CAJ | 학술논문

以壳聚糖为载体,通过正交实验确定了用壳聚糖凝胶微球固定多功能淀粉酶OPMA-N的最适条件：以20 mg/mL戊二醛交联,在25℃及pH=6.5条件下固定1.5 h,但需在引入底物淀粉介导的保护效应后,才能获得良好稳定性的固定化OPMA-N( IOPMA-N),显示了底物保护效应在催化部位占分子中较大区域的酶(如OPMA-N)固定化过程中的重要性.对比分析游离酶与固定化酶的性质与功能发现,在50℃及pH=6.0~7.0条件下, IOPMA-N的表观比活力比游离酶OPMA-N提高3倍以上,催化效率( kcat/Km )提高4倍以上,对弱酸性至中性及常温(30~50℃)环境的耐受性明显高于OPMA-N,并具有良好的操作稳定性和储存稳定性,重复使用15次后,酶活力仍然能够保持75%,在4℃下储存半衰期达31 d,而OPMA-N在4℃下储存5 d后活力基本丧失.催化产物的对比分析结果表明, OPMA-N固定化后,水解活性明显提高,但同时转苷活性有所下降,这与已报道的OPMA-N分子中2种催化活力存在平衡机制的结论一致,同时也表明在OPMA-N的固定化中底物淀粉对其水解活性有明显的保护作用,但对其转苷活性几乎无贡献.
이각취당위재체,통과정교실험학정료용각취당응효미구고정다공능정분매OPMA-N적최괄조건：이20 mg/mL무이철교련,재25℃급pH=6.5조건하고정1.5 h,단수재인입저물정분개도적보호효응후,재능획득량호은정성적고정화OPMA-N( IOPMA-N),현시료저물보호효응재최화부위점분자중교대구역적매(여OPMA-N)고정화과정중적중요성.대비분석유리매여고정화매적성질여공능발현,재50℃급pH=6.0~7.0조건하, IOPMA-N적표관비활력비유리매OPMA-N제고3배이상,최화효솔( kcat/Km )제고4배이상,대약산성지중성급상온(30~50℃)배경적내수성명현고우OPMA-N,병구유량호적조작은정성화저존은정성,중복사용15차후,매활력잉연능구보지75%,재4℃하저존반쇠기체31 d,이OPMA-N재4℃하저존5 d후활력기본상실.최화산물적대비분석결과표명, OPMA-N고정화후,수해활성명현제고,단동시전감활성유소하강,저여이보도적OPMA-N분자중2충최화활력존재평형궤제적결론일치,동시야표명재OPMA-N적고정화중저물정분대기수해활성유명현적보호작용,단대기전감활성궤호무공헌.
The catalytic site of the multifunctional amylase OPMA-N occupied a relatively large region in OPMA-N molecule. The optimal conditions to immobilize OPMA-N with hydrogel microspheres of chitosan as a carrier were established by orthogonal experiment as follows:20 mg/mL glutaraldehyde served as cross-linking agent, at 25 ℃, pH=6.5 for 1.5 h, but as long as after the introduction of the substrate-mediated protective effect and conformational memory in order to obtain a stabilized immobilized OPMA-N( IOPMA-N) . This high-lighted the importance of the protective effect of the substrate in the immobilization process of the multifunc-tional enzymes such as OPMA-N in which the catalytic site-covering region generally occupies a larger region in OPMA-N molecule. Compared with the free OPMA-N, IOPMA-N showed more than 3 times apparent activi-ty and more than 4 times catalytic efficiency(kcat/Km) at 50 ℃, pH=6.0—7.0, and had a significantly bet-ter tolerance to the pH from neutral to weakly acidic under moderate temperatures ( 30—50 ℃) . IOPMA-N also exhibited a better operational stability and storage stability by its more than 75% of the apparent activity after 15 times repeated use and 31 d half-life when stored at 4 ℃, while OPMA-N would lose its activity com-pletely after stored 5 d at 4 ℃. Comparative analysis of the catalytic product showed that IOPMA-N exhibited higher hydrolytic activity but slightly lower transglycosyl activity than OPMA-N, which is consistent with the fact that there is a functional( active) balance between hydrolytic and transglycosyl activities in OPMA-N mole-cule, and meanwhile, it also indicated that its substrate had a significant protective effect on its hydrolytic ac-tivity but hardly on its transglycosyl activity in its immobilization.