双酶法制备小麦微孔淀粉的动力学

摘要

In order to understand the kinetic characteristics of the synergistic hydrolysis of wheat starch by α-amylase and glucoamylase for preparing microporous starch,the effects of pH,reaction temperature and enzyme dosage on hydrolysis rate during the hydrolysis of wheat type A granular starch by both enzymes were investigated and the Michaelis constants(Km) for the individual and combined enzymatic hydrolysis of wheat starch were determined.Both individual enzymatic hydrolysis processes obeyed the Michaelis-Menten equation,and the Km and Vmax(maximum reaction velocity) were 9.548 mg/mL and 0.659 mg/(mL.min) for and α-amylase,and 12.676 mg/mL and 0.555 mg/(mL min) for glucoamylase,respectively.The hydrolysis product glucose was a competitive inhibitor against the enzymes with an inhibitory constant(Ki) of 4.288 mg/mL.Maximum hydrolysis was reached when 5 mg/mL of wheat type A granular starch was hydrolyzed with 10 U/mL of α-amylase and 20 U/mLof glucoamylase under the conditions of 55 ℃ and pH 4.5 for 25 min.As a result,2.54 mg/mLof reducing sugar was produced.In conclusion,α-amylase and glucoamylase can be used together to hydrolyze wheat starch for the preparation of microporous starch and their combination shows significantly higher hydrolysis efficiency than individual use.%为明确α-淀粉酶和糖化酶协同水解小麦淀粉制备微孔淀粉的动力学特征,以小麦A淀粉为材料,系统地分析pH值、反应温度、α-淀粉酶及糖化酶用量对水解速率的影响,并确定α-淀粉酶、糖化酶单一酶的米氏常数以及双酶协同效应。结果表明：在单一水解体系中,α-淀粉酶和糖化酶对小麦A淀粉的降解均遵循Michaelis-Menten方程,α-淀粉酶的米氏常数Km为9.548mg/mL,最大反应初速率（Vmax）为0.659mg/（mL.min）,糖化酶以淀粉为底物的米氏常数（Km）为12.676mg/mL,最大反应初速率（Vmax）为0.555mg/（mL.min）。水解产物葡萄糖对反应体系具有竞争性抑制剂的作用,其抑制常数（Ki）为4.288mg/mL。在小麦A淀粉质量浓度为5mg/mL、α-淀粉酶10U/mL、糖化酶20U/mL、反应温度55℃、pH4.5、水解时间为25min的条件下,可达到淀粉的水解极限即还原糖生成质量浓度为2.54mg/mL。α-淀粉酶和糖化酶可协同水解小麦A淀粉制备微孔淀粉,双酶协同作用的水解效率明显高于单酶的水解效率。