摘要
利用内切β-1,4-半乳聚糖酶(endo-β-1,4-galactanase,EC 3. 2. 1. 89)水解土豆浆制备低聚半乳糖,有望解决产品回收率低的问题。为此,该研究通过分子克隆技术,将黑曲霉β-1,4-半乳聚糖酶基因agh A在毕赤酵母中进行克隆表达,构建获得了重组菌GS115(p PIC-aghA)。摇瓶培养条件下,重组酶Agh A的酶活为130 U/m L,高于大多数已报道的半乳聚糖酶的酶活。酶学性质的研究表明,该酶的最适反应pH值和温度分别为4. 5和45℃,且在pH 4. 0~6. 0或30~50℃具有较好稳定性。大部分金属离子和EDTA对重组酶Agh A的酶活没有显著影响; Fe3+对其活性有强烈的抑制作用;而Hg2+则可使Agh A几乎完全失活。该酶对半乳聚糖的最大反应速率Vmax和米氏常数Km分别为400 mg/(m L·min)和0. 08 mg/m L。此外,重组酶Agh A还可以水解土豆浆,产生半乳二糖、半乳三糖和少量半乳四糖等低聚半乳糖。
High yields of galactooligosaccharides may be obtained by enzymatic hydrolysis of potato pulp using endo-β-1,4-galactanase(EC 3. 2. 1. 89). In present study,the gene aghA encoding endo-β-1,4-galactanase from Aspergillus niger CICIM F0510 was thus cloned and expressed in Pichia pastoris,which generated the recombinant strain GS115(pPIC-aghA). In shaking flask experiments,the activity of recombinant enzyme AghA was 130 U/m L. The optimal pH and temperature of AghA were 4. 5 and 45 ℃,respectively,and it was stable at pH 4. 0-6. 0 or at 30-50 ℃. Most of chemical ions and EDTA had no significant effects on the activity of AghA,while it was strongly inhibited by Fe3 +and nearly completely lost in the presence of Hg2 +. The Vmaxand Kmof AghA towards galactan(potato)were determined to be 400 mg/(m L·min) and 0. 08 mg/m L,respectively. Besides,potato pulp was hydrolyzed by AghA into galactobiose,galactotrioses and a small amount of galactotetraose. The results obtained paved the way for large scale production of galactanase AghA and its applications in preparing galactooligosaccharides from potato pulp.
引文
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