ABSTRACT
Isomalto-oligosaccharides (IMO) have good physiochemical properties and excellent physiological functions to make it widely used in food, medicine, feed, cosmetics and other industries. However, the procedures for industrial production of IMO are complicated. Therefore, it is necessary to develop an economical and easy-to-operate method. The genes encoding for β-amylase and α-transglucosidase were fused and co-displayed on the yeast cell surface of Yarrowia lipolytica which can convert liquefied starch to IMO in one step. The highest IMO purity of 75.3% was obtained using the displayed fusion-enzyme at 50 °C. This method showed potential application in IMO production.
Subject(s)
Oligosaccharides , Starch , Yarrowia , beta-AmylaseABSTRACT
We used in vitro molecular evolution technology by error-prone PCR and high-throughput screening to improve thermostability of Bacillus flexus CCTCC 2015368 β-amylase. Mutant D476N with significant thermostability increase was selected by LB agar starch plate colorimetric assay and 96-well plate enzyme activity assay. The optimum pH was 6.5 for the mutant D476N, compared to 7.0 of the wild type. The optimal temperature was 55 ℃ for both mutant D476N and the wild type. The T₅₀ value of the mutant D476N was 4 ℃ higher than that of the wild type. The half-life of mutant D476N at 55 ℃ was 35 min, 95% higher than that of the wild type. The Km of the mutant D476N was 97.98 μmol/L, 1.14 times of that of the wild type (85.86 μmol/L). The thermostability of the mutant D476N was slightly lower than that of the wild type. The three-dimensional structure of wild type and mutant D476N was simulated by SWISS-MODEL and analyzed by PyMol software. The mutated amino acid residue Asn476 was located on the loop of protein surface. The molecular free energy(ΔG) of D476N was calculated by MOE software was 106.0 kcal/mol, reduced by 10.3% compared to the wild enzyme. These results were consistent with the theory that the protein molecular free energy and thermostability were negatively correlated.
ABSTRACT
This study was carried out after a five day germination period on TZEE*TZEEW* DEMARSCUS*TZEE-W one of the most recommended high amylolytic Nigerian maize cultivar. Purification steps comprising of fractional precipitation by ammonium sulphate, gel filtration and anion exchange chromatography, was used respectively to purify β amylase (EC 3.2.1.2) from the malt. The sensitivity of the beta amylase indicated that it has serine at its active site. An apparent 60KDa monomeric protein was detected by one dimensional sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE). Identity assigned to the purified protein by Matrix-assisted laser desorption ionization time –of- flight mass spectrometry ( MALDI-ToF) reference to electronic protein data base is a 58542Da high putative beta amylase – Q9AV88-ORSA from Oryza sativa. Complete primary structure thereafter deduced with the aid of MS/MS MALDI- ToF showed a signature of a highly conserved ubiquitous not yet reported beta amylase. This study paved an insight to the gene encoding the β amylase in TZEE*TZEEW* DEMARSCUS*TZEE-W and better understanding of the activity of the enzyme at molecular level.