Ortiz-Soto Maria Elena, Rudiño-Piñera Enrique, Rodriguez-Alegria Maria Elena, Munguia Agustin Lopez
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, UNAM, Cuernavaca, Morelos, 62210, México.
BMC Biotechnol. 2009 Jul 27;9:68. doi: 10.1186/1472-6750-9-68.
Increasing attention has been focused on inulin and levan-type oligosaccharides, including fructosyl-xylosides and other fructosides due to their nutraceutical properties. Bacillus subtilis levansucrase (LS) catalyzes the synthesis of levan from sucrose, but it may also transfer the fructosyl moiety from sucrose to acceptor molecules included in the reaction medium. To study transfructosylation reactions with highly active and robust derivatives, cross-linked enzyme aggregates (CLEAs) were prepared from wild LS and two mutants. CLEAs combine the catalytic features of pure protein preparations in terms of specific activity with the mechanical behavior of industrial biocatalysts.
Two types of procedures were used for the preparation of biocatalysts from purified wild type LS (WT LS) B. subtilis and the R360K and Y429N LS mutants: purified enzymes aggregated with glutaraldehyde (cross-linked enzyme aggregates: CLEAs), and covalently immobilized enzymes in Eupergit C. The biocatalysts were characterized and used for fructoside synthesis using xylose as an acceptor model. CLEAs were able to catalyze the synthesis of fructosides as efficiently as soluble enzymes. The specific activity of CLEAs prepared from wild type LS (44.9 U/mg of CLEA), R360K (56.5 U/mg of CLEA) and Y429N (1.2 U/mg of CLEA) mutants were approximately 70, 40 and 200-fold higher, respectively, than equivalent Eupergit C immobilized enzyme preparations (U/mg of Eupergit), where units refer to global LS activity. In contrast, the specific activity of the free enzymes was 160, 171.2 and 1.5 U/mg of protein, respectively. Moreover, all CLEAs had higher thermal stability than corresponding soluble enzymes. In the long term, the operational stability was affected by levan synthesis.
This is the first report of cross-linked transglycosidases aggregates. CLEAs prepared from purified LS and mutants have the highest specific activity for immobilized fructosyltransferases (FTFs) reported in the literature. CLEAs from R360K and Y429N LS mutants were particularly suitable for fructosyl-xyloside synthesis as the absence of levan synthesis decreases diffusion limitation and increases operational stability.
由于其营养特性，菊粉和果聚糖型低聚糖，包括果糖基木糖苷和其他果糖苷，受到越来越多的关注。枯草芽孢杆菌果聚糖蔗糖酶（LS）催化由蔗糖合成果聚糖，但它也可能将蔗糖的果糖基部分转移到反应介质中的受体分子上。为了研究具有高活性和稳健性的衍生物的转果糖基化反应，从野生型LS和两个突变体中制备了交联酶聚集体（CLEAs）。CLEAs在比活性方面结合了纯蛋白质制剂的催化特性和工业生物催化剂的机械性能。
使用两种类型的程序从纯化的枯草芽孢杆菌野生型LS（WT LS）以及R360K和Y429N LS突变体中制备生物催化剂：用戊二醛聚集纯化的酶（交联酶聚集体：CLEAs），以及在Eupergit C中共价固定化酶。对生物催化剂进行了表征，并使用木糖作为受体模型用于果糖苷合成。CLEAs能够像可溶性酶一样有效地催化果糖苷的合成。由野生型LS（44.9 U/mg CLEA）、R360K（56.5 U/mg CLEA）和Y429N（1.2 U/mg CLEA）突变体制备的CLEAs的比活性分别比等效的Eupergit C固定化酶制剂（U/mg Eupergit）高约70、40和200倍，其中单位指的是总体LS活性。相比之下，游离酶的比活性分别为160、171.2和1.5 U/mg蛋白质。此外，所有CLEAs都比相应的可溶性酶具有更高的热稳定性。从长期来看，操作稳定性受果聚糖合成的影响。
这是交联转糖苷酶聚集体的首次报道。从纯化的LS和突变体制备的CLEAs具有文献中报道的固定化果糖基转移酶（FTFs）的最高比活性。来自R360K和Y429N LS突变体的CLEAs特别适合果糖基木糖苷的合成，因为果聚糖合成的缺失减少了扩散限制并提高了操作稳定性。
