Highly thermostable and surfactant-activated chitinase from a subseafloor bacterium, Laceyella putida

Abstract

A novel chitinase (LpChiA) was purified to homogeneity from a culture of Laceyella putida JAM FM3001. LpChiA hydrolyzed colloidal chitin optimally at a pH of 4 in an acetate buffer and temperature of 75 ºC. The enzyme was remarkably stable to incubation at 70 ºC up to 1 h at pH 5.2, and its activity half-life was 3 days. The molecular mass of the enzyme was around 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and around 75 kDa by gel filtration, suggesting it is a homodimer. The enzyme activity was enhanced about 60% when pre-incubated with anionic, cationic, and nonionic surfactants. The gene for LpChiA was cloned by PCR and sequenced. The nucleotide sequence of the gene consisted of 1,683 bp encoding 560 amino acids. The N-terminal and internal amino acid sequences of the purified LpChiA from L. putida suggested that the mature enzyme was composed of 384 amino acids after cleaving its 176 N-terminal amino acids and dimerized to express its activity. The deduced amino acid sequence of the mature enzyme showed the highest similarity to chitinase of Laceyella sacchari with 79% identity.

Biochemical and genetic characterization of β-1,3 glucanase from a deep subseafloor Laceyella putida

“…putida JAM FM3001 is one of thermophiles isolated from deep subseafloor (Kobayashi et al 2008). As expected, LpGluA showed a high optimal temperature of 80°C like as LpChiA (75°C) (Shibasaki et al 2014). The optimal temperature of LpGluA is the highest value among reported β-1,3-glucanases from eubacteria and plants.…”

“…The primer sets used for amplification of the gene encoding LpGluA including two upstream regions are shown by converging arrows with a dotted line. The Nterminal and internal amino acid sequences are indicated by double underlines beneath the deduced amino acid sequence Induction of LpGluA and recombinant enzyme L. putida JAM FM3001 produces chinase (LpChiA)(Shibasaki et al 2014) and LpGluA. GluNAc was the best inducer of LpChiA production, and LpGluA was also induced by it, as well as by laminarin.…”

“…We have isolated many aerobic and heterotrophic bacteria from deep subseafloor core sediments collected by the deep-sea drilling vessel BChikyu^off Shimokita Peninsula of Japan at a water depth of 1180 m (Kobayashi et al 2008). We investigated several enzymes of the isolates, namely an endo-1,5-α-Larabinanase from Bacillus subtilis JAM A-3-6 (Fukada et al 2011), a chitosanase from Bacillus thuringiensis JAM GG01 ), a chitinase from Laceyella putida JAM FM3001 (Shibasaki et al 2014), and a pectate lyase from Georgenia muralis from deep subseafloor sediments of Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6983-5) contains supplementary material, which is available to authorized users. Suruga Bay of Japan at a water depth of 756 m (Sasaki et al 2015).…”

Cloning, expression, purification and application of a novel chitinase from a thermophilic marine bacterium Paenibacillus barengoltzii

“…To date, a number of chitinases have been isolated and characterized from bacteria such as Bacillus cereus (Hammami et al, 2013), Bacillus licheniformis (LaribiHabchi, Bouanane-Darenfed, Drouiche, Pauss, & Mameri, 2015), Chitinibacter sp. GC72 (Gao, Zhang, Chen, Hao, Tong, & Ouyang, 2015), Laceyella putida (Shibasaki, Uchimura, Miura, Kobayashi, Usami, & Horikoshi, 2014) and Massilia timonae (Adrangi, Faramarzi, Shahverdi, & Sepehrizadeh, 2010). Moreover, many chitinase genes from Bacillus halodurans (da Silva, García-Fraga, López-Seijas, & Sieiro, 2014), B. licheniformis (Sandalli, Kacagan, Canakci, & Belduz, 2008;Songsiriritthigul, Lapboonrueng, Pechsrichuang, Pesatcha, & Yamabhai, 2010), Chitiniphilus shinanonensis (Huang, Shizume, Nogawa, Taguchi, & Shimosaka, 2012), Paenibacillus barengoltzii (Fu, Yan, Yang, Yang, Guo, & Jiang, 2014), Pseudoalteromonas tunicate (García-Fraga et al, 2015), Sanguibacter antarcticus (Lee et al, 2010), Serratia proteamaculans (Purushotham & Podile, 2012), and Vibrio sp.…”

An acidic, thermostable exochitinase with β-N-acetylglucosaminidase activity from Paenibacillus barengoltzii converting chitin to N-acetyl glucosamine

“…Among them, bacterial chitinases gained more research interest due to their diverse properties and potential industrial applications [6]. To date, a number of bacterial chitinases have been isolated and characterized [3,7-15]. Many chitinase genes have also been cloned and expressed from bacteria such as Bacillus sp.…”