Production of Useful Oligosaccharides by Lytic Enzymes

从东方诺卡氏菌IFO 12806或里氏木霉的培养滤液中提取的几丁质酶，通过连续柱层析得到了明显的均一。该酶本质上是一种水解酶，它还催化四-N-乙酰基壳四糖(GlcNAc)_4和五-N-乙酰基壳五糖(GlcNAc)_5的转糖基化反应。该酶将四糖转化为六-N-乙酰基壳六糖(GlcNAc)_6和二-N-乙酰基壳二糖(GlcNAc)_2。五-N-乙酰基壳五糖(GlcNAc)_5的转糖速率依赖于温度、底物浓度和p H。在含有高浓度甲醇的水溶液中，溶菌酶催化可有效地诱导从(GlcNAc)_5到对-硝基苯基2-acetamido-2-deoxy-&lt；beta&gt；-D-glucopyranoside(PNP-GlcNAc)4位的转壳低聚糖。在该反应中采用甲醇水溶液体系，不仅保证了底物的溶解性，而且显著提高了p-nitrophenyl-penta-N-acetyl-&lt；beta&gt；-chitopentaoside(PNP-(GlcNAc)_5)的产量。与p-nitrophenyl-tetra-N-acetyl-&lt；beta&gt；-chitotetraoside(PNP-(GlcNAc)4)相比，PnP-(GlcNAc)_5是溶菌酶测定的底物。

Chitinase from the culture filtrates of Nocardia orientalis IFO 12806 or Trichoderma reesei was purified to apparent homogeneity by successive column chromatographies. The enzyme, which is essentially a hydrolase, also catalyzed a transglycosylation reaction on tetra-N-acetyl chitotetraose(GlcNAc)_4 and penta N-acetyl-chitopentaose(GLcNAc)_5. The enzyme converted the tetrasaccharide into hexa-N-acetyl-chitohexaose(GlcNAc)_6 and di-N-acetyl-chitobiose(GLcNAc)_2 as the major products. The corresponding values for penta-N-acetyl-chitopentaose(GLcNAc)_5 were hepta-N-acetyl-chitoheptaose(GLcNAc)_3. The rate of the transglycosylation depended on the temperature, the concentration of substrate and the pH.Transchitooligosylation from (GlcNAc)_5 to the 4-position of p-nitrophenyl 2-acetamido-2-deoxy-<beta>-D-glucopyranoside(PNP-GlcNAc) was efficiently induced through lysozyme catalysis in an aqueous solution containing methanol with a high concentration. Use of the aqueous methanol system in this reaction not only guaranteed solubility of PNP-GlcNAc substrate, but also resulted in a remarkable increase in p-nitrophenyl-penta-N-acetyl-<beta>-chitopentaoside(PNP-(GlcNAc)_5) production. PNP-(GlcNAc)_5 was substrate for lysozyme assay compared with p-nitrophenyl-tetra-N-acetyl-<beta>-chitotetraoside (PNP-(GlcNAc)_4).