Purification, Characterization, Gene Cloning And Expression Of An α-Galactosidase From Penicillium Sp. F63

a-galactosidases (a-D-galactoside galactohydrolase, EC 3.2.1.22), which are distributed widely in microorganisms, plants and animals, catalyze the hydrolysis of a-1,6 linked a-galactoside residues from polymeric galacto(gluco)mannans and from oligosaccharides such as melibose, raffinose and stachyose. a-galactosidase is widely used in industrial fields, such as feed and food additive, pulp and paper processing, medicine and pharmaceutical industry. However, a-galactosidases are mainly produced from naturally microbial strains and the expression level of a-galactosidases is too low for commercial use. An effective approach to solving the problem is to clone an a-galactosidase coding gene and to construct recombinant strains with high-level expression of a-galactosidase to improve the production and to low the price of the a-galactosidase.An extracellular a-galactosidase from Penicillium sp F63, designated as Agll, was purified to homogeneity by ammonium sulfate precipitation, ion-exchange and gel filtration chromatography with a molecular mass of 82 kDa on SDS-PAGE while 330 kDa on native gradient PAGE, which indicated that Agll is a tetramer of identical subunits. Identification of MALDI-TOF-MS and sequencing of ESI-MS/MS suggested that the protein is a novel a-galactosidase. Based on amino acid sequences of the internal peptides, degenerated primers were synthesized and used to amplify specific chromosomal DNA sequence encoding the partial fragment of the target a-galactosidase. Subsequently, inverse PCR was performed to clone the flanking sequences of the fragment and to acquire the whole genomic sequence of the a-galactosidase. A cDNA encoding the a-galactosidase was isolated by RT-PCR. The ORF of Agll consists of 2205 nucleotides encoding a protein of 734 amino acids including 21 residues as a putative signal peptide in its N-terminal. Agll was intron-less, and a GUG codon was considered as the start codon of the gene. The theoretical molecular mass and pI of the protein were 78.5 kDa and 5.07, respectively. Seven potential N-glycosylation sites (N-X-S/T) could be identified in the amino acid sequence of the mature protein. Comparison of the deduced amino acid sequence of Agll with those of other a-galactosidase genes showed that Agll has the highest overall similarity to AglC of A. niger with a sequence identity of 69.6%. However, a-galactosidases of family 27 such as those of P. purpurogenum (identity, 7.5%) and P. simplicissimum (identity, 8.2%) showed little homology to Agll. To our knowledge, this is the first report of gene cloning for Penicillium a-galactosidase belonging to family 36 of glycosyl hydrolases.The gene encoding mature protein of Agll was cloned into expression vector pPIC9. The recombinant vector was then linearized and transformed into strain GS115 by electroporation. a-galactosidase-expressing transformants were selected by their ability to degradate the substrate pNPG and the recombinant colonies were colored yellow. There were 23 recombinants in the 100 transformants, of which the recombinant 56# produced the highest activity reached lllU/mL inhigh cell density fermentation carried out in a 3 L fermenter, which was known to be the highest expression level with determination of enzyme activity against pNPG as substrate.The recombinant and native a-galactosidase were purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion-exchange and gel filtration chromatography and were characterized. The optimal temperature of native enzyme is 45 °C while that of recombinant enzyme is 40°C. The optimal pH values of the native and the recombinant enzyme were both 5.0. Both of the enzymes are sensitive to high temperature. The native enzyme displays good pH stability after 12 hour in the range from 5.0 to 6.0 while the recombinant enzyme does after 12 hours from 6.0 to 6.5, which retain 88% of their activity. The specific activity of the native enzyme is 106.4 U/mg while that of the recombiant enzyme is 925.1 U/mg. The native and the recombinant enzymes are completely inhibited by Hg2+. The native a-galactosidase is partially inhibited by Ag+ and the recombinant a-galactosidase is totally inhibited by Ag+. While other metal ions and chemicals have no or little effect on the activity of the a-galactosidases. The native and recombiant a-galactosidases catalyze the release of galactose from natural oligosaccharides, such as melibiose, raffinose, and stachyose.Because of the higher specific activity and the better optimal temperature and pH, which are nearly in accordance with the physicochemical conditions of the intestines of monogastric animals, the recombinant a-galactosidase is more advantageous to be used in animal feed additive than the native one.