ABSTRACT
A cDNA from Penicillium minioluteum HI-4 encoding a dextranase (1,6-alpha-glucan hydrolase, EC 3.2.1.11) was isolated and characterized. cDNA clones corresponding to genes expressed in dextran-induced cultures were identified by differential hybridization. Southern hybridization and restriction mapping analysis of selected clones revealed four different groups of cDNAs. The dextranase cDNA was identified after expressing a cDNA fragment from each of the isolated groups of cDNA clones in the Escherichia coli T7 system. The expression of a 2 kb cDNA fragment in E. coli led to the production of a 67 kDa protein which was recognized by an anti-dextranase polyclonal antibody. The cDNA contains 2109 bp plus a poly(A) tail, coding for a protein of 608 amino acids, including 20 N-terminal amino acid residues which might correspond to a signal peptide. There was 29% sequence identity between the P. minioluteum dextranase and the dextranase from Arthrobacter sp. CB-8.
Subject(s)
DNA, Complementary/genetics , DNA, Fungal/genetics , Dextranase/genetics , Penicillium/enzymology , Penicillium/genetics , Amino Acid Sequence , Arthrobacter/enzymology , Arthrobacter/genetics , Base Sequence , Cloning, Molecular , Dextranase/chemistry , Escherichia coli/genetics , Gene Expression , Gene Library , Genes, Fungal , Molecular Sequence Data , Molecular Weight , Nucleic Acid Hybridization , Protein Biosynthesis , Protein Sorting Signals/chemistry , Protein Sorting Signals/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Sequence Homology, Amino AcidABSTRACT
We have cloned and expressed a bacterial thermostable alpha amylase gene in Pichia pastoris using the methanol-controlled alcohol oxidase (AOX1) promoter. Two integrative vectors were constructed with two different secretion signal sequences in order to obtain efficient secretion of the protein. One vector contains the structural gene encoding the mature alpha amylase fused to the SUC2 gene signal sequence from Saccharomyces cerevisiae. In the other vector, the alpha amylase is expressed with its own signal sequence. In both cases, the alpha amylase were secreted into the culture medium with high efficiency, around 2.5 and 0.9 g/l respectively.
Subject(s)
Amylases/biosynthesis , Bacillus/enzymology , Gene Expression , Pichia/genetics , Protein Sorting Signals/metabolism , Alcohol Oxidoreductases/genetics , Amylases/genetics , Amylases/isolation & purification , Amylases/metabolism , Bacillus/genetics , Base Sequence , Cloning, Molecular , Gene Expression Regulation, Enzymologic , Genes, Bacterial , Genetic Vectors , Molecular Sequence Data , Promoter Regions, Genetic , Protein Sorting Signals/biosynthesis , Recombinant Proteins/biosynthesis , Transformation, GeneticABSTRACT
Data on blood glucose concentration in bulls affected with molasses associated polioencephalomalacia are controversial. It has been suggested that the brain lesions are related to a "hypoglycemic state" during the development of polioencephalomalacia. This paper reports the mobilization of glucose by means of the epinephrine test in three bulls fed two diets, one forage based and the other molasses based. The results showed significantly greater hyperglycemic responses in the animals during the molasses diet than during the forage one. This probably means that glucose stores (as glycogen) are higher in cattle consuming molasses than those consuming forage. Such hepatic glucose output is in disagreement with the hypoglycemia theory as the cause of the early stages of brain lesions and focuses the probable cause as being related to glucose utilization.
