Development and characterization of an α-l-rhamnosidase mutant with improved thermostability and a higher efficiency for debittering orange juice

Li Jun Li; Zhe Yu Wu; Yue Yu; Lu Jia Zhang; Yan Bing Zhu; Hui Ni; Feng Chen

doi:10.1016/j.foodchem.2017.11.064

Development and characterization of an α-l-rhamnosidase mutant with improved thermostability and a higher efficiency for debittering orange juice

Food Chem. 2018 Apr 15:245:1070-1078. doi: 10.1016/j.foodchem.2017.11.064. Epub 2017 Nov 16.

Authors

Li Jun Li¹, Zhe Yu Wu², Yue Yu², Lu Jia Zhang³, Yan Bing Zhu¹, Hui Ni⁴, Feng Chen⁵

Affiliations

¹ College of Food and Biology Engineering, Jimei University, Xiamen, Fujian Province 361021, China; Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen, Fujian Province 361021, China; Research Center of Food Biotechnology of Xiamen City, Xiamen, Fujian Province 361021, China.
² College of Food and Biology Engineering, Jimei University, Xiamen, Fujian Province 361021, China.
³ College of Chemistry and Molecular Engineering, East China Normal University, Shanghai 201100, China.
⁴ College of Food and Biology Engineering, Jimei University, Xiamen, Fujian Province 361021, China; Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen, Fujian Province 361021, China; Research Center of Food Biotechnology of Xiamen City, Xiamen, Fujian Province 361021, China. Electronic address: nihui1973@yahoo.com.cn.
⁵ College of Food and Biology Engineering, Jimei University, Xiamen, Fujian Province 361021, China; Department of Food, Nutrition and Packaging Sciences, Clemson University, Clemson, SC 29634, USA.

PMID: 29287324
DOI: 10.1016/j.foodchem.2017.11.064

Abstract

The glycoside hydrolase, α-l-rhamnosidase, could remove the bitter taste of naringin from citrus juices. However, most α-l-rhamnosidases are easily deactivated at high temperatures, limiting the practice in debittering citrus juices. The V529A mutant of the α-l-rhamnosidase r-Rha1 from Aspergillus niger JMU-TS528 was developed with improved thermostability using directed evolution technology and site-directed mutagenesis. The enzyme mutant had a half-live of thermal inactivation T_(1/2) of 1.92 h, 25.00 min, and 2 min at 60, 65, and 70 °C, respectively. In addition, it had improved substrate affinity and better resistance to the inhibition of glucose. The improved substrate affinity was related to its lowered binding energy. Most significantly, the naringin content was reduced to below the bitter taste threshold by treatment with 75 U/mL of the mutant during the preheating process of orange juice production. The comprehensive results indicate that thermostability improvement could promote the practical value of α-l-rhamnosidase in citrus juice processing.

Keywords: 4-Nitrophenyl-alpha-l-rhamnoside (PubChem CID: 3082141); Alanine (PubChem CID: 5950); Characterization; Error-prone PCR; Hesperidin (PubChem CID: 10621); Juice debittering; Naringin (PubChem CID: 442428); Prunin (PubChem CID: 92794); Rutin (PubChem CID: 5280805); Thermostability; Valine ((PubChem CID: 6287); l-Rhamnose (PubChem CID: 25310); α-l-Rhamnosidase.

MeSH terms

Aspergillus niger / enzymology
Citrus sinensis / chemistry*
Directed Molecular Evolution
Fruit and Vegetable Juices / analysis*
Glycoside Hydrolases / chemistry*
Glycoside Hydrolases / genetics*
Glycoside Hydrolases / metabolism
Mutagenesis, Site-Directed
Protein Engineering*
Taste*
Temperature

Substances

Glycoside Hydrolases
alpha-L-rhamnosidase