title: Gene cloning, expression, and biochemical characterization of an alkali-tolerant β-mannanase from Humicola                      insolens Y1.
abstract: In this article, we firstly report a highly alkali-tolerant fungal β-mannanase from Humicola insolens Y1. The full-length cDNA of the β-mannanase, designated as man5A, has an open reading frame of 1,233 bp that encodes a 411-amino acid polypeptide (Man5A) with a calculated molecular mass of 42.3 kDa. The deduced sequence of Man5A comprises a putative 20-residue signal peptide and a catalytic domain belonging to glycoside hydrolase family 5, and displays 61-85% identities with hypothetical proteins and 32-39% with experimentally verified fungal β-mannanases. Purified recombinant Man5A produced by Pichia pastoris has a specific activity of 1,122 U mg⁻¹ and exhibits optimal activity at pH 5.5 and 70°C. Distinct from other reported fungal β-mannanases, Man5A is highly alkali tolerant, exhibiting 45 and 36% of the maximal activity at pH 8.0 and 9.0, respectively, and more than 10% activity even at pH 10.0. Moreover, Man5A has excellent pH stability at pH 5.0-12.0 and is highly thermostable at 50°C. The higher frequency of alkaline amino acids (Arg and Lys), greater pKa values of the catalytic residues, and more positively charged residues on the surface of Man5A might be the causes. Man5A has strong resistance to various neutral and alkaline proteases, retaining more than 97% of the activity after proteolytic treatment for 1 h. The superior characteristics of Man5A make it more advantageous for the application in the kraft pulp industry.
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: Huiying
authors.initials: H
authors.lastname: Luo
authors.forename: Kun
authors.initials: K
authors.lastname: Wang
authors.forename: Huoqing
authors.initials: H
authors.lastname: Huang
authors.forename: Pengjun
authors.initials: P
authors.lastname: Shi
authors.forename: Peilong
authors.initials: P
authors.lastname: Yang
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D044902:beta-Mannosidase