琼胶酶水解工艺条件的优化及产物分析

Condition Optimization and Product Analysis of Agarase Hydrolysis Process

为探讨琼胶酶水解琼胶的动态过程变化,采用DNS法测定还原糖,结合苯酚-硫酸法测定体系总糖,根据二者比例,得出体系总糖随时间变化的平均聚合度,从而得出各因素对琼胶水解过程的影响。结果表明,来源于厦门红树林的海洋弧菌JMUAZ6菌株产生的琼胶酶,其水解琼胶的适宜工艺条件为:水解温度40℃,p H 7.0,初始底物质量浓度12 g/L,加酶量25%,在120 r/min振荡条件下酶解反应90 min。此工艺条件下产生还原糖的质量浓度2.179 g/L,平均聚合度最低值(DP)3。通过20 L罐放大试验验证小试优化工艺条件具有良好的重现性。采用薄层色谱分析酶解产物的降解情况,结果显示:反应48 h后,终产物为二糖及少量的四糖。

To study dynamic changing process and technical conditions of agar hydrolysis by agarase, the content of reducing sugar was determined by a 3,5-dinitroalicylic acid assay, as well as the content of total polysaccharide was detected by phenol-sulfuric acid method. According to the ratio of reducing sugar and total sugar, the variation regularity of average polymeric degree of total sugar in reaction system has been achieved. Thus the influence of enzymatic hydrolysis affected by the reaction factors was abtained. The optimum condtions for agar hydrolysis by agarase which came from marine Vibrio JMUAZ6 were achieved by a series of single-factor experiments. The results showed that the concentration of reducing sugar reached 2.179 g/L and the average polymetric degree fell to 3, when substrate at the concentration of 12 g/L was dissolved in buffer solution at pH 7.0, adding 25% enzyme dosage and the agar hydrolysis was carried out in a shaking bath at 40 ℃ and 120 r/min for 90 min. In addition, the optimal synthesizing parameters mentioned above were verified in a way of continuous and stable operation for 20 L tank magnified experiment. Furthermore, the enzymatic hydrolysate was identified by thin-layer chromatography and it was shown that the intermediate products mainly include neoagarobiose, neoagaroetraose and neoagarohexaose. The end product of degradation is neoagarobiose after a 48 hour period enzymatic hydrolysis process.