title: Cloning, over-expression and characterization of an alkali-tolerant endo-β-1,4-mannanase from Penicillium freii F63.
abstract: A glycosyl hydrolase family 5 endo-β-mannanase gene (man5F63) was cloned from Penicillium freii F63 and overexpressed in Pichia pastoris. man5F63 contained an open reading frame of 1260 bp that encoded a polypeptide of 419 amino acids including a putative 18-residue signal peptide. The recombinant enzyme (rMan5F63) was secreted into the culture supernatant to near electrophoretic homogeneity with a high yield (1.1 gl(-1) in flask). Its apparent molecular weight was approximately 72.0 kDa, 29.0 kDa higher than the theoretical molecular mass. rMan5F63 was optimal at pH 4.5 and 60 °C and exhibited good stability over a broad pH range from acidic to alkaline (>85.0% activity at pH 4.0-9.0, >70.0% activity at pH 10.0 and 43.7% even at pH 12.0). The activity of rMan5F63 was significantly enhanced in the presence of Co(2+), Cu(2+), Mn(2+) and β-mercaptoethanol and was strongly inhibited by Hg(2+) and SDS. The specific activity, K(m) and V(max) values were 47.5 U mg(-1), 7.8 mg ml(-1) and 70.4 μmol min(-1)mg(-1), respectively, for locust bean gum, and 40.3 U mg(-1), 2.3 mg ml(-1) and 61.7 μmol min(-1)mg(-1), respectively, for konjac flour. All these favorable enzymatic properties make it cost-effective to commercialization and valuable in various industries.
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China.
authors.forename: Yawei
authors.initials: Y
authors.lastname: Wang
authors.forename: Pengjun
authors.initials: P
authors.lastname: Shi
authors.forename: Huiying
authors.initials: H
authors.lastname: Luo
authors.forename: Yingguo
authors.initials: Y
authors.lastname: Bai
authors.forename: Huoqing
authors.initials: H
authors.lastname: Huang
authors.forename: Peilong
authors.initials: P
authors.lastname: Yang
authors.forename: Hairong
authors.initials: H
authors.lastname: Xiong
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D000222:Adaptation, Physiological; D000468:Alkalies; D000595:Amino Acid Sequence; D001483:Base Sequence; D057005:Bioengineering; D019976:Cloning, Organism; D008361:Mannosidases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010407:Penicillium / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism
chemical_list: D000468:Alkalies; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase