title: A novel low-temperature-active β-glucosidase from symbiotic Serratia sp. TN49 reveals four essential positions for substrate accommodation.
abstract: A 2,373-bp full-length gene (bglA49) encoding a 790-residue polypeptide (BglA49) with a calculated mass of 87.8 kDa was cloned from Serratia sp. TN49, a symbiotic bacterium isolated from the gut of longhorned beetle (Batocera horsfieldi) larvae. The deduced amino acid sequence of BglA49 showed the highest identities of 80.1% with a conceptually translated protein from Pantoea sp. At-9b (EEW02556), 38.3% with the identified glycoside hydrolase (GH) family 3 β-glucosidase from Clostridium stercorarium NCBI 11754 (CAB08072), and <15.0% with the low-temperature-active GH 3 β-glucosidases from Shewanella sp. G5 (ABL09836) and Paenibacillus sp. C7 (AAX35883). The recombinant enzyme (r-BglA49) was expressed in Escherichia coli and displayed the typical characteristics of low-temperature-active enzymes, such as low temperature optimum (showing apparent optimal activity at 35°C), activity at low temperatures (retaining approximately 60% of its maximum activity at 20°C and approximately 25% at 10°C). Compared with the thermophilic GH 3 β-glucosidase, r-BglA49 had fewer hydrogen bonds and salt bridges and less proline residues. These features might relate to the increased structure flexibility and higher catalytic activity at low temperatures of r-BglA49. The molecular docking study of four GH 3 β-glucosidases revealed five conserved positions contributing to substrate accommodation, among which four positions of r-BglA49 (R192, Y228, D260, and E449) were identified to be essential based on site-directed mutagenesis analysis.
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: Junpei
authors.initials: J
authors.lastname: Zhou
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
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: Tiezheng
authors.initials: T
authors.lastname: Yuan
authors.forename: Peilong
authors.initials: P
authors.lastname: Yang
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003080:Cold Temperature; D001517:Coleoptera / Q000382:microbiology* / Q000502:physiology; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D012705:Serratia / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D013379:Substrate Specificity; D013559:Symbiosis; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001617:beta-Glucosidase