title: Cloning, evaluation, and high-level expression of a thermo-alkaline pectate lyase from alkaliphilic Bacillus clausii with potential in ramie degumming.
abstract: Alkaline pectate lyases (Pels) have potential application in bioscouring of the textile industry. In this study, a thermo-alkaline Pel (BacPelA) gene from an alkaliphilic Bacillus clausii strain was cloned and overexpressed in Escherichia coli. The mature BacPelA exhibited maximum activity at pH 10.5 and 70 °C and showed high cleavage capability on methylated pectins. BacPelA showed the highest specific activity of 936.2 U mg-1on ≥85% methylated pectin and 675.5 U mg-1on standard substrate polygalacturonic acid (PGA) upon evaluation of the absorbance at 235 nm (A235). The Kmand kcatvalues for PGA were 0.54 g l-1and 346.5 s-1, respectively. Moreover, the 3,5-dinitrosalicylic acid (DNS) assay, which detects the released reducing oligogalacturonic acids, was confirmed to be inaccurate and unsuitable for endo-acting pectinase activity assay because of the difference in the reducibility by DNS reagent between the standard galacturonic acid and the catalytic oligomer products. Significant ramie fiber weight loss was observed following treatment with BacPelA (24.8%) and combined enzyme-chemical method (30.9%), which indicated that the degumming efficiency of BacPelA was the highest of all alkaline and thermostable Pels reported to date. The total activity of the recombinant mature BacPelA reached 8378.2 U ml-1(A235) by high-cell-density cultivation in fed-batch fermentation with productivity of 239.4 U ml-1h-1using E. coli as host, which represents the highest Pel yield reported to date. Therefore, BacPelA, with promising properties for bioscouring, shows potential applications for ramie degumming in the textile industry.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, People's Republic of China.|National Engineering Laboratory for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, People's Republic of China.|National Engineering Laboratory for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Yanfen
authors.initials: Y
authors.lastname: Xue
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, People's Republic of China. mayanhe@im.ac.cn.|National Engineering Laboratory for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. mayanhe@im.ac.cn.
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
mesh_terms: D000070019:Bacillus clausii / Q000201:enzymology*; D001711:Biotransformation; D032402:Boehmeria / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010368:Pectins / Q000378:metabolism*; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D010368:Pectins; D011133:Polysaccharide-Lyases; C023911:pectate lyase; C003181:polygalacturonic acid
keywords: Alkaline pectate lyase; Bacillus clausii; Characterization; Overproduction; Ramie degumming