Studies Of The Polysaccharide Hydrolases From Abalone(Haliotis Discus Hannai) Viscera And Enzymatic Modification For Konjac Gum

Konjac gum(KGM)has good water holding capacity,gelling,thickening and film forming properties,and it has been used in medical,chemical and food industry.However,KGM is one of the highest viscosity water-soluble gums,so its application is limited.In the present study,KGM was degraded using crude enzyme of abalone viscera from Haliotis discus hannai.The physicochemical properties of degraed KGM samples(DKGMs)were investigated.The result showed that the crude enzyme of abalone viscera showed an optimal activity about 50? and pH6.0,and exhibited strong activity at 30-50? and pH 5.0-7.0.Substrate specificity showed that the activity of crude enzyme of abalone viscera to konjac gum was 18.5 times that of carboxymethylcellulose sodium.After enzymatic hydrolysis,the viscosity of KGM(1%)decreased from 15500 m Pa.s to 398 m Pa.s and the M_w decreased from 1.80×10⁶ Da to 4.48×10⁵Da.Both KGM and DKGMs were pseudoplastic fluids,and the apparent viscosity was decreased with increasing enzymatic degradation time.The viscoelasticity,melting temperature(T_m)and gelling temperature(T_g)were significantly decreased after enzymatic hydrolysis.The gel structure of DKGMs was damaged seriously after enzymatic hydrolysis,and the microstructure of DKGMs was fragmented.These results suggested that there are enzymes with strong enzymatic activity to konjac gum in abalone viscera,which has potential application value in biological methods to degrade konjac gum.According to above results,two polysaccharide hydrolases which could significantly degrade konjac gum were highly purified from abalone viscera via a series of chromatographies over Phenyl-HP hydrophobic column,Capto-S cation exchange column,affinity column and Sephacryl S-200HR gel exclusion column.The molecular weight was estimated to be 50 k Da and 40 k Da as determined by SDS-PAGE.LC-MS/MS analysis confirming the purified protein was endoglucanase and endo-beta-1,4-mannanase,respectively.The glucanase showed an optimal activity at pH 50? and 6.0,was comparably stable at pH 20-50? and 5.5-7.5.The mannanase showed an optimal activity at pH 50? and 6.0,was comparably stable at pH 20-45? and 4.5-6.0.The metal ions such as Mg²⁺,Cu²⁺,Al³⁺,Zn²⁺,Mn²⁺,Fe³⁺and Fe²⁺could strongly suppress the glucanase activity while Ca²⁺and Co²⁺showed obvious activated effect on enzyme activity.The metal ions such as Cu²⁺,Zn²⁺,Mn²⁺and Fe³⁺could strongly suppress the mannanase activity while Ca²⁺,Al³⁺,Co²⁺,Mg²⁺,Ba²⁺and Fe²⁺showed obvious activated effect on enzyme activity.Moreover,after the glucanase and mannanase were mixed in different proportions,it was found that the two enzymes had a synergistic effect on the degradation of konjac gum.When the ratio of glucanase to mannanase was 2:3,the enzymatic hydrolysis activity of konjac gum was the strongest.The DKGMs were added to myofibrillar protein form Nemipterus virgatus in a certain proportion,and the effect of konjac gum with different molecular weight on the gel properties of myofibrillar protein were investigated.The results showed that the turbidity,particle size and surface hydrophobicity of the composite system increased at first and then decreased with the decrease of molecular weight of konjac gum,and MP/KGM-150 exhibited the highest turbidity,particle size and surface hydrophobicity.Dynamic rheology show that DKGMs increased the storage modulus of myofibrillar protein gel after heating,and MP/KGM-150 exhibited the highest torage modulus.SDS-PAGE analysis indicated that the myosin heavy chain content first decreased and then increased,and the MP/KGM-150 had the shallowest bands.The thermal stability of myofibrillar protein increased with the addition of DKGMs,which may be related to the stabilization of the water content of the composite system.Moreover,the gel network structure of myofibrillar protein was more compact and uniform with the addition of DKGMs,and the gel network structure of MP/KGM-150 was most compact and uniform and moisture channels was smallest,indicating appropriate degradation for KGM could improve the gel properties of myofibrillar protein.