title: Recombinant production and characterization of full-length and truncated β-1,3-glucanase PglA from Paenibacillus sp. S09.
abstract: BACKGROUND
  β-1,3-Glucanases catalyze the hydrolysis of glucan polymers containing β-1,3-linkages. These enzymes are of great biotechnological, agricultural and industrial interest. The applications of β-1,3-glucanases is well established in fungal disease biocontrol, yeast extract production and wine extract clarification. Thus, the identification and characterization of novel β-1,3-glucanases with high catalytic efficiency and stability is of particular interest.
  
  
  RESULTS
  A β-1,3-glucanase gene designated PglA was cloned from a newly isolated strain Paenibacillus sp. S09. The gene PglA contained a 2631-bp open reading frame encoding a polypeptide of 876 amino acids which shows 76% identity with the β-1,3-glucanase (BglH) from Bacillus circulans IAM1165. The encoded protein PglA is composed of a signal peptide, an N-terminal leader region, a glycoside hydrolase family 16 (GH16) catalytic domain and a C-terminal immunoglobulin like (Ig-like) domain. The Escherichia coli expression system of PglA and five truncated derivatives containing one or two modules was constructed to investigate the role of catalytic and non-catalytic modules. The pH for optimal activity of the enzymes was slightly affected (pH 5.5-6.5) by the presence of different modules. However, the temperature for optimal activity was strongly influenced by the C-terminal domain and ranged from 50 to 60°C. Deletion of C-terminal domain resulted in obviously enhancing enzymatic thermostability. Specific activity assay indicated that PglA specifically hydrolyzes β-1,3-glucan. Insoluble β-1,3-glucan binding and hydrolysis were boosted by the presence of N-and C-terminal domains. Kinetic analysis showed that the presence of N-and C-terminus enhances the substrate affinity and catalytic efficiency of the catalytic domain toward laminarin. Carbohydrate-binding assay directly confirmed the binding capabilities of the N-and C-terminal domains.
  
  
  CONCLUSIONS
  This study provides new insight into the impacts of non-catalytic modules on enzymatic properties of β-1,3-glucanase. Activity comparison of full-length PglA and truncated forms revealed the negative effect of C-terminal region on thermal stability of the enzyme. Both the N-and C-terminal domains exerted strong binding activity toward insoluble β-1,3-glucan, and could be classified into CBM families.
authors.affiliation: Center for Molecular Metabolism, Nanjing University of Science & Technology, 200 Xiaolingwei, Nanjing 210094, China. jfzhang@mail.njust.edu.cn.
authors.forename: Rui
authors.initials: R
authors.lastname: Cheng
authors.forename: Jinping
authors.initials: J
authors.lastname: Chen
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Yu
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.forename: Shiming
authors.initials: S
authors.lastname: Wang
authors.forename: Jianfa
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D043326:Glucan 1,3-beta-Glucosidase / Q000096:biosynthesis* / Q000235:genetics; D005936:Glucans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D056507:Paenibacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D011134:Polysaccharides / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature; D047071:beta-Glucans / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005936:Glucans; D011134:Polysaccharides; D011994:Recombinant Proteins; D047071:beta-Glucans; C008247:laminaran; C033363:beta-1,3-glucan; D043326:Glucan 1,3-beta-Glucosidase