Genetic and biochemical characterization of a protease-resistant mesophilic β-mannanase from Streptomyces sp. S27.
A β-mannanase gene, designated as man5S27, was cloned from Streptomyces sp. S27 using the colony polymerase chain reaction (PCR) method and expressed in Escherichia coli BL21 (DE3). The open reading frame consisted of 1,161 bp and encoded a 386-amino-acid polypeptide (Man5S27) with calculated molecular mass of 37.2 kDa. The encoded protein comprised a putative 38-residue signal peptide, a family 5 glycoside hydrolase domain, and a family 10 carbohydrate-binding module. Purified recombinant Man5S27 had high specific activity of 2,107 U mg⁻¹ and showed optimal activity at pH 7.0 and 65 °C. The enzyme remained stable at pH 5.0-9.0 and had good thermostability at 50°C. The K (m) values for locust bean gum and konjac flour were 0.16 and 0.41 mg ml⁻¹, with V(max) values of 3,739 and 1,653 μmol min⁻¹ mg⁻¹, respectively. Divalent metal ions such as Mn²+, Zn²+, Ca²+, Pb²+, and Fe²+ enhanced the enzyme activity, but Ag+ and Hg²+ strongly inhibited the activity. Man5S27 also showed resistance to various neutral proteases (retaining >95% activity after proteolytic treatment for 2 h).
Shi
Pengjun
P
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.
Yuan
Tiezheng
T
Zhao
Junqi
J
Huang
Huoqing
H
Luo
Huiying
H
Meng
Kun
K
Wang
Yaru
Y
Yao
Bin
B
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D005798:Genes, Bacterial; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D044902:beta-Mannosidase / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D044902:beta-Mannosidase; D010450:Endopeptidases