Thermostability improvement of sucrose isomerase PalI NX-5: a comprehensive strategy.

OBJECTIVE: To increase the thermal stability of sucrose isomerase from Erwinia rhapontici NX-5, we designed a comprehensive strategy that combines different thermostabilizing elements. RESULTS: We identified 19 high B value amino acid residues for site-directed mutagenesis. An in silico evaluation of the influence of post-translational modifications on the thermostability was also carried out. The sucrose isomerase variants were expressed in Pichia pastoris X33. Thus, for the first time, we report the expression and characterization of glycosylated sucrose isomerases. The designed mutants K174Q, L202E and K174Q/L202E, showed an increase in their optimal temperature of 5 °C, while their half-lives increased 2.21, 1.73 and 2.89 times, respectively. The mutants showed an increase in activity of 20.3% up to 25.3%. The Km values for the K174Q, L202E, and K174Q/L202E mutants decreased by 5.1%, 7.9%, and 9.4%, respectively; furthermore, the catalytic efficiency increased by up to 16%. CONCLUSIONS: With the comprehensive strategy followed, we successfully obtain engineered mutants more suitable for industrial applications than their counterparts: native (this research) and wild-type from E. rhapontici NX-5, without compromising the catalytic activity of the molecule.

Accumulation, assembly, and digestibility of amarantin expressed in transgenic tropical maize.

An amaranth ( Amaranthus hypochondriacus) 11S globulin cDNA, encoding one of the most important storage proteins (amarantin) of the seed, with a high content of essential amino acids, was used in the transformation of CIMMYT tropical maize genotype. Constructs contained the amarantin cDNA under the control of a tissue-specific promoter from rice glutelin-1 ( osGT1) or a constitutive ( CaMV 35S) promoter with and without the first maize alcohol dehydrogenase intron ( AdH). Southern-blot analysis confirmed the integration of the amarantin cDNA, and copy number ranged from one to more than ten copies per maize genome. Western-blot and ultracentrifugation analyses of transgenic maize indicate that the expressed recombinant amarantin precursors were processed into the mature form, and accumulated stably in maize endosperm. Total protein and some essential amino acids of the best expressing maize augmented 32% and 8-44%, respectively, compared to non-transformed samples. The soluble expressed proteins were susceptible to digestion by simulated gastric and intestinal fluids, and it is suggested that they show no allergenic activity. These findings demonstrate the feasibility of using genetic engineering to improve the amino acid composition of grain crops.

Transgenic plants of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. ex Steud., from microprojectile bombardment of highly chlorophyllous embryogenic cells.

For the first time, the production of transgenic plants of the forage grass blue grama, Bouteloua gracilis [H.B.K.] Lag. ex Steud., is reported. Transgenic plants containing a gus Colon, two colons nptll fusion driven by a double CaMV35S promoter were obtained by microprojectile bombardment of the highly chlorophyllous embryogenic cell line 'TIANSJ98'. Transformed B. gracilis cell lines resisted a lethal concentration of 160 mg/l of kanamycin for at least 8 months. Chlorophyll development and growth rate were used as useful criteria for discriminating transformed from non-transformed clones. Stable integration of the transgene in the blue grama genome was demonstrated by PCR and Southern-hybridization analysis. Expression of the NPTll protein in transgenic plants grown under greenhouse conditions was confirmed indirectly by spraying kanamycin (150-250 mg/l) on plant foliage, and directly by ELISA immunological tests. Control plants sprayed with kanamycin showed foliar necrosis and reduced growth (tillering) compared to plants containing the transgene. NPTll was found in transgenic plants in levels ranging between 12.6 and 29.6 ng/mg FW of cells, as determined by ELISA reactions.

Establishment, characterization and plant regeneration from highly chlorophyllous embryogenic cell cultures of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. ex Steud.

A finely dispersed, homogeneous and highly chlorophyllous cell suspension (TIANSJ98 cell line) was obtained from shoot apices of Bouteloua gracilis (H.B.K.) Lag. ex Steud. cultured on MPC medium containing MS salts supplemented with 2,4-D (1 mg/l), BAP (2 mg/l) and adenine (40 mg/l). When the TIANSJ98 cell line was grown in this medium with shaking at 180 rpm it had doubling times of 7.2 and 3.7 days in terms of fresh and dry weight, respectively. Total chlorophyll content in this cell culture ranged from 121.6 to 18.3 µg/g FW at 12 and 21 days following culture initiation. Plants regenerated from the TIANSJ98 cell line, via somatic embryogenesis, were grown to maturity and produced seeds. Although different cell culture systems have been described for cereals and grasses, to the best of our knowledge this is the first report of a highly chlorophyllous and regenerable cell suspension in Poaceae.

Overexpression, purification, and in vitro refolding of the 11S globulin from amaranth seed in Escherichia coli.

An amarantin 11S globulin cDNA encoding one of the most important storage proteins of amaranth seeds, with a high content of essential amino acids, was expressed in Escherichia coli. A good level of expression of recombinant amarantin with a molecular weight of 59 kDa was obtained. The recombinant protein was extracted by ammonium sulfate precipitation and purified to homogeneity using ion-exchange chromatography and reversed phase high-performance liquid chromatography. The expressed protein exhibited electrophoretic, immunochemical, and surface hydrophobicity properties similar to those of native amarantin from amaranth seed. Also, the recombinant protein was refolded in vitro using two different methods.