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通过改良托伦斯反应比色法测定淀粉脱支酶的活性

Colorimetric Determination of the Activity of Starch-Debranching Enzyme via Modified Tollens' Reaction.

作者信息

Luo Ke, Kim Nack-Geun, You Sang-Mook, Kim Young-Rok

机构信息

Graduate School of Biotechnology & Department of Food Science and Biotechnology, College of Life Sciences, Kyung Hee University, Yongin 17104, Korea.

出版信息

Nanomaterials (Basel). 2019 Sep 10;9(9):1291. doi: 10.3390/nano9091291.

DOI:10.3390/nano9091291
PMID:31509936
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6781065/
Abstract

Nelson-Somogyi and 3,5-dinitrosalicylic acid (DNS) assays are the classical analytical methods for the determination of activity of starch-debranching enzymes, however, they have a narrow detection range and do not adapt to the quantitative measurement of linear polysaccharides. Herein, we developed a simple and accurate colorimetric assay for determining the activity of starch-debranching pullulanase through the modified Tollens' reaction in combination with UV irradiation. Silver nanoparticles (AgNPs) were formed by reducing aldehyde groups in short-chain glucans (SCGs) generated by debranching of waxy maize starch using pullulanase through the modified Tollens' reaction. In addition to providing a reducing moiety to the Tollens' reaction, the debranching product, SCGs, also enhanced the colloidal stability of synthesized AgNPs, of which the amplitude of its surface plasmon resonance (SPR) absorbance peak was proportional to the concentration of SCGs ranging from 0.01-10 mg/mL. The detection limit of this system was 0.01 mg/mL, which was found to be 100 times higher than that of the conventional DNS assay. The purification of SCGs by recrystallization and gelatinization improved the selectivity of this colorimetric assay for debranching products, which provides a simple and accurate means of monitoring the debranching process and characterizing the activity of starch-debranching enzymes.

摘要

尼尔森-索莫吉法和3,5-二硝基水杨酸（DNS）法是测定淀粉脱支酶活性的经典分析方法，然而，它们的检测范围较窄，不适用于线性多糖的定量测定。在此，我们开发了一种简单准确的比色法，通过改良的托伦斯反应结合紫外线照射来测定淀粉脱支酶普鲁兰酶的活性。使用普鲁兰酶对糯玉米淀粉进行脱支反应生成的短链葡聚糖（SCG）中的醛基，通过改良的托伦斯反应还原形成银纳米颗粒（AgNP）。除了为托伦斯反应提供还原部分外，脱支产物SCG还增强了合成的AgNP的胶体稳定性，其表面等离子体共振（SPR）吸光度峰值的幅度与0.01-10 mg/mL范围内的SCG浓度成正比。该系统的检测限为0.01 mg/mL，发现比传统DNS法高100倍。通过重结晶和糊化对SCG进行纯化，提高了该比色法对脱支产物的选择性，为监测脱支过程和表征淀粉脱支酶的活性提供了一种简单准确的方法。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d963/6781065/eaaec2cd59cd/nanomaterials-09-01291-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d963/6781065/de1b33b87359/nanomaterials-09-01291-sch001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d963/6781065/1cf9c925de47/nanomaterials-09-01291-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d963/6781065/77f4b4e94296/nanomaterials-09-01291-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d963/6781065/0a5fafcf7cdf/nanomaterials-09-01291-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d963/6781065/2d41afd79b3a/nanomaterials-09-01291-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d963/6781065/a5e9ca028b2e/nanomaterials-09-01291-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d963/6781065/eaaec2cd59cd/nanomaterials-09-01291-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d963/6781065/de1b33b87359/nanomaterials-09-01291-sch001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d963/6781065/1cf9c925de47/nanomaterials-09-01291-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d963/6781065/77f4b4e94296/nanomaterials-09-01291-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d963/6781065/0a5fafcf7cdf/nanomaterials-09-01291-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d963/6781065/2d41afd79b3a/nanomaterials-09-01291-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d963/6781065/a5e9ca028b2e/nanomaterials-09-01291-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d963/6781065/eaaec2cd59cd/nanomaterials-09-01291-g006.jpg

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本文引用的文献

1
Synthesis of monodisperse starch microparticles through molecular rearrangement of short-chain glucans from natural waxy maize starch.通过天然糯玉米淀粉短链葡聚糖的分子重排合成单分散淀粉微粒。
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Effect of Lecithin on the Spontaneous Crystallization of Enzymatically Synthesized Short-Chain Amylose Molecules into Spherical Microparticles.卵磷脂对酶促合成的短链直链淀粉分子自发结晶形成球形微粒的影响。
Polymers (Basel). 2019 Feb 4;11(2):264. doi: 10.3390/polym11020264.
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Self-assembly kinetics of debranched short-chain glucans from waxy maize starch to form spherical microparticles and its applications.
支链短链葡聚糖从蜡质玉米淀粉的自组装动力学形成球形微粒及其应用。
Colloids Surf B Biointerfaces. 2019 Apr 1;176:352-359. doi: 10.1016/j.colsurfb.2019.01.002. Epub 2019 Jan 8.
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