Expression of exo-inulinase gene from Aspergillus niger 12 in E. coli strain Rosetta-gami B (DE3) and its characterization.

Inulin is a linear carbohydrate polymer of fructose subunits (2-60) with terminal glucose units, produced as carbon storage in selected plants. It cannot directly be taken up by most microorganisms due to its large size, unless prior hydrolysis through inulinase enzymes occurs. The hydrolyzed inulin can be taken up by microbes and/or recovered and used industrially for the production of high fructose syrup, inulo-oligosaccharides, biofuel, and nutraceuticals. Cell-free enzymatic hydrolysis would be desirable for industrial applications, hence the recombinant expression, purification and characterization of an Aspergillus niger derived exo-inulinase was investigated in this study. The eukaroyototic exo-inulinase of Aspergillus niger 12 has been expressed, for the first time, in an E. coli strain [Rosetta-gami B (DE3)]. The molecular weight of recombinant exo-inulinase was estimated to be ∼81 kDa. The values of Km and Vmax of the recombinant exo-inulinase toward inulin were 5.3 ± 1.1 mM and 402.1 ± 53.1 µmol min(-1)  mg(-1) protein, respectively. Towards sucrose the corresponding values were 12.20 ± 1.6 mM and 902.8 ± 40.2 µmol min(-1)  mg(-1) protein towards sucrose. The S/I ratio was 2.24 ± 0.7, which is in the range of native inulinase. The optimum temperature and pH of the recombinant exo-inulinase towards inulin was 55°C and 5.0, while they were 50°C and 5.5 towards sucrose. The recombinant exo-inulinase activity towards inulin was enhanced by Cu(2+) and reduced by Fe(2+) , while its activity towards sucrose was enhanced by Co(2+) and reduced by Zn(2+) . © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:629-637, 2016.

Bioprocess development for production, purification, and structural characterization of recombinant hCD83ext as a potential therapeutic protein.

An effective bioprocess for the production of hCD83ext (i.e. the extracytoplasmic domain of human CD83) as a potential therapeutic protein was developed. It primarily consists of (1) cell cultivation for the production of recombinant glutathione-S-transferase-hCD83ext (GST-hCD83ext) fusion protein and (2) downstream processing for purification of hCD83ext. The developed bioprocess is robust, reproducible, easy to operate, and, most importantly, can generate hCD83ext with a high yield and purity. For cell cultivation, a high GST-hCD83ext expression level, estimated to be more than 10% of total cellular protein, with a cell density of 8 OD(600) was obtained by tuning several culture parameters, including medium recipe, host/vector system, induction condition, temperature, and aeration. For downstream processing, milligrams of very pure and low-endotoxin hCD83ext was obtained through simultaneous binding and cleavage of GST-hCD83ext in a GST affinity chromatographic column followed by a polishing step using anion exchange chromatography. To identify potential factors associated with bioactivity consistency, structural changes for the final product of hCD83ext were characterized and monitored. Formation of various hCD83ext multimeric forms, including dimer, trimer, and tetramer, via intermolecular disulfide bonds was observed.