Archives of microbiology: screening of pectinase-producing bacteria from citrus peel and characterization of a recombinant pectate lyase with applied potential

• Published in: Archives of microbiology Vol. 202; no. 5; pp. 1005 - 1013
• Main Authors: Guan, Yi, Wang, Donghuang, Lv, Chao, Zhang, Yuewen, Gelbic, Ivan, Ye, Xiuyun
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-07-2020; Springer Nature B.V
• Subjects: Affinity chromatography; Archives & records; Assaying; Bacillus; Bacteria; Biochemistry; Biomedical and Life Sciences; Biotechnology; Calcium; Calcium ions; Cell Biology; Citrus fruits; E coli; Ecology; Enzymatic activity; Enzyme activity; Enzyme kinetics; Escherichia coli; Food industry; Fruits; Industrial applications; Life Sciences; Microbial Ecology; Microbiology; Original Paper; Pectate lyase; Pectin; Pectinase; pH effects; Pulp & paper industry; Substrates; Thermal stability; Three dimensional models; Wine; Pectate lyase; Enzyme properties; sp; PelB; Heterologous expression; Bacillus sp
• ISSN: 0302-8933; 1432-072X
• DOI: 10.1007/s00203-020-01807-0

Pectinase is widely used in numerous industrial fields, including the food, wine, and paper industries. In this work, seven bacteria were isolated from orange peel and their pectinase production activity was assayed. One bacterium (OR-B2) identified as a
Bacillus
sp. showed the highest enzyme activity towards others. A gene encoding a pectate lyase designed as PelB-B2 in this work was amplified and heterogeneous expressed in
E.
coli
. PelB-B2 was defined as a member of the PelB pectate lyase family after phylogenic tree analysis. 3D model of PelB-B2 was constructed by SWISS-MODEL and PelB-B2 showed conserved para
-β
structure. After inducing culture and purified by Ni-affinity chromatography, the properties of the purified PelB-B2 were assayed. Optimal pH and temperature for PelB-B2 was pH 8.0 and 50 °C, respectively. PelB-B2 showed excellent pH stability and thermostability. It was stable within pH range 3.0–11.0 and retained more than 51% activity after incubation at 40 °C, 50 °C, or 60 °C for 1 h. Furthermore, we determined that PelB-B2 was a Ca
2+
-dependent pectinase and the pectin extracted from citrus was the benefit substrate for PelB-B2. The
K
m
and
V
max
of PelB-B2 were 1.64 g/L and 232.56 mol/(L min), respectively. The OR-B2 can be a new resource for pectinase production and the PelB-B2 has potential for industrial application.
Graphic abstract
7 bacteria were isolated from orange peel, namely OR-B1 to OR-B7 and their pectinase activities were assayed. One pectate lyase belongs to PelB family was cloned from OR-B2 and heterogeneous expressed in E. coli. Purified PelB-B2 was further studied with its properties. Effects of pH, temperature, chemicals, substrate on the enzyme activity were assayed and the enzyme kinetic was also measured.