壳聚糖接枝双醛淀粉固定化黑曲霉木聚糖酶的研究
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摘要
不同的酶由于其结构和特性都不相同,因此选择的载体和固定化方法也各有不同。为了所研究的酶能够很好的适应所选的的固定化载体和采用的固定化方法,载体的选择仍然是固定化酶首要解决的问题。对于木聚糖酶来说,由于底物的一些限制,固定化方法上多采用吸附和载体结合法。本文在此基础上选取了两种无机插层材料水滑石和磷酸锆以及天然材料壳聚糖通过接枝双醛淀粉这些载体,筛选出对黑曲霉A-25来源的木聚糖酶固定化效果较好的载体,并对其固定化工艺进行了优化,进一步研究了固定化酶的酶学特性。研究结果如下:
1.寻找合适的载体是固定化研究的重要课题之一。通过对所选的几种载体的固定化试验,筛选出了固定化木聚糖酶效果较好的壳聚糖接枝双醛淀粉。然后对壳聚糖接枝双醛淀粉材料固定化木聚糖酶的条件进行了优化,结果表明:3.0g壳聚糖球添加氧化度为95%的双醛淀粉1.0g在70℃条件下处理16h,然后在此条件下,1.0g的载体在添加0.2mo/l pH5.6醋酸缓冲液、温度30℃,加酶量为4ml的条件下进行8h固定化处理效果好,获得的固定化酶回收率可达63%。
2.与游离酶相比,壳聚糖固定化酶和壳聚糖接枝双醛淀粉固定化酶,其最适pH没有变化,均在4.6,pH的稳定性变化不大,在pH4.6-8.6之间固定化酶稳定性很好,总体壳聚糖接枝双醛淀粉固定化酶的pH稳定性好于壳聚糖直接吸附固定化酶。酶的最适温度提高了,壳聚糖接枝双醛淀粉固定化酶和壳聚糖固定化酶的最适温度分别由游离酶的50℃提高到55℃和60℃,温度稳定性都明显增加了,壳聚糖接枝双醛淀粉以及壳聚糖固定化酶在50℃下半衰期分别是游离酶的2.35倍和1.51倍。同时固定化酶在4℃冰箱内的贮存稳定性比游离酶要好,贮存76天后固定化酶残余活力还保持在85%,游离酶仅为60%。供试的所有离子对壳聚糖固定化酶和壳聚糖接枝双醛淀粉固定化酶的影响有些区别,K~+、Mg~(2+)和EDTA,对壳聚糖固定化酶的影响是部分失活的,但是对壳聚糖接枝双醛淀粉固定化酶是稍微激活的;除Ag~+以外,其它离子对固定化酶都没有激活作用。壳聚糖和壳聚糖接枝双醛淀粉固定化酶对桦木木聚糖的K_m值分别由原酶的1.14mg/ml稍微增加到1.17mg/ml和1.39mg/ml,表明采用壳聚糖或者是壳聚糖接枝双醛淀粉作为固定化材料基本保持了原有酶和底物的亲和性,但是采用壳聚糖接枝双醛淀粉固定化后V_(max)稍有增大。
3.采用壳聚糖接枝双醛淀粉载体固定化木聚糖酶水解木聚糖得到的水解液成分通过高效液相色谱分析发现,获得了以木二糖和木三糖为主的低聚木糖。
The enzyme because the different of its structure and the characteristic are not all same,therefore the immobilized carrier and the fixed method also respectively have the difference.It is important to look for good characteristic carrier and the best immobilized method,thesedays,people still commit themselves to develop new type carriers and the new pattern.As for the xylanase enzyme,as a result of substrate some limits,the enzyme usually immobilized by the adsorption or covalent. The paper selected the best carrier from two layer inorganic materials(layered Double Hydroxide andα-Zirconium phosphate)and natural polymer- Chitosan,The conditions for immobilization were optimized.The immobilized enzymes were further characterized in terms of their catalytic activity,kinetic parameters and thermal stability, etc.The main results were as follow:
1.Developing an ideal carrier is one of the important topics of immobilization study. Chitosan grafting Dialdehydle the Starch which was selected from three kinds of different materials showed good immobilization effect on the xylanase from Aspergillus niger-25.The optimal condition for xylanase immobilization were as follow: Dialdehydle the Starch(the oxidized degree is 90%),1.0g;the time pretreated Chitosan at 70℃,16h;the amount xylanase used,4ml(0.2M,pH5.6 acetic- sodium acetic buffer);the temperature and time for immobilization,30℃and 8h,respectively. The activity yield was 63%.
