Celińska Ewelina, Białas Wojciech, Borkowska Monika, Grajek Włodzimierz
Department of Biotechnology and Food Microbiology, Poznań University of Life Sciences, ul. Wojska Polskiego 48, 60-627, Poznań, Poland,
Appl Microbiol Biotechnol. 2015 Mar;99(6):2727-39. doi: 10.1007/s00253-014-6314-2. Epub 2014 Dec 31.
Raw-starch-digesting enzymes (RSDE) are of major importance for industrial applications, as their usage greatly simplifies the starch processing pipeline. To date, only microbial RSDE have gained considerable attention, since only microbial production of enzymes meets industrial demands. In this study, α-amylase from rice weevil (Sitophilus oryzae), the major rice pest, was cloned and expressed in Yarrowia lipolytica Po1g strain. The enzyme was secreted into the culture medium, and the peak activity (81 AU/L) was reached after only 29 h of culturing in 5-L bioreactors. Through simple purification procedure of ammonium sulfate precipitation and affinity chromatography, it was possible to purify the enzyme to apparent homogeneity (25-fold purification factor, at 5 % yield). The optimal conditions for the α-amylase activity were pH 5.0 and a temperature of 40 °C. The α-amylase studied here did not show any obligate requirement for Ca(2+) ions. The recombinant α-amylase appeared to efficiently digest granular starch from pea, amaranth, waxy corn, and waxy rice.
生淀粉消化酶（RSDE）在工业应用中具有重要意义，因为它们的使用极大地简化了淀粉加工流程。迄今为止，只有微生物RSDE受到了广泛关注，因为只有微生物产生的酶才能满足工业需求。在本研究中，从主要的水稻害虫米象（Sitophilus oryzae）中克隆了α-淀粉酶，并在解脂耶氏酵母Po1g菌株中进行表达。该酶被分泌到培养基中，在5-L生物反应器中培养仅29小时后就达到了峰值活性（81 AU/L）。通过硫酸铵沉淀和亲和色谱的简单纯化程序，有可能将该酶纯化至表观均一性（纯化倍数为25倍，产率为5%）。α-淀粉酶活性的最佳条件是pH 5.0和温度40°C。此处研究的α-淀粉酶对Ca(2+)离子没有任何绝对需求。重组α-淀粉酶似乎能有效消化豌豆、苋菜、糯玉米和糯米中的颗粒淀粉。
