Production of Useful Oligosaccharides by Lytic Enzymes

从东方诺卡氏菌（Nocardia orientalis）IFO 12806或里氏木霉（Trichoderma reesei）的培养液中提取几丁质酶，经柱层析纯化至表观均一。该酶是一种水解酶，它也催化四-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。该酶将四糖转化为主要产物六-N-乙酰壳六糖（GlcNAc）_6和二-N-乙酰壳二糖（GlcNAc）_2。五-N-乙酰基-壳五糖（GLcNAc）_5的相应值为七-N-乙酰基-壳七糖（GLcNAc）_3。在<beta>高浓度甲醇水溶液中，利用溶菌酶的催化作用，可有效地将对硝基苯基2-乙酰氨基-2-脱氧-β-D-吡喃葡萄糖苷（PNP-GlcNAc）5转糖基至4位。在此反应中使用甲醇水溶液体系不仅保证了PNP-GlcNAc底物的溶解性，而且导致了对硝基苯基-五-N-乙酰基-β<beta>-壳戊糖苷（PNP-（GlcNAc）_5）产量的显著增加。PNP-（GlcNAc）_5与对硝基苯基-四-N-乙酰基-β<beta>-壳聚糖四糖苷（PNP-（GlcNAc）_4）相比，是溶菌酶活性测定的底物。

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).