title: A new acidophilic thermostable endo-1,4-β-mannanase from Penicillium oxalicum GZ-2: cloning, characterization and functional expression in Pichia pastoris.
abstract: BACKGROUND
  Endo-1,4-β-mannanase is an enzyme that can catalyze the random hydrolysis of β-1, 4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans and has a number of applications in different biotechnology industries. Penicillium oxalicum is a powerful hemicellulase-producing fungus (Bioresour Technol 123:117-124, 2012); however, few previous studies have focused on the cloning and expression of the endo-1,4-β-mannanase gene from Penicillium oxalicum.
  
  
  RESULTS
  A gene encoding an acidophilic thermostable endo-1,4-β-mannanase (E.C. 3.2.1.78) from Penicillium oxalicum GZ-2, which belongs to glycoside hydrolase family 5, was cloned and successfully expressed in Pichia pastoris GS115. A high enzyme activity (84.4 U mL(-1)) was detected in the culture supernatant. The recombinant endo-1,4-β-mannanase (rPoMan5A) was tagged with 6 × His at its C-terminus and purified using a Ni-NTA Sepharose column to apparent homogeneity. The purified rPoMan5A showed a single band on SDS-PAGE with a molecular mass of approximately 61.6 kDa. The specific activity of the purified rPoMan5A was 420.9 U mg(-1) using locust bean gum as substrate. The optimal catalytic temperature (10 min assay) and pH value for rPoMan5A are 80 °C and pH 4.0, respectively. The rPoMan5A is highly thermostable with a half-life of approximately 58 h at 60 °C at pH 4.0. The K m and V max values for locust bean gum, konjac mannan, and guar gum are 7.6 mg mL(-1) and 1425.5 μmol min(-1) mg(-1), 2.1 mg mL(-1) and 154.8 μmol min(-1) mg(-1), and 2.3 mg mL(-1) and 18.9 μmol min(-1) mg(-1), respectively. The enzymatic activity of rPoMan5A was not significantly affected by an array of metal ions, but was inhibited by Fe(3+) and Hg(2+). Analytical results of hydrolytic products showed that rPoMan5A could hydrolyze various types of mannan polymers and released various mannose and manno-oligosaccharides, with the main products being mannobiose, mannotriose, and mannopentaose.
  
  
  CONCLUSIONS
  Our study demonstrated that the high-efficient expression and secretion of acid stable and thermostable recombinant endo-1, 4-β-mannanase in Pichia pastoris is suitable for various biotechnology applications.
authors.affiliation: Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization, College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing, 210095, China. 2012203049@njau.edu.cn.
authors.forename: Hanpeng
authors.initials: H
authors.lastname: Liao
authors.affiliation: Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization, College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing, 210095, China. 995151498@qq.com.
authors.forename: Shuixian
authors.initials: S
authors.lastname: Li
authors.affiliation: Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization, College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing, 210095, China. 260241395@qq.com.
authors.forename: Haiping
authors.initials: H
authors.lastname: Zheng
authors.affiliation: Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization, College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing, 210095, China. weizhong@njau.edu.cn.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Wei
authors.affiliation: Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization, College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing, 210095, China. liudongyang@njau.edu.cn.
authors.forename: Dongyang
authors.initials: D
authors.lastname: Liu
authors.affiliation: Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization, College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing, 210095, China. waseem@njau.edu.cn.
authors.forename: Waseem
authors.initials: W
authors.lastname: Raza
authors.affiliation: Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization, College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing, 210095, China. lhp91@126.com.
authors.forename: Qirong
authors.initials: Q
authors.lastname: Shen
authors.affiliation: Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization, College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing, 210095, China. ycxu@njau.edu.cn.
authors.forename: Yangchun
authors.initials: Y
authors.lastname: Xu
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005685:Galactans / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008351:Mannans / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010407:Penicillium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D010802:Phylogeny; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D053149:Plant Gums / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D005685:Galactans; D008351:Mannans; D053149:Plant Gums; C007894:guar gum; D044902:beta-Mannosidase; C017471:locust bean gum