Study Of Key Enzymes Related To Formaldehyde Production In Odor-Forming Pathway Of Shiitake Mushroom (Lentinus Edodes)

Formaldehyde is one of by-products of odor-formed pathway where GGT and C-S lyase act as key enzymes. The aim of this study is to understand the contribution of odor-formed pathway to endogenous formaldehyde formation in shiitake mushroom (Lentinus edodes) by regulating activity or expression of GGT and C-S lyase. First, C-S lyase was purified from shiitake mushroom and its structures and properties were studied; second, the full-length cDNA sequence of GGT was cloned, and the relationship of GGT gene transcription and fonnaldehyde production in shiitake mushroom was discovered; third, effects of GGT and C-S lyase on the formaldehyde formation were studied by promoting or inhibiting activity of these two enzymes with their promoters and inhibitors addition into shiitake homogenate. Effects of MgSO4in medium on characteristic odors fonnation, odor precursors formation, formaldehyde formation, GGT gene expression, GGT and C-S lyase activity were studied. All results are shown as follows. 1Rapid purification and characterization of C-S lyase in shiitake mushroom(1) Rapid purification and structures of C-S lyaseC-S lyase was purified from shiitake mushroom through two key steps, protein precipitation by PEG6000and C-S lyase elution by hydroxyapatite column, reaching to234.82purified fold and12.36%recovery. These results of SDS-PAGE, Native-PAGE and HPLC assays suggest the high purity of C-S lyase which consists of two identical subunits of44KDa. Amino acid composition of C-S lyase was analyzed, and17kinds of amino acids were detected. Of these amino acids, tyrosine accounts the highest with55%, and the ratio of the acidic amino acid and basic amino acid is13.06/3.14.(2) Enzyme properties of C-S lyaseThe result of isoelectric focusing electrophoresis (IEF) assay shows that the isoelectric point of C-S lyase is at pH4.75. The optimal temperature and pH value for enzymatic catalyzation are around35℃and7.5, respectively. C-S lyase is of good thermal stability. The catalytic efficiency of C-S lyase acting on S-methyl-L-cysteine sulfoxide is significantly higher than that acting on S-allyl-L-cysteine sulfoxide and S-methyl-D-cysteine sulfoxide. In addition, C-S lyase activity can be inhibited by Cys, GSH, and Zn2+, and be promoted by Lys.2Cloning and analysis of GGT cDNA full-length sequence (1) Cloning of GGT cDNA full-length sequenceThe full-length cDNA sequence of GGT was obtained by RACE technology. The full-length cDNA sequence of GGT is2063bp (Gene bank accession NO. JX123478), including36bp of5’-untranslated region (UTR),113bp of3’-UTR and1914bp of open reading frame (ORF) that encodes a637-amino acid protein. The calculated molecular mass of this protein is70KDa, contained a GGT familial conserved domain. The BLAST analyasis of the deduced amino acid sequences of GGT with those of other species indicated that shiitake mushroom GGT sharing94%similarity with Schizophyllum commune,94%with Sporisorium reiliaimm,92%with Auricularia delicate.(2) Expression pattern of GGT mRNA in shiitake mushroomThe developmental expression pattern of different growth stage study results show that in stalks and caps, GGT gene expression increases from the first stage to the third stage and then decreases significantly. Tissue-specific study results show that the GGT mRNA levels are higher expressed in stalks than in caps significantly. Strains study results show that GGT mRNA levels are higher in806than in808. Stored temperatures (4℃,20℃,30℃) study results show that the GGT mRNA level is the highest in shiitake mushroom stored at4℃and the lowest at30℃. Correspondingly, formaldehyde content was determined in growth process of shiitake mushroom. All these results indicate that the GGT gene expression positively affect formaldehyde formation.3The contribution of odor-formed pathway to fonnaldehyde fonnation(1) Effect of GGT and C-S lyase on formaldehyde fonnationTo homogenate of shiitake mushroom, adding purified GGT and C-S lyase, Na+and K+can promote the fonnaldehyde fonnation, and adding Ser-borate, GSH, Cys and Zn2+can inhibit the fonnaldehyde fonnation. In addition, as regulating the homogenate pH value from4to10, the formaldehyde formation increase gradually until reaches the highest at pH8and then decreases from pH9to10.(2) Effect of MgSO4in medium on fonnaldehyde and odor fonnationOf shiitake mushrooms cultured in medium with MgSO4level of0.1%,0.2%,0.4%, respectively, formaldehyde, odor compounds (1,2,4,5-Tetrathiane and Lenthionine), odor precursors (S-allyl-L-cysteine sulfoxide and S-methyl-L-cysteine sulfoxide), GGT mRNA level, GGT activity, and C-S lyase activity were measured. Results show that, as MgSO4content increases the fonnation of fonnaldehyde, odor compounds and odor precursors rises following the enhance of GGT gene expression and enzyme activity of GGT and C-S lyase.As a conclusion, the odor-formed pathway is the main pathway of fonnaldehyde fonnation in shiitake mushroom.