. It inhibited proliferation of HepG2 hepatoma and MCF 7 breast cancer cells, with an IC50 value of 79.2 and 63.8 µM, respectively. It potently inhibited HIV-1 reverse transcriptase activity with an IC50 of 48 µM. However, there were no antifungal and mitogenic activities.

The immobilization of hepatocytes on 24 nm-sized gold colloid for enhanced hepatocytes proliferation.

Bioartificial liver and hepatocyte transplantation is anticipated to supply a temporary metabolic support for candidates of liver transplantation or for patients with fulminant liver failure. An essential restriction of this form is the inability to acquire an enough amount of hepatocytes. Enhancement of the proliferation and differentiated function of hepatocytes is becoming a pursued target. Here, porcine hepatocytes were successfully immobilized on nano-sized gold colloid particles to construct a "hepatocyte/gold colloid" interface at which hepatocytes can be quickly proliferated. The properties of this resulting interface were characterized and confirmed by scanning electron microscopy and atomic force microscopy. The proliferative mechanism of hepatocytes was also discussed. The proliferated hepatocytes could be applied to the clinic based on their excellent functions for the synthesis of protein, glucose and urea as well as lower lactate dehydrogenase release.