2.Compared to the free enzyme,Immobilization of xylanase from Aspergillus niger-25 on Chitosan grafting Dialdehydle the Starch and Chitosan showed that:both of the optimum pH were 4.6,pH stability did not change significantly by the immobilization, and was stable at pH 4.6～8.6,however,the immobilization effect by Chitosan grafting Dialdehydle the Starch was better than the Chitosan.The optimum temperature of the immobilized enzyme by Chitosan grafting Dialdehydle the Starch and Chitosan increased from 50℃to 55℃and 60℃,respectively,the thermalstability of the immobilized enzyme increased significantly and for Chitosan grafting Dialdehydle and Chitosan immobilized enzyme at 50℃which their half life were 2.35 and 1.51 times compared to the free enzyme,respectively.The storage stability in 4℃refrigerator was improved after immobilization.,the relativity activity was retained at 85%while the free enzyme was 60%after storaged 76 days.To the both immoblized enzymes,the affect of all ions used in the experiments were different,the metal irons K、Mg and EDTA had activation the immobilized enzyme by the Chitosan grafting Dialdehydle the Starch,but to the Chitosan immobilized enzyme which were inhibited.The other irons could not enhance the activity of the xylanase except Ag iron.For Chitosan grafting Dialdehydle the Starch and Chitosan immobilized enzyme,the K_m values for birch xylan slightly increased from 1.14 mg/ml to 1.39 and 1.17 mg/ml,respectively,which was indicated immobilization of xylanase essentially conserved the affinity of the enzyme towards its substrate.Increases in V_(max)upon immobilization are generally not observed.However,V_(max)values of the immobilized enzyme by Chitosan grafting Dialdehydle the Starch was slightly enhanced in this study.
3.Immobilized xylanase by the Chitosan grafting Dialdehydle the starch carrier hydrolyzed the Birchwood xylan,the component of the hydrolyzed solution by the HPLC analysed indicated a good result.
1.寻找合适的载体是固定化研究的重要课题之一。通过对所选的几种载体的固定化试验,筛选出了固定化木聚糖酶效果较好的壳聚糖接枝双醛淀粉。然后对壳聚糖接枝双醛淀粉材料固定化木聚糖酶的条件进行了优化,结果表明:3.0g壳聚糖球添加氧化度为95%的双醛淀粉1.0g在70℃条件下处理16h,然后在此条件下,1.0g的载体在添加0.2mo/l pH5.6醋酸缓冲液、温度30℃,加酶量为4ml的条件下进行8h固定化处理效果好,获得的固定化酶回收率可达63%。
2.与游离酶相比,壳聚糖固定化酶和壳聚糖接枝双醛淀粉固定化酶,其最适pH没有变化,均在4.6,pH的稳定性变化不大,在pH4.6-8.6之间固定化酶稳定性很好,总体壳聚糖接枝双醛淀粉固定化酶的pH稳定性好于壳聚糖直接吸附固定化酶。酶的最适温度提高了,壳聚糖接枝双醛淀粉固定化酶和壳聚糖固定化酶的最适温度分别由游离酶的50℃提高到55℃和60℃,温度稳定性都明显增加了,壳聚糖接枝双醛淀粉以及壳聚糖固定化酶在50℃下半衰期分别是游离酶的2.35倍和1.51倍。同时固定化酶在4℃冰箱内的贮存稳定性比游离酶要好,贮存76天后固定化酶残余活力还保持在85%,游离酶仅为60%。供试的所有离子对壳聚糖固定化酶和壳聚糖接枝双醛淀粉固定化酶的影响有些区别,K~+、Mg~(2+)和EDTA,对壳聚糖固定化酶的影响是部分失活的,但是对壳聚糖接枝双醛淀粉固定化酶是稍微激活的;除Ag~+以外,其它离子对固定化酶都没有激活作用。壳聚糖和壳聚糖接枝双醛淀粉固定化酶对桦木木聚糖的K_m值分别由原酶的1.14mg/ml稍微增加到1.17mg/ml和1.39mg/ml,表明采用壳聚糖或者是壳聚糖接枝双醛淀粉作为固定化材料基本保持了原有酶和底物的亲和性,但是采用壳聚糖接枝双醛淀粉固定化后V_(max)稍有增大。
3.采用壳聚糖接枝双醛淀粉载体固定化木聚糖酶水解木聚糖得到的水解液成分通过高效液相色谱分析发现,获得了以木二糖和木三糖为主的低聚木糖。
The enzyme because the different of its structure and the characteristic are not all same,therefore the immobilized carrier and the fixed method also respectively have the difference.It is important to look for good characteristic carrier and the best immobilized method,thesedays,people still commit themselves to develop new type carriers and the new pattern.As for the xylanase enzyme,as a result of substrate some limits,the enzyme usually immobilized by the adsorption or covalent. The paper selected the best carrier from two layer inorganic materials(layered Double Hydroxide andα-Zirconium phosphate)and natural polymer- Chitosan,The conditions for immobilization were optimized.The immobilized enzymes were further characterized in terms of their catalytic activity,kinetic parameters and thermal stability, etc.The main results were as follow:
1.Developing an ideal carrier is one of the important topics of immobilization study. Chitosan grafting Dialdehydle the Starch which was selected from three kinds of different materials showed good immobilization effect on the xylanase from Aspergillus niger-25.The optimal condition for xylanase immobilization were as follow: Dialdehydle the Starch(the oxidized degree is 90%),1.0g;the time pretreated Chitosan at 70℃,16h;the amount xylanase used,4ml(0.2M,pH5.6 acetic- sodium acetic buffer);the temperature and time for immobilization,30℃and 8h,respectively. The activity yield was 63%.
2.Compared to the free enzyme,Immobilization of xylanase from Aspergillus niger-25 on Chitosan grafting Dialdehydle the Starch and Chitosan showed that:both of the optimum pH were 4.6,pH stability did not change significantly by the immobilization, and was stable at pH 4.6～8.6,however,the immobilization effect by Chitosan grafting Dialdehydle the Starch was better than the Chitosan.The optimum temperature of the immobilized enzyme by Chitosan grafting Dialdehydle the Starch and Chitosan increased from 50℃to 55℃and 60℃,respectively,the thermalstability of the immobilized enzyme increased significantly and for Chitosan grafting Dialdehydle and Chitosan immobilized enzyme at 50℃which their half life were 2.35 and 1.51 times compared to the free enzyme,respectively.The storage stability in 4℃refrigerator was improved after immobilization.,the relativity activity was retained at 85%while the free enzyme was 60%after storaged 76 days.To the both immoblized enzymes,the affect of all ions used in the experiments were different,the metal irons K、Mg and EDTA had activation the immobilized enzyme by the Chitosan grafting Dialdehydle the Starch,but to the Chitosan immobilized enzyme which were inhibited.The other irons could not enhance the activity of the xylanase except Ag iron.For Chitosan grafting Dialdehydle the Starch and Chitosan immobilized enzyme,the K_m values for birch xylan slightly increased from 1.14 mg/ml to 1.39 and 1.17 mg/ml,respectively,which was indicated immobilization of xylanase essentially conserved the affinity of the enzyme towards its substrate.Increases in V_(max)upon immobilization are generally not observed.However,V_(max)values of the immobilized enzyme by Chitosan grafting Dialdehydle the Starch was slightly enhanced in this study.
3.Immobilized xylanase by the Chitosan grafting Dialdehydle the starch carrier hydrolyzed the Birchwood xylan,the component of the hydrolyzed solution by the HPLC analysed indicated a good result.
引文
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