Puirfication And Characteirzation Of A Raw Starch Digesting Glucoamylase

Raw starch digesting amylase, which belongs to amylase family, can directly process orhydrolyze ungelatinized starch. With its ability to directly degrade raw starch materials, rawstarch digesting amylase shows advantages in saving energy and reducing costs when used inindustry. At present, the researches on raw starch amylase are mainly focused on the enzymesfrom microbe. And there are detailed studies on its enzymatic properties, characteristics, thestructure of enzyme, the expression of gene and so on. However, glucoamylases from fungallack such elaborate research and need exploring further in certain aspects such as enzymeactivities and stability, due to which, they can’t be put into practical use. Therefore,importance should be attached to study on raw starch digesting amylases with newfunctionality.This article was started from a crude enzyme which was from a pre-screened strain, andan excellent raw-starch digesting glucoamylase was purified from this crude enzyme, then theenzyme properties were deeply studied, including the comparation with commercialglucoamylase about hydrolysis characteristics on raw starch and alcoholic fermention, theenzymatic properties, and then had a primary understanding on the enzyme characteristics,which determined that it had an excellent hydrolysis function on raw starch. The maincontents are as follows:Firstly, the crude enzyme was compared with a commericial glucoamylase on raw starchdigesting properties, the results showed it was far superior to the commercial enzyme underthe same conditions, and then we separated and purified the active ingredient （RG2） from thecrude enzym using the combination of HiPrep DEAE FF16/10anion exchange, Mono Qanion exchange and Gel filtration chromatography. The crude enzyme was purified4.58timeswith31.68%recovery of enzyme activity. The molecular weight of the purified raw starchglucoamylase was about82KDa identified by SDS-PAGE. The pure enzyme’s glucoamylasespecific activity was about1257.36U/mg as well as its raw starch digesting glucoamylasespecific activity was345.15U/mg.Subsequently, the study of hydrolysis characterstics on raw starch showed that:1) theratio of the activities of RG2acted on ungelatinized soluble starch, corn starch and corn flourrespected to the activity of gelatinized starch were27.19%、17.27%、41.80%, which werehigher than Glucoamylase G1, Glucoamylase G2and Solid glucoamylase SG1. The producttrends of RG2acted on three kinds of substrates were accumulated faster and more than otherthree enzymes;2) through the experiments of adsorption rates and hydrolyzate degree, theeffects of RG2acted on three kinds of substrates showed that: corn flour> soluble starch>cornstarch;3) the concentration of corn flour had no significant influence on the hydrolyzatedegree; under the concentration of8%, the soluble starch were hydrolyzated well; at the rangeof8%-28%, the degree of hydrolysis was relatively stable; while the degree was decreasedwhen the concentration was higher than32%; when acted on corn starch, the overall degreewas relatively lower, and the degree was dropped and reached steady when the substrateconcentration was higher than4%. After that, the study on enzymatic properties of pure enzyme showed that:1) theoptimum temperature of RG2was50°C, and there was a good stability under50°C which90%activity were still reserved after heated for2h at50°C;2) the optimum pH was4.6, andthere was a good stability between pH3.5and7.0;3) the RG2was activated by Fe²⁺, Mg²⁺,Mn²⁺, Co²⁺and was strongly activated by Fe²⁺, however, EDTA, Cu²⁺and K+inhibited theenzyme activity at various extents;4) its Kmand V_maxfor soluble starch were7.436mg/mLand1.449mg/（mL·min）;5) the substrate specificity of RG2showed that the enzyme hadhigher enzyme activity on malt dextrin and next was soluble starch;6) through the HPLCanalysis of the hydrolyzates on ungelatinized soluble starch and gelatinized starch showedthat the main product was glucose.7) the mass spectrum was obtained through the massspectrometry and it was proved that the enzyme had the highest homology with α-1,4glucosidase from Aspergillus sp.Then, the study of RG2applied to alcoholic fermention showed that: RG2had theadvantages of shorter fermentation time, lower residual tota l sugar and higher ethanolproduction than G2and SG1, which had a good application value.