Screening Of Acid-stable Amylase Producing Strain From Distillers Yeast And Enzymatic Property

The process of making traditional Chinese liquor is primarily through solid-statefermentation or brewing, the characteristic is that saccharification and fermentation can beconducted at the same time during brewing. As fermentation proceeds, a variety of byproducts（lactate, succinate, acetate etal） will drive the pH of vinasse very low, consequently theactivity of common α-amylase will be impaired and result in incomplete hydrolysis of starch,low utilization rate of raw material. The application of acid-stable α-amylase becomes a veryimportant technological consideration in terms of making full use of surplus starch, improvingthe output rate of final products and reducing the total cost.Contraposing the problem in the utilization of vinasse, an acid-stable α-amylaseproducing strain was screened from the solid culture of Heng-shui-Lao-Bai-Gan, it wasidentified as Rhizopus microsporus var by morphological observation and26S rDNA, andthe compositions of culture medium and the conditions of fermentation were optimized, andthen separation and purification of acid-stable α-amylase and enzymatic properties werestudied in this paper, the results provided valuable references for the improvement of theliquor production technology.The main contents of research were as follows:The high producing strain （S7） was screened by screening and rescreening, the activityof enzyme reached225.76U/g dry substance. It was identified as Rhizopus microsporus varby morphological observation and26S rDNA, the sequence showed99%homogeneity withthe large rRNA subunit of the controlled strain Rhizopus microsporus var（AB363776.1）.Single-factor experiment and response surface analysis were used to optimize thisstrain’s composition of culture medium, the optimal compositions of culture medium werewater content11.156mL, the proportion of bran to bean cake4.145, MgCl20.0772%. On thiscondition, the activity of enzyme increased from225.76U/g to259.902U/g dry substance.The conditions of fermentation were optimized from initial pH, temperature, chargeamount. The optimal fermentation conditions were initial pH5.0, temperature32℃, chargeamount10g. On this condition, enzyme activity finally reached269.8U/g when fermentation time reached72h.The acid-stable α-amylase was purified through ammonium sulfate precipitation,Sephadex G-25desalination and decoloration, DEAE-52cellulose chromatography and itsmolecular weight was estimated to be about75kD by SDS-PAGE.The enzymatic properties of acid-stable α-amylase were studied, the results showed that itsoptimal pH was5.0, optimal reactive temperature was70℃. The enzyme had strong acidstability and heat stability, the relatively activity was still above80%after the enzyme thatwas added in buffer solution of pH4.0was kept at40℃for1h; the relatively activity thatwas determined by starch solution of pH4.0still above50%after the enzyme was kept at60℃for1h. The activation energy Ea=34.3kJ/mol, and the pre-exponential constantA=4.5x10⁴. Mg²⁺, K⁺, Ca²⁺, Fe²⁺had remarkable stimulation on enzyme activity, whereasMn²⁺, Zn²⁺, Cu²⁺had significant inhibitory effect on enzyme activity. Km was estimated as5.2g/L by Lineweaver-Burk double-reciprocal plot.