title: Biochemical characterization of a thermophilic β-mannanase from Talaromyces leycettanus JCM12802 with high specific activity.
abstract: Thermophilic β-mannanases are of increasing importance for wide industrial applications. In the current study, gene cloning, functional expression in Pichia pastoris, and characterization of a thermophilic β-mannanase (Man5A) from thermophilic Talaromyces leycettanus JCM12802 are reported. Deduced Man5A exhibits the highest identity with a putative β-mannanase from Talaromyces stipitatus ATCC10500 (70.3 %) and is composed of an N-terminal signal peptide, a fungal-type carbohydrate-binding module (CBM) of family 1, and a catalytic domain of glycosyl hydrolase (GH) family 5 at the C-terminus. Two recombinant proteins with different glycosylation levels, termed Man5A1 (72 kDa) and Man5A2 (60 kDa), were identified after purification. Both enzymes were thermophilic, exhibiting optimal activity at 85-90 °C, and were highly stable at 70 °C. Man5A1 and Man5A2 had a pH optimum of 4.5 and 4.0, respectively, and were highly stable over the broad pH range of 3.0-10.0. Most metal ions and sodium dodecyl sulfate (SDS) had no effect on the enzymatic activities. Man5A1 and Man5A2 exhibited high specific activity (2,160 and 1,800 U/mg, respectively) when using locust bean gum as the substrate. The CBM1 and two key residues D191 and R286 were found to affect Man5A thermostability. Man5A displays a classical four-site-binding mode, hydrolyzing mannooligosaccharides into smaller units, galactomannan into mannose and mannobiose, and glucomanman into mannose, mannobiose, and mannopentaose, respectively. All these properties make Man5A a good candidate for extensive applications in the bioconversion, pulp bleaching, textile, food, and feed industries.
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081, People's Republic of China.
authors.forename: Caihong
authors.initials: C
authors.lastname: Wang
authors.forename: Huiying
authors.initials: H
authors.lastname: Luo
authors.forename: Canfang
authors.initials: C
authors.lastname: Niu
authors.forename: Pengjun
authors.initials: P
authors.lastname: Shi
authors.forename: Huoqing
authors.initials: H
authors.lastname: Huang
authors.forename: Kun
authors.initials: K
authors.lastname: Meng
authors.forename: Yingguo
authors.initials: Y
authors.lastname: Bai
authors.forename: Kun
authors.initials: K
authors.lastname: Wang
authors.forename: Huifang
authors.initials: H
authors.lastname: Hua
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D032901:Talaromyces / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D044902:beta-Mannosidase