title: High-level expression of two thermophilic β-mannanases in Yarrowialipolytica.
abstract: Two thermophilic β-mannanases (ManA and ManB)were successfully expressed in Yarrowialipolytica using vector pINA1296I. The sequences of manA from Aspergillus niger CBS 513.88 and manB from Bacillus subtilis BCC41051 were optimized based on codon-usage bias in Y.lipolytica and synthesized by overlapping polymerase chain reaction (PCR). We utilized the pINA1296I vector, which allows inserting and expression of multiple copies of an expression cassette, to engineer recombinant strains containing multiple copies of manA or manB. Following verification of target-gene expression by quantitative PCR, fermentation experiments indicated that recombinant protein levels and enzyme activity increased along with increasing manA/manB copy number.After production in a 10 l fermenter, we obtained maximum enzyme activity from strains YLA6 and YLB6 of3024 U/mL and 1024 U/mL, respectively. Additionally, purification and characterization results revealed that the optimum pH and temperature for manA activity were pH∼5 and ∼70 °C, and for manB activity were pH∼7 and 60 °C, respectively. These results indicated that the thermo stabilities of these two enzymes were higher than most other mannanases, making them potentially useful for industrial applications.
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, Hubei Province 430062, People's Republic of China.
authors.forename: Wang
authors.initials: W
authors.lastname: YaPing
authors.affiliation: National Biopesticide Engineering Research Center, Hubei Academy of Agricultural, Wuhan, Hubei Province 430064, People's Republic of China.
authors.forename: Rao
authors.initials: R
authors.lastname: Ben
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, Hubei Province 430062, People's Republic of China.
authors.forename: Zhang
authors.initials: Z
authors.lastname: Ling
authors.affiliation: Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei University, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, Hubei Province 430062, People's Republic of China. Electronic address: malixing98@163.com.
authors.forename: Ma
authors.initials: M
authors.lastname: Lixin
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics*; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D025062:Yarrowia / Q000235:genetics / Q000378:metabolism*; D044902:beta-Mannosidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D044902:beta-Mannosidase
keywords: High-density fermentation; Mannanse; Multi-copy expression cassette; Thermophilic; Yarrowialipolytica