A novel highly acidic beta-mannanase from the acidophilic fungus Bispora sp. MEY-1: gene cloning and overexpression in Pichia pastoris.
Using degenerate polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR, a 1,347-bp full-length complementary DNA fragment encompassing the gene man5A, which encodes a 429-amino acid beta-mannanase with a calculated mass of 46.8 kDa, was cloned from acidophilic Bispora sp. MEY-1. The deduced amino acid sequence (catalytic domain) displayed highest identity (54.1%) with the Emericella nidulans endo-beta-1,4-D-mannanase, a member of the glycoside hydrolase family 5. Recombinant MAN5A was overexpressed in Pichia pastoris, and its activity in the culture medium reached 500 U ml(-1). The enzyme was acidophilic, with highest activity at pH 1.0-1.5, lower than any known mannanases, and optimal temperature for activity was 65 degrees C. MAN5A had good pH adaptability, excellent thermal and pH stability, and high resistance to both pepsin and trypsin. The specific activity, K(m), and V(max) for locust bean gum substrate was 3,373 U mg(-1), 1.56 mg ml(-1), and 6,587.6 micromol min(-1) mg(-1), respectively. The enzymatic activity was not significantly affected by ions such as Ca(2+), Cr(3+), Co(2+), Zn(2+), Na(+), K(+), and Mg(2+) and enhanced by Ni(2+), Fe(3+), Mn(2+) and Ag(+). These favorable properties make MAN5A a potential candidate for use in various industrial applications.
Luo
Huiying
H
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.
Wang
Yaru
Y
Wang
Hui
H
Yang
Jun
J
Yang
Yuhui
Y
Huang
Huoqing
H
Yang
Peilong
P
Bai
Yingguo
Y
Shi
Pengjun
P
Fan
Yunliu
Y
Yao
Bin
B
D000143:Acids / Q000378:metabolism*; D001203:Ascomycota / Q000201:enzymology* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D013696:Temperature; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000143:Acids; D005656:Fungal Proteins; D044902:beta-Mannosidase