Cloning, Sequence, And Expression Of α-D-Galactosidase From Coffee Bean (Coffea Liberica & Coffea Canephora)

a-D-galactosidase ( a-Gal, R. C. 3. 2. 1. 22 ) i s an exo-glycosidaso, widely present in the bio-resources. The enzyme isolated from coffee beans has been well characterized and it has high activity in hydrolyzing the terminal a -Gal residues from glycoconjugates on human blood group B erythrocytes to demonstrate group 0 serotype. a -Gal from coffee bean has been successf u 11 y used i n the serologicl conversion of red blood cells in clinical studies of blood transfusion. The molecular weight of different sources of a-Gal is between 28 000-370 000, and the molecular weight of a-Gal from coffee bean(Coffea arabicd) is 41 000. The whole Tenth cDNA of a -Gal from coffee bean(C. arabicd) is 1384bp and that of the mat peptides cDNA is 1089bp.The results of this study are as follows:l.A Simple, fast, and efficiency method for extracting of total RNA from coffee bean has been used by RNA extracting Kit which were improved in this paper.2. Two mat_peptides cDNA encoding n -Gal have been cloned by RT-PCR from coffee bean ( C. liberica & C. canephora ) . The mat_peptidcs cDNA are 1089bp, encoded 364 amino acid(AA). The cDNA from C. liberica is 98.7% homologous to the published C. arabica sequence. It has been found that 14bp of DNA sequence or 8 AA of protein sequence were changed. The cDNA of u -Gal gene from C. canephora is 99.27% homologous to the published C. arabica sequence,while 8bp of DNA sequence or 4 AA of protein sequence were changed.3. Four expression vectors, pPICZ a A/Gal-Z, pPICZ a A/Ga1-D, pGAPZ a A/Gal-Z, pGAPZ a A/Gal-D have been constructed successfully with the a-Gal gene from coffee bean and the vectors, pPICZ a A and pGAPZ a of Pichia pasloris.4. One Excherichia coli expression vector, pFT-22/Gal-D has been constructed with the a -Gal gene isolated from C. liberica and the E coli secretion vector pET-22b ( + ) .5. The three constructed vectors pPICZ a A/Gal-Z, pPICZa A/Gal-D, pGAPZ a A/Gal-D were transferred into P. pastoris stain GS115. The shake-flask expression with recombinant strain of P. pastoris, pPICZ a A/Gal-Z/GS115, pPICZ a A/Gal-D/GS115, pGAPZ a A/Gal-D/GSl15; the fermentation expression with recombinant strain of P. pastoris , pPICZ a A/Gal-D/GSl15 have been made. The products of shake-flask and fermentation expression showed a main band of 40KD on SDS-PAGE.6. The enzyme assay showed the high enzyme activity of r a -galactosidase when using pPICZ a A vector.The enzyme activity from C. liberica was 19.11 (U/ml)at 48 h and up to 48.22( U/ml )at 108 h. the en/yme activity from C. cancphora was 15.93 (U/ml) at 48 h and up to 18.18 (U/ml) at 108 h.7. There were some differences between the r a -Gal quantities of expression with different vectors, the methanol inducing vector, pPICZ a A was better than that of the constitute vector pGAPZ a A.8. The constructed vector pPICZa A/Gal-D has been transferred into P. pastoris stain GS115, followed by fermentation, a single protein band of 40 KD showed on SDS-PAGE. The enzyme assay showed the activity of a -galactosidase.9. The constructed vector pET-22/Gal-D was transferred into E. coli stain DL21(DE3)pLysS, followed by shake-flask expression. There was not obviously objective protein band has been seen on SDS-PAGE. The enzyme assay showed a light activity of a -Gal. The result showed that the a -Gal gene was not well expression in this expression system.