Study On Biologically Active Substances From Mung Bean

Purification, characterization, crystallization, X-ray diffraction were carried out on biologically active substances from mung bean (Phaseolus mungo) seeds, as well as the investigation of the relationship between structure and function. Malate dehydrogenase, chitinase, nonspecific lipid transfer protein(nsLTP) and lysozyme were isolated by a combination of extraction, ammonium sulfate precipitation, ion exchange chromatography on CM-sephadex C-50 column, High Performance Liquid Chromatography (HPLC) on POROS HS-20 column and gel filtration on Sephadex-G75. The purity of biological proteins was estimated by SDS-polyacrylamide gel electrophoresis, CLC chromatography, mass spectrometry and N-terminal amino acid sequence analysis, respectively. The purified malate dehydrogenase (MDH) exhibited a molecular mass of 38.0 kDa in SDS-polyacrylamide gel electrophoresis (SDS-PAGE )both under non-reduced and reduced conditions. The pI was 9.7 by isoelectric focusing. The enzyme expressed its optimum activity at pH 7.2, 35℃, the Km for oxaloacetate was 112 μM. The partial N-terminal amino acid sequence data analysis of the first 20 amino acids of the MDH was determined to be 1-ASEPGPERKVAVLGAAGGIG-20, and revealed 75% to 95% homology with other reported plant MDHs. K⁺ activated the enzyme, Ca²⁺ and Mg²⁺ had seldom action, the enzyme activity was inhibited in various degree by Zn²⁺, Pb²⁺ and Cu²⁺. The effect of Zn²⁺ was classified as noncompetitive type. A chitinase exhibited a molecular mass of 30.8kDa in SDS-PAGE both under non-reduced and reduced conditions. The pI was 6.3 by isoelectric focusing. The enzyme expressed its optimum activity at pH 5.4, and showed stable activity from 40℃to 50℃. It exerted antifungal action toward Fusarium solani, Fusarium oxysporum, Mycosphaerella arachidicola, Pythium aphanidermatum and Sclerotium rolfsii. A lysozyme exhibited a molecular mass of 14.4 kDa in SDS-PAGE, and a monomer with intramolecular disulfide bridges. The enzyme expressed its optimum activity at pH 5.5, 55℃. The N-terminal amino acid sequence was determined to be 1-DMPGKVALTAQSF-13, and revealed 23% homology with known reported lysozyme from hen egg white (HWEL). The lysozyme exerted antifungal action toward Fusarium oxysporum, Fusarium solani, Pythium aphanidermatum, Sclerotium rolfsii and Botrytis cinere, as well as showing antibacterial action against Staphylococcus aureus. The IC50 to Fusarium solani was 11μM and the minimum inhibitory concentration (MIC) to Staphylococcus aureus was 0.26μM. The active peptide nonspecific lipid transfer protein (nsLTP) was a monomer with intramolecular disulfide bridges in SDS-PAGE, and exhibited a molecular mass of 9292Da in mass spectrometry. The lipid binding of nsLTP was investigated in aqueous solution by fluorescence spectroscopy. It exerted strong growth inhibitory activity against plant pathogenic fungi Fusarium solani, Fusarium oxysporum, Pythium aphanidermatum, and Sclerotium rolfsii. Furthermore, it also showed inhibitory activity against Staphylococcus aureus with minimum inhibitory concentration of 0.06 mmol/ml. The peptide partial N-terminal amino acid sequence was identified as 1-MTCGQVQGNLAQCIGFLEKGG-21, which displayed a high degree homology with other antifungal peptides isolated from plants. A crystallization strategy was designed for nsLTP according to the Crystal Screen. NsLTP was crystallized at 297K using ammonium sulfate as a precipitant, by means of the hanging-drop vapour-diffusion method. The optimized crystals were obtained with a reservoir solution containing 1.5 M ammonium sulfate as a precipitant, 0.1 M Tris-HCl buffer pH 8.5, 8% (v/v) glycerol and 0.5% (w/v) octyl ?-D-glucopyranoside as a detergent. The crystals are rhombohedral, belonging to the space group P212121, with unit-cell parameters a=38.671, b= 51.785, c= 55.925 ?. Native X-ray diffraction data were collected to a resolution of 2.4 ?. Assuming one molecule in the crystallographic asymmetric unit results in a Matthews coefficient (VM) of approximately 3.0 ? 3 Da-1 corresponding to a solvent content of about 58%. The feature of nsLTP structure was a single compact domain with four α-helices and a long carboxyl terminal region, with the four disulfide bonds interconnecting the secondary structure elements. It shows a hydrophobic cavity surrounded by α-helices connected through disulfide bonds, while the hydrophobic cavity is the binding pocket for lipid or fatty acid molecules. A probable mechanism was that the lipid-binding activity and the structure with hydrophobic cavity would have attributed to its antibacterial and antifungal activity.