Purification and biochemical characterization of a novel thermostable and halotolerant subtilisin SAPN, a serine protease from Melghiribacillus thermohalophilus Nari2AT for chitin extraction from crab and shrimp shell by-products.

The present study investigates the purification and biochemical characterization of a novel extracellular serine alkaline protease, subtilisin (called SAPN) from Melghiribacillus thermohalophilus Nari2AT. The highest yield of protease (395 IU/g) with white shrimp shell by-product (40 g/L) as a unique source of nutriments in the growth medium was achieved after 52 h at 55 °C. The monomeric enzyme of about 30 kDa was purified to homogeneity by ammonium sulfate fractionation, heat treatment, followed by sequential column chromatographies. The optimum pH and temperature values for subtilisin activity were pH 10 and 75 °C, respectively, and half lives of 9 and 5 h at 80 and 90 °C, respectively. The sequence of the 25 NH2-terminal residues pertaining of SAPN exhibited a high homology with those of Bacillus subtilisins. The inhibition by DFP and PMSF indicates that this enzyme belongs to the serine proteases family. SAPN was found to be effective in the deproteinization (DDP %) of blue swimming crab (Portunus segnis) and white shrimp (Metapenaeus monoceros) by-products, with a degree of 65 and 82%, respectively. The commercial and the two chitins obtained in this work showed a similar peak pattern in Fourier-Transform Infrared (FTIR) analysis, suggesting that SAPN is suitable for the bio-production of chitin from shell by-products.

Purification and biochemical characterization of a novel acido-halotolerant and thermostable endochitinase from Melghiribacillus thermohalophilus strain Nari2AT.

An extracellular acido-thermostable endochitinase (called ChiA-Mt45) from thermohalophilic Melghiribacillus thermohalophilus strain Nari2AT gen. nov. sp. nov., was purified and biochemically characterized. The maximum chitinase activity recorded after 48-h of incubation at 55 °C was 9000 U/mL. Pure enzyme was obtained after heat treatment (20 min at 90 °C) followed by sequential column chromatographies on fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC). Based on MALDI-TOF/MS analysis, the purified enzyme is a monomer with a molecular mass of 45201.10 Da. The 27 residue NH2-terminal sequence of the enzyme showed high homology with Bacillus GH-18 chitinases family. The optimum pH and temperature values for chitinase activity were pH 3.5 and 90 °C, respectively. In addition, the enzyme was halotolerant and can be classified as an extremozyme. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB) and N-ethylmaleimide (NEM). Its Km and kcat values were 0.253 mg colloidal chitin/mL and 47000 s-1, respectively. Interestingly, its catalytic efficiency was higher than those of chitinases ChiA-Hh59 from Hydrogenophilus hirchii KB-DZ44 and chitodextrinase from Streptomyces griseus, and N-acetyl-ß-glucosaminidase from Trichoderma viride. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Additionally, thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Mt45 acted as an endosplitting enzyme. Overall, the chitinase ChiA-Mt45 may have great potential for the enzymatic degradation of chitin.

Melghiribacillus thermohalophilus gen. nov., sp. nov., a novel filamentous, endospore-forming, thermophilic and halophilic bacterium.

A novel filamentous, endospore-forming, thermophilic and moderately halophilic bacterium designated strain Nari2A(T) was isolated from soil collected from an Algerian salt lake, Chott Melghir. The novel isolate was Gram-staining-positive, aerobic, catalase-negative and oxidase-positive. Optimum growth occurred at 50-55 °C, 7-10% (w/v) NaCl and pH 7-8. The strain exhibited 95.4, 95.4 and 95.2% 16S rRNA gene sequence similarity to Thalassobacillus devorans G19.1(T), Sediminibacillus halophilus EN8d(T) and Virgibacillus kekensis YIM-kkny16(T), respectively. The major menaquinone was MK-7. The polar lipid profile consisted of phosphatidylglycerol, diphosphatidylglycerol, three unknown phosphoglycolipids and two unknown phospholipids. The predominant cellular fatty acids were iso-C(15 : 0) and iso-C(17 : 0). The DNA G+C content was 41.9 mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain Nari2A(T) is considered to represent a novel species of a new genus in the family Bacillaceae , order Bacillales , for which the name Melghiribacillus thermohalophilus gen. nov., sp. nov. is proposed. The type strain of Melghiribacillus thermohalophilus is Nari2A(T) ( = DSM 25894(T) = CCUG 62543(T)).