title: Cloning and characterization of a new β-mannosidase from Streptomyces sp. S27.
abstract: A new β-mannosidase gene, designated as man2S27, was cloned from Streptomyces sp. S27 using the colony PCR method and expressed in Escherichia coli BL21 (DE3). The full-length gene consists of 2499 bp and encodes 832 amino acids with a calculated molecular mass of 92.6 kDa. The amino acid sequence shares highest identity of 62.6% with the mannosidase Man2A from Cellulomonas fimi which belongs to the glycoside hydrolase family 2. Purified recombinant Man2S27 showed optimal activity at pH 7.0 and 50 °C. The specific activity, K(m), and k(cat) values for p-nitrophenyl-β-d-mannopyranoside (p-NP-β-MP) were 35.3 U mg(-1), 0.23 mM, and 305 s(-1), respectively. Low transglycosylation activity was observed when Man2S27 was incubated with p-NP-β-MP (glycosyl donor) and methyl-α-d-mannopyranoside (p-NP-α-MP) (acceptor) at 50 °C and pH 7.0, and a small amount of methylmannobioside was synthesized. Using locust bean gum as the substrate, more reducing sugars were liberated by the synergistic action of Man2S27 and β-mannanase (Man5S27), and the synergy degree in sequential reactions with Man5S27 firstly and Man2S27 secondly was higher than that in the simultaneous reactions.
authors: 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, PR China.
authors: Pengjun
authors: P
authors: Shi
authors: Guoyu
authors: G
authors: Yao
authors: Yanan
authors: Y
authors: Cao
authors: Peilong
authors: P
authors: Yang
authors: Tiezheng
authors: T
authors: Yuan
authors: Huoqing
authors: H
authors: Huang
authors: Yingguo
authors: Y
authors: Bai
authors: Bin
authors: B
authors: Yao
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004357:Drug Synergism; D004926:Escherichia coli; D005685:Galactans / Q000378:metabolism; D005798:Genes, Bacterial; D015698:Genomic Library; D006868:Hydrolysis; D007218:Industrial Microbiology; D008351:Mannans / Q000378:metabolism; D008969:Molecular Sequence Data; D053149:Plant Gums / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005685:Galactans; D008351:Mannans; D053149:Plant Gums; D011993:Recombinant Fusion Proteins; C014647:mannobiose; D044902:beta-Mannosidase; C447084:galactomannanase Man2, Thermotoga neapolitana; C017471:locust bean gum