Expanded bed adsorption (EBA) has been widely used in industrial downstream bioprocessing. Solid matrix is the principal pillar supporting the successful application of EBA. A novel spherical cellulose-titanium dioxid...Expanded bed adsorption (EBA) has been widely used in industrial downstream bioprocessing. Solid matrix is the principal pillar supporting the successful application of EBA. A novel spherical cellulose-titanium dioxide composite matrix was prepared through the method of water-in-oil suspension thermal regeneration. Its typical physical properties were wet density 1.18g.cm-3, diameters in the range of 100-300um, porosity 85.5%, and water content 72.3%. Expansion characteristics and liquid mixing performance of the matrix in expanded bed were investigated using water and 10% (by mass) glycerol solution as mobile phases. The results indicate that the custom-assembled matrix has a stable flow hydrodynamics and exhibits the same degree of liquid-phase mixing or column efficiency as the commercially available Streamline adsorbent.展开更多
Glutamate decarboxylase(GAD, EC4.1.1.15) can catalyze the decarboxylation of L-glutamate to form γ-aminobutyrate(GABA), which is in great demand in some foods and pharmaceuticals. In our previous study,gad, the gene ...Glutamate decarboxylase(GAD, EC4.1.1.15) can catalyze the decarboxylation of L-glutamate to form γ-aminobutyrate(GABA), which is in great demand in some foods and pharmaceuticals. In our previous study,gad, the gene coding glutamate decarboxylase from Lactobacillus brevis CGMCC 1306, was cloned and its soluble expression was realized. In this study, error-prone PCR was conducted to improve its activity, followed by a screening. Mutant Q51 H with high activity [55.4 mmol·L-1·min-1·(mg protein)-1, 120% higher than that of the wild type at p H 4.8] was screened out from the mutant library. In order to investigate the potential role of this site in the regulation of enzymatic activity, site-directed saturation mutagenesis at site 51 was carried out,and three specific mutants, N-terminal truncated GAD, Q51 P, and Q51 L, were identified. The kinetic parameters of the three mutants and Q51 H were characterized. The results reveal that aspartic acid at site 88 and N-terminal domain are essential to the activity as well as correct folding of GAD. This study not only improves the activity of GAD, but also sheds new light on the structure–function relationship of GAD.展开更多
基金Supported by the National Natural Science Foundation of China(No.20076042,No.20206029)and the Scientific Research Foundation of the State Education Ministry for the Returned Overseas Chinese Scholars(No.2002-247).
文摘Expanded bed adsorption (EBA) has been widely used in industrial downstream bioprocessing. Solid matrix is the principal pillar supporting the successful application of EBA. A novel spherical cellulose-titanium dioxide composite matrix was prepared through the method of water-in-oil suspension thermal regeneration. Its typical physical properties were wet density 1.18g.cm-3, diameters in the range of 100-300um, porosity 85.5%, and water content 72.3%. Expansion characteristics and liquid mixing performance of the matrix in expanded bed were investigated using water and 10% (by mass) glycerol solution as mobile phases. The results indicate that the custom-assembled matrix has a stable flow hydrodynamics and exhibits the same degree of liquid-phase mixing or column efficiency as the commercially available Streamline adsorbent.
基金Supported by the National Natural Science Foundation of China(30970638,21176220,and 31240054)the National Natural Science Foundation of Zhejiang Province(LZ13B060002)
文摘Glutamate decarboxylase(GAD, EC4.1.1.15) can catalyze the decarboxylation of L-glutamate to form γ-aminobutyrate(GABA), which is in great demand in some foods and pharmaceuticals. In our previous study,gad, the gene coding glutamate decarboxylase from Lactobacillus brevis CGMCC 1306, was cloned and its soluble expression was realized. In this study, error-prone PCR was conducted to improve its activity, followed by a screening. Mutant Q51 H with high activity [55.4 mmol·L-1·min-1·(mg protein)-1, 120% higher than that of the wild type at p H 4.8] was screened out from the mutant library. In order to investigate the potential role of this site in the regulation of enzymatic activity, site-directed saturation mutagenesis at site 51 was carried out,and three specific mutants, N-terminal truncated GAD, Q51 P, and Q51 L, were identified. The kinetic parameters of the three mutants and Q51 H were characterized. The results reveal that aspartic acid at site 88 and N-terminal domain are essential to the activity as well as correct folding of GAD. This study not only improves the activity of GAD, but also sheds new light on the structure–function relationship of GAD.
