抗真菌内切几丁质酶酶学性质及制备低聚壳寡糖功能分析

Enzymatic Properties of Antifungal Endochitinase and Its Function in the Preparation of Chitosan Oligosaccharides

为开发几丁质及壳聚糖生物转化活性寡聚糖并实现几丁质酶在食品防腐及生防方面的应用,从金色链霉菌(Streptomyces aureofaciens)XZ-Sa62中分离纯化几丁质酶(ChiA-Sa62),并研究其生物转化及抗菌功能。本实验采用Q-Sepharose Fast Flow、Sephadex G-100层析和反相高效液相色谱纯化ChiA-Sa62,经纯化后ChiA-Sa62纯化倍数为41.3倍,比活力为1119.8 U/mg。并采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和基质辅助激光解吸电离-飞行时间质谱测得ChiA-Sa62是分子质量为62.55 kDa的单亚基蛋白。ChiA-Sa62在50℃和pH 5.0的条件下,活性最高,并在60℃以下及pH 3.0~9.0范围内有良好稳定性。金属盐离子Ca^(2+)、Mn^(2+)、Mg^(2+)和Co^(2+)可激活酶活性。ChiA-Sa62对几丁质及脱乙酰度75%的壳聚糖具有专一内切水解活性,薄层色谱显示,水解产物分别为2~5个N-乙酰氨基葡萄糖单位的几丁寡糖和2~4个D-氨基葡萄糖单位的壳寡糖。ChiA-Sa62对底物胶体几丁质的米氏常数K_m和最大反应速率V_(max)值分别为2.75 mg/mL和64.52 U/mg。ChiA-Sa62可强烈抑制所试病原真菌菌丝生长;碘化丙啶染色显示,经ChiA-Sa62处理后的灰葡萄孢菌丝其细胞膜受到破坏。水解产物壳寡糖对致病性G^(-)菌(大肠杆菌和铜绿假单胞菌)和G~+菌(金黄色葡萄球菌和蜡样芽孢杆菌)有抑制作用,并且LIVE/DEAD染色显示壳寡糖可致使金黄色葡萄球菌死亡。本实验结果表明,Chi A-Sa62几丁质及壳聚糖在生物转化以及食品防腐及生物防治方面具有较大的应用潜力。

In order to develop chitin and chitosan bioconversion of active oligosaccharides and achieve the application of chitinase in food preservation and biocontrol,chitinase(ChiA-Sa62)was isolated and purified from Streptomyces aureofaciens XZ-Sa62,and its bioconversion and antibacterial function were studied.ChiA-Sa62 was purified by Q-Sepharose Fast Flow,Sephadex G^(-)100 chromatography,and reverse phase high performance liquid chromatography(RP-HPLC),with a purification factor of 41.3 and a specific activity of 1119.8 U/mg.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)showed that ChiA-Sa62 was a single subunit protein with a molecular weight of 62.55 kDa.ChiA-Sa62 exhibited maximum activity at 50℃and pH 5,and exhibited good stability below 60℃and within pH range of 3.0-9.0.Ca^(2+),Mn^(2+),Mg^(2+),and Co^(2+)could activate enzyme activity.ChiA-Sa62 had a specific endonucleating hydrolysis activity on chitin and chitosan with a degree of deacetylation of 75%.Thin layer chromatography(TLC)showed that the hydrolysis products were 2-5 N-acetylglucosamine(GlcNAc)units of chitosan oligosaccharides and 2-4 D-glucosamine(GlcN)units of chitosan oligosaccharides,respectively.The colloidal chitin was used as the substrate,with ChiA-Sa62 Km and Vmax values of 2.75 mg/mL and 64.52 U/mg,respectively.ChiA-Sa62 strongly inhibited the tested pathogenic fungi,and propidium iodide(PI)staining showed that ChiA-Sa62 disrupted the integrity of the cell membrane of Botrytis cinerea.The hydrolyzed chitosan oligosaccharide had antibacterial activity against pathogenic G^(-)bacteria(Escherichia coli and Pseudomonas aeruginosa)and G+bacteria(Staphylococcus aureus and Bacillus cereus).LIVE/DEAD staining showed that chitosan oligosaccharide could cause the death of S.aureus.ChiA-Sa62 chitin and chitosan have great potentials for application in biotransformation,food preservation,and biological control.