Nemukula A, Mutanda T, Wilhelmi B S, Whiteley C G
Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa.
Bioresour Technol. 2009 Mar;100(6):2040-5. doi: 10.1016/j.biortech.2008.10.022. Epub 2008 Nov 22.
A transferase was isolated, purified and characterised from Aspergillus aculeatus. The enzyme exhibited a pH and temperature optima of 6.0 and 60 degrees C, respectively and under such conditions remained stable with no decrease in activity after 5h. The enzyme was purified 7.1 fold with a yield of 22.3% and specific activity of 486.1Umg(-1) after dialysis, concentration with polyethyleneglycol (30%) and DEAE-Sephacel chromatography. It was monomeric with a molecular mass of 85kDa and K(m) and V(max) values of 272.3mM and 166.7micromolmin(-1)ml(-1). The influence of pH, temperature, reaction time, and enzyme and sucrose concentration on the formation of short-chain fructooligosaccharides (FOS) was examined by statistical response surface methodology (RSM). The enzyme showed both transfructosylation and hydrolytic activity with the transfructosylation ratio increasing to 88% at a sucrose concentration of 600mgml(-1). Sucrose concentration (400mgml(-1)) temperature (60 degrees C), and pH (5.6) favoured the synthesis of high levels of GF(3) and GF(4). Incubation time had a critical effect on the yield of FOS as the major products were GF(2) after 4h and GF(4) after 8h. A prolonged incubation of 16h resulted in the conversion of GF(4) into GF(2) as a result of self hydrolase activity.
从棘孢曲霉中分离、纯化并表征了一种转移酶。该酶的最适pH和温度分别为6.0和60℃，在此条件下保持稳定，5小时后活性无下降。经透析、聚乙二醇（30%）浓缩和DEAE-琼脂糖凝胶层析后，该酶纯化了7.1倍，产率为22.3%，比活性为486.1Umg(-1)。它是单体，分子量为85kDa，K(m)和V(max)值分别为272.3mM和166.7micromolmin(-1)ml(-1)。采用统计响应面法（RSM）研究了pH、温度、反应时间、酶和蔗糖浓度对短链低聚果糖（FOS）形成的影响。该酶同时具有转果糖基化和水解活性，在蔗糖浓度为600mgml(-1)时，转果糖基化率提高到88%。蔗糖浓度（400mgml(-1)）、温度（60℃）和pH（5.6）有利于高水平合成GF(3)和GF(4)。孵育时间对FOS的产量有关键影响，因为4小时后的主要产物是GF(2)，8小时后的主要产物是GF(4)。由于自身水解酶活性，延长孵育16小时会导致GF(4)转化为GF(2)。
