title: Identification of a novel alkaline amylopullulanase from a gut metagenome of Hermetia illucens.
abstract: A novel pullulanase gene, PulSS4, was identified from the gut microflora of Hermetia illucens by a function-based metagenome screening. The PulSS4 gene had an open reading frame of 4455 base pairs, and encoded a mature protein of 1484 amino acids, with a signal peptide sequence of 44 amino acids. The deduced amino acid sequence of PulSS4 gene showed 51% identity with that of the amylopullulanase of Amphibacillus xylanus, exhibiting no significant sequence homology to already known pullulanases. A conserved domain analysis revealed it to be a pullulanase type II with respective active sites at the N-terminal pullulanase and C-terminal amylase domain. PulSS4 was active in the temperature range of 10-50°C, with an optimum activity at 40°C. It was active in the pH range of 6.5-10.5, with optimum pH at 9.0, and retained more than 80% of its original activity in a broad pH range of 5-11 for 24h at 30°C. Also, PulSS4 was highly stable against many different chemical reagents, including 10% polar organic solvents and 1% non-ionic detergents. Overall, PulSS4 is expected to have the strong potential for application in biotechnological industries that require high activity at moderate temperature and alkaline conditions.
authors.affiliation: Metabolic Engineering Division, National Academy of Agricultural Science, Rural Development Administration, Jeonju 560-500, Republic of Korea.
authors.forename: Young-Seok
authors.initials: YS
authors.lastname: Lee
authors.affiliation: Metabolic Engineering Division, National Academy of Agricultural Science, Rural Development Administration, Jeonju 560-500, Republic of Korea.
authors.forename: So-Hyeon
authors.initials: SH
authors.lastname: Seo
authors.affiliation: Metabolic Engineering Division, National Academy of Agricultural Science, Rural Development Administration, Jeonju 560-500, Republic of Korea.
authors.forename: Sang-Hong
authors.initials: SH
authors.lastname: Yoon
authors.affiliation: Metabolic Engineering Division, National Academy of Agricultural Science, Rural Development Administration, Jeonju 560-500, Republic of Korea.
authors.forename: Su-Yeon
authors.initials: SY
authors.lastname: Kim
authors.affiliation: Metabolic Engineering Division, National Academy of Agricultural Science, Rural Development Administration, Jeonju 560-500, Republic of Korea.
authors.forename: Bum-Soo
authors.initials: BS
authors.lastname: Hahn
authors.affiliation: Metabolic Engineering Division, National Academy of Agricultural Science, Rural Development Administration, Jeonju 560-500, Republic of Korea.
authors.forename: Joon-Soo
authors.initials: JS
authors.lastname: Sim
authors.affiliation: Metabolic Engineering Division, National Academy of Agricultural Science, Rural Development Administration, Jeonju 560-500, Republic of Korea.
authors.forename: Bon-Sung
authors.initials: BS
authors.lastname: Koo
authors.affiliation: Metabolic Engineering Division, National Academy of Agricultural Science, Rural Development Administration, Jeonju 560-500, Republic of Korea. Electronic address: changmuk@rda.go.kr.
authors.forename: Chang-Muk
authors.initials: CM
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004175:Diptera / Q000382:microbiology*; D004789:Enzyme Activation; D000069196:Gastrointestinal Microbiome; D015698:Genomic Library; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D054892:Metagenome; D056186:Metagenomics; D008969:Molecular Sequence Data; D010802:Phylogeny; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins; D013213:Starch / Q000737:chemistry; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D013213:Starch; D006026:Glycoside Hydrolases; C082507:amylopullulanase
keywords: Characterization; Hermetia illucens; Metagenomic library; Pullulanase; Purification