辣根过氧化物酶的稳定性及结构变化：微波加热与传统加热处理的比较

Stability and structural changes of horseradish peroxidase: microwave versus conventional heating treatment.

作者信息

Lopes Lucas C, Barreto Maria T M, Gonçalves Karen M, Alvarez Heiddy M, Heredia Montserrat Fortuny, de Souza Rodrigo Octavio M A, Cordeiro Yraima, Dariva Cláudio, Fricks Alini T

机构信息

Programa de Pós-Graduação em Biotecnologia Industrial, Universidade Tiradentes, Instituto de Tecnologia e Pesquisa. Laboratório de Engenharia de Bioprocessos, Av. Murilo Dantas, 300, Farolândia, 49032-490 Aracaju, SE, Brazil.

Departamento de Biotecnologia Farmacêutica, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-902, Brazil.

出版信息

Enzyme Microb Technol. 2015 Feb;69:10-8. doi: 10.1016/j.enzmictec.2014.11.002. Epub 2014 Nov 24.

DOI:10.1016/j.enzmictec.2014.11.002

PMID:25640719

Abstract

Effects of conventional heating (CH) and microwave (MW) on the structure and activity of horseradish peroxidase (HRP) in buffer solution were studied. CH incubation between 30 and 45 °C increased activity of HRP, reaching 170% of residual activity (RA) after 4-6 h at 45 °C. CH treatment at 50 and 60 °C caused HRP inactivation: RA was 5.7 and 16.7% after 12 h, respectively. Secondary and tertiary HRP structural changes were analyzed by circular dichroism (CD) and intrinsic fluorescence emission, respectively. Under CH, activation of the enzyme was attributed to conformational changes in secondary and tertiary structures. MW treatment had significant effects on the residual activity of HRP. MW treatment at 45 °C/30W followed by CH treatment 45 °C regenerated the enzyme activity. The greatest loss in activity occurred at 60 °C/60 W/30 min (RA 16.9%); without recovery of the original activity. The inactivation of MW-treated HRP was related to the loss of tertiary structure, indicating changes around the tryptophan environment.

摘要

研究了传统加热（CH）和微波（MW）对缓冲溶液中辣根过氧化物酶（HRP）结构和活性的影响。在30至45°C之间进行CH孵育可提高HRP的活性，在45°C下孵育4至6小时后，残余活性（RA）达到170%。在50和60°C下进行CH处理会导致HRP失活：12小时后的RA分别为5.7%和16.7%。分别通过圆二色性（CD）和内源荧光发射分析了HRP二级和三级结构的变化。在CH条件下，酶的激活归因于二级和三级结构的构象变化。MW处理对HRP的残余活性有显著影响。在45°C/30W下进行MW处理，然后在45°C下进行CH处理可使酶活性恢复。在60°C/60W/30分钟时活性损失最大（RA为16.9%）；无法恢复到原始活性。MW处理的HRP失活与三级结构的丧失有关，表明色氨酸环境周围发生了变化。