Molecular cloning, heterologous expression, and in silico sequence analysis of Enterobacter GH19 class I chitinase (chiRAM gene).

BACKGROUND: Using in silico sequence analyses, the present study aims to clone and express the gene-encoding sequence of a GH19 chitinase from Enterobacter sp. in Escherichia coli. METHODS AND RESULTS: The putative open reading frame of a GH19 chitinase from Enterobacter sp. strain EGY1 was cloned and expressed into pGEM®-T and pET-28a (+) vectors, respectively using a degenerate primer. The isolated nucleotide sequence (1821 bp, GenBank accession no.: MK533791.2) was translated to a chiRAM protein (606 amino acids, UniProt accession no.: A0A4D6J2L9). The in silico protein sequence analysis of chiRAM revealed a class I GH19 chitinase: an N-terminus signal peptide (Met1-Ala23), a catalytic domain (Val83-Glu347 and the catalytic triad Glu149, Glu171, and Ser218), a proline-rich hinge region (Pro414 -Pro450), a polycystic kidney disease protein motif (Gly 465-Ser 533), a C-terminus chitin-binding domain (Ala553- Glu593), and conserved class I motifs (NYNY and AQETGG). A three-dimensional model was constructed by LOMETS MODELLER of PDB template: 2dkvA (class I chitinase of Oryza sativa L. japonica). Recombinant chiRAM was overexpressed as inclusion bodies (IBs) (~ 72 kDa; SDS-PAGE) in 1.0 mM IPTG induced E. coli BL21 (DE3) Rosetta strain at room temperature 18 h after induction. Optimized expression yielded active chiRAM with 1.974 ± 0.0002 U/mL, on shrimp colloidal chitin (SCC), in induced E. coli BL21 (DE3) Rosetta cells growing in SB medium. LC-MS/MS identified a band of 72 kDa in the soluble fraction with a 52.3% coverage sequence exclusive to the GH19 chitinase of Enterobacter cloacae (WP_063869339.1). CONCLUSIONS: Although chiRAM of Enterobacter sp. was successfully cloned and expressed in E. coli with appreciable chitinase activity, future studies should focus on minimizing IBs to facilitate chiRAM purification and characterization.

Degradation of polyesters by a novel marine Nocardiopsis aegyptia sp. nov.: application of Plackett-Burman experimental design for the improvement of PHB depolymerase activity.

This is the first report on the degradation of poly(3-hydroxybutyrate) (PHB), and its copolymers poly(3-hydroxyvalerate) P(3HB-co-10-20% HV) by Nocardiopsis aegyptia, a new species isolated from marine seashore sediments. The strain excreted an extracellular PHB depolymerase and grew efficiently on PHB or its copolymers as the sole carbon sources. The degradation activity was detectable by the formation of a transparent clearing zone around the colony on an agar Petri plate after 25 days, or a clearing depth under the colony in test tubes within 3 weeks. The previous techniques proved that the bacterium was able to assimilate the monomeric components of the shorter alkyl groups of the polymers. Nocardiopsis aegyptia hydrolyzed copolymers 10-20% PHBV more rapidly than the homopolymer PHB. The bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate), and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). The samples were degraded at the surface and proceeded to the inner part of the materials. Clear morphological alterations of the polymers were noticed, indicating the degradative capability of the bacterium. Plackett-Burman statistical experimental design has been employed to optimize culture conditions for maximal enzyme activity. The main factors that had significant positive effects on PHB depolymerase activity of Nocardiopsis aegyptia were sodium gluconate, volume of medium/flask and age of inoculum. On the other hand, MgSO4.7H2O, KH2PO4, K2HPO4 and NH4NO3 exhibited negative effects. Under optimized culture conditions, the highest activity (0.664 U/mg protein) was achieved in a medium predicted to be near optimum containing (in g/L): PHB, 0.5; C6H11O7Na, 7.5; MgSO4.7H2O, 0.35; K2HPO4, 0.35; NH4NO3, 0.5; KH2PO4, 0.35; malt extract, 0.5 and prepared with 50% seawater. The medium was inoculated with 1% (v/v) spore suspension of 7 days old culture. Complete clarity of the medium was achieved after 3 days at 30 degrees C.

Nocardiopsis aegyptia sp. nov., isolated from marine sediment.

An actinomycete, strain SNG49(T), was isolated from marine sediment of Abu Qir Bay, on the western seashore of Alexandria, Egypt. The bacterium was aerobic and Gram-positive. It produced beige to light-yellow aerial mycelium, brown substrate mycelium and straight to flexuous hyphae, but no specific spore chains. 16S rDNA sequence analysis and chemotaxonomic markers were consistent with classification of strain SNG49(T) in the genus Nocardiopsis, i.e. meso-diaminopimelic acid; no diagnostic sugars; phosphatidylcholine, phosphatidylmethylethanolamine, phosphatidylinositol, phosphