通过固定在活化和非活化琼脂糖凝胶上稳定来自炭黑曲霉的生淀粉消化淀粉酶。
Stabilization of a raw starch digesting amylase from Aspergillus carbonarius via immobilization on activated and non-activated agarose gel.
机构信息
Microbiology Department, Faculty of Biological Sciences, University of Nigeria Nsukka, Nsukka, Nigeria.
出版信息
World J Microbiol Biotechnol. 2012 Jan;28(1):335-45. doi: 10.1007/s11274-011-0824-1. Epub 2011 Jun 28.
Applications of raw starch digesting amylases (RSDAs) are limited due to instability, product inhibition of enzyme and contamination. RSDA from Aspergillus carbonarius was stabilized through immobilization on agarose gel by adsorption, spontaneous crosslinking and conjugation using glycidol, glutaraldehyde or polyglutaraldehyde. Effects of immobilization on kinetics, catalytic, storage and operational stability of immobilized enzyme were evaluated. Polyglutaraldehyde activated agarose RSDA (PGAg-RSDA) gave the highest immobilization yield (100%) with expressed activity of 86.7% while that of glycidol activated RSDA (GlyAg-RSDA) was 80.4%. A shift in pH from optimum of 5 for the soluble enzyme to 6 for RSDA adsorbed on agarose followed by crosslinking with glutaraldehyde (AgRSDA-CROSS) and simultaneous adsorption and crosslinking (AgRSDA-RET), and pH 7 for PGAg-RSDA was seen. PGAg-RSDA and AgRSDA-CROSS were most pH stable and retained over 82% of their activities between pH 3.5 and 9 compared to 59% for the soluble enzyme. Thermoinactivation studies showed that immobilized RSDAs with the exception of GAg-RSDA retained over 90% of their activities at 60°C for 120 min while soluble enzyme retained only 76% activity under the same condition. AgRSDA-CROSS, PGAg-RSDA, Gly-RSDA and GAg-RSDA retained approximately 100% of their activities after 30 days storage at 4°C. GlyAg-RSDA retained 99.6%, PGAg-RSDA 94%, AgRSDA-CROSS 90%, GAg-RSDA 86.5% and Ag-RSDA-RET 80% activity after 10 batch reactions. Immobilization stabilized RSDA and permits processing at higher temperatures to reduce contamination.
生淀粉糖化酶（RSDA）的应用由于其不稳定性、产物对酶的抑制和污染而受到限制。通过吸附、自发交联和使用缩水甘油、戊二醛或聚戊二醛进行接枝，将黑曲霉来源的 RSDA 固定在琼脂糖凝胶上。评价了固定化对固定化酶动力学、催化、储存和操作稳定性的影响。聚戊二醛活化琼脂糖 RSDA（PGAg-RSDA）的固定化产率最高（100%），表达活性为 86.7%，而缩水甘油活化 RSDA（GlyAg-RSDA）为 80.4%。与游离酶相比，吸附在琼脂糖上的 RSDA 的最适 pH 从 5 转移到 6，然后用戊二醛交联（AgRSDA-CROSS），同时吸附和交联（AgRSDA-RET），PGAg-RSDA 的最适 pH 为 7。PGAg-RSDA 和 AgRSDA-CROSS 的 pH 稳定性最高，在 pH 3.5 至 9 之间保留了超过 82%的活性，而游离酶的活性保留了 59%。热失活动力学研究表明，除了 GlyAg-RSDA 之外，固定化 RSDAs 在 60°C 下保持超过 90%的活性 120 分钟，而在相同条件下，游离酶仅保留 76%的活性。AgRSDA-CROSS、PGAg-RSDA、Gly-RSDA 和 GAg-RSDA 在 4°C 下储存 30 天后保留了大约 100%的活性。GlyAg-RSDA 保留了 99.6%、PGAg-RSDA 保留了 94%、AgRSDA-CROSS 保留了 90%、GAg-RSDA 保留了 86.5%、Ag-RSDA-RET 保留了 80%的活性，在 10 批反应后。固定化稳定了 RSDA，并允许在更高的温度下进行处理，以减少污染。
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