The Study on Fermentation and α-galactosidase of Panaeolus Fimicola and Irpex Lacteus

Author:Guo Ya Jie

Supervisor:song yuan shao xiao ming


Degree Year:2016





Macrofungi is an important resource,containing a variety of physiologically active substances.a-Galactosidases are a group of exoglycosidases catalyzing the cleavage of terminal α-l,6-linked galactosyl residues of a-galactosides,including raffimose family oligosaccharides(RFOs),galacto(gluco)-mannans,and galactolipids.In the present study,two novel a-galactosidases from Panaeolus fimicola and Irpex lacteus are purified and characterized.And the fermentation conditions of the two mushrooms are optimized.The main findings are as follows:Twenty-three mushroom strains were isolated and then identified by ITS sequence analysis.Sixteen strains can be identified to species.Seven strains are only identified to genus.Increased a-galactosidase activity were detected from P.fimicola and I.lacteus in submerged culture,thus the two mushrooms were used as materials to obtain a-galactosidases.Submerged culture conditions of P.fimicola were optimized.Optimal seed medium was:corn starch 20 g/L,yeast 7 g/L,K2HPO4-3H2O1 g/L\MgSO4-7H2O 0.5 g/L,cultured for 5 days.Optimum fermentation medium for a-galactosidase production was:soluble starch 90 g/L,yeast 40 g/L,K2HPO4·3H2O1 g/L\MgSO4·7H2O 0.5 g/L,cultured for 9 days.a-Galactosidase activity in culture filtrate was 0.93 U/mL after optimization,31 times higher than initial activity 0.03 U/mL.A monomeric a-galactosidase was isolated from the culture filtrate of P.fimicola,to date a-galactosidase has not been reported from the genus Panaeolus.It was purified 64.4-fold to electrophoretic homogeneity and exhibiting a specific activity of 0.3166 U/mg by ion exchange chromatography and gel filtration.It demonstrated a molecular mass of 66 kDa in SDS-PAGE.The enzyme exhibited optimal activity at 60℃ and pH 4.0.It was stable over a temperature range of 4-40℃ and a wide pH range of 3.0-10.0.The enzyme was completely inactivated by Ag+ and Fe3+ions and partially inhibited by Cu2+,Cd2+,Hg2+,Pb2+ and Al3+ ions.In contrast,it was stimulated in the presence of Mn2+ and Fe2+ ions at 5 mM and 10 mM,respectively.The complete inhibition of the enzyme by N-bromosuccinimide(NBS)signified the presence of tryptophan residue(s)at or near the active site.The a-galactosidase had broad substrate specificity(4-nitrophenyl-a-D-galactopyranoside,melibiose,raffinose,stachyose,locust bean gum and guar gum).These features of the enzyme suggest a potential value in some production processes.Submerged culture conditions of L lacteus were optimized.Optimum fermentation medium for a-galactosidase production was:soluble starch 20 g/L,sucrose 30 g/L,soybean meal 80 g/L,CaCl2 3 g/L,K2HPO4·3H2O 1 g/L\MgSO4·7H20 0.5 g/L,(NH4)2S04 0.5 g/L,cultured for 11 days.a-Galactosidase activity in culture filtrate was 5.75 U/mL after optimization,205 times higher than initial activity 0.028 U/mL.A monomeric a-galactosidase from the culture filtrate of I.lacteus was purified 94.19-fold to electrophoretic homogeneity.It exhibited a specific activity of 18.36 U/mg and demonstrated a molecular mass of 60 kDa in SDS-PAGE.It was optimally active at 80℃ and pH 5.0,and it was stable over a temperature range of 4℃ to 70℃ and a wide pH range of 2.0 to 12.0.It was completely inactivated by Ag+ and Hg2+ ions and NBS.Moreover,it exhibited good resistance to proteases.Galactose acted as a noncompetitive inhibitor with Ki and Kis of 3.34 and 0.29 mM,respectively.Theα-galactosidase presented a broad substrate specificity,which included pNPG,melibiose,stachyose,and raffinose with Km values of 1.27,3.24,7.1,and 22.12 mM,correspondingly.ILGI exhibited efficient and complete hydrolysis to raffinose and stachyose.These results indicate that,mushroom,especially I.lacteus is an appropriate source for enzyme production because it is edible and grows rapidly.α-Galactosidase from I.lacteus exhibits distinctive features such as thermal and pH stabilities,protease resistance,and effective degradation on RFOs.These findings suggest that ILGI is a good choice as a promising product with potential applications in industry and research.