Cloning And Expression Of An α-galactosidase And An Acid Phosphatase Genes

α-galactosidase (α-D-galactoside galactohydrolase, EC 3.2.1.22)which is distributedwidely in microorganisms, plants and animals catalyzes hydrolysis ofα-1,6 linkedα-galactoside residues from oligosaccharides such as melibose, raffinose and stachyose,and from polymeric galacto (gluco) mannans,α-galactosidase is widely used in manyfields, such as feed additive, food additive, pulp and paper processing, and medicine andpharmaceutical industry. However,α-galactosidases are mainly produced from naturallymicrobial strains at levels that are too low for commercial use. An effective approach tosolve the problem is to clone anα-galactosidase coding gene and to construct recombinantstrains with high-level expression ofα-galactosidase by improving the production andreducing the price of theα-galactosidase.We isolated a new strain of Geobacillus kaustophilus from a geothermic.colliery inthe middle reaches and upriver of the Yangtze River. Based on the genomic sequenceanalysis of another strain of the same genus, we successfully cloned a putativeα-gal-ggene.The ORF of Agll consisted of 2190 nucleotides encoding a protein of 729 aminoacids. The theoretical molecular mass and pI of the protein were 83.5 kDa and 6.595,respectively.The gene,α-gal-g, was cloned into expression vector pPIC9. The recombinant vectorwas linearized and transformed into yeast strain GS115 by electroporation. There were 13recombinants in the 100 transformants. The highest activity produced by the engineeredstrain 92~# reached 80 U/ml in high cell density fed-batch fermentation carried out in a 3Lfermentor.A group of thermophilic microorganisms were isolated from hotspring mud with atemperature of 40～85℃. By extracting the genomic DNA and sequencing randomly, wefound there was very high identity between these sequences and the genomic sequence ofThermus bacteria. According to the putative acid phosphatase gene sequence (TTHA1616)of Thermus thermophilus HB8 from Genbank (Accession No: AP008226), we designedprimers and amplified the sequence by PCR. A 798 bp DNA sequence with putative entiredacid phosphatase conserved domain, 98％similar to that of TTHA1616 from Thermus thermophilus, was gotten. Then we expressed it in E.coli, and got a 33 kDa protein whichwe deduced that it had the activity of acid phosphatase.