N 端七氨基酸延伸同时提高了壳聚糖酶 CsnA 的 pH 稳定性、最适温度、热稳定性和催化效率。

N-Terminal seven-amino-acid extension simultaneously improves the pH stability, optimal temperature, thermostability and catalytic efficiency of chitosanase CsnA.

作者信息

Han Yujuan, Gao Peixin, Yu Wengong, Lu Xinzhi

机构信息

Key Laboratory of Glycoscience & Glycotechnology of Shandong Province; Key Laboratory of Marine Drugs, Chinese Ministry of Education; Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology; Department of Glycobiology; School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao, 266003, China.

出版信息

Biotechnol Lett. 2018 Jan;40(1):75-82. doi: 10.1007/s10529-017-2436-9. Epub 2017 Sep 13.

DOI:10.1007/s10529-017-2436-9

PMID:28905142

Abstract

OBJECTIVE

To determine the effects of the extra N-terminal seven-amino-acid sequence on the function of chitosanase CsnA.

RESULTS

Sequence and structure analysis indicated that the mature CsnA contains a seven-amino-acid extension in a disordered form at the N-terminus. To determine the function of this sequence, both mature CsnA and its N-terminus-truncated mutant, CsnAΔN, were expressed in Escherichia coli and characterized. Compared with CsnAΔN, CsnA exhibited a 15 °C higher temperature optimum, enhanced pH stability, thermostability and catalytic efficiency. The underlying mechanisms responsible for these changes were analyzed by circular dichroism (CD) spectroscopy. CD analysis revealed that the deletion of the N-terminal sequence resulted in a decrease in the T of 4.3 °C and this sequence altered the secondary structure of the enzyme.

CONCLUSIONS

The N-terminal sequence is essential for the stability and activity of chitosanase CsnA.

摘要

目的

确定额外的N端七氨基酸序列对壳聚糖酶CsnA功能的影响。

结果

序列和结构分析表明，成熟的CsnA在N端含有一个无序形式的七氨基酸延伸。为了确定该序列的功能，成熟的CsnA及其N端截短突变体CsnAΔN均在大肠杆菌中表达并进行了表征。与CsnAΔN相比，CsnA表现出高15°C的最适温度、增强的pH稳定性、热稳定性和催化效率。通过圆二色性（CD）光谱分析了导致这些变化的潜在机制。CD分析表明，N端序列的缺失导致熔解温度（Tm）降低4.3°C，并且该序列改变了酶的二级结构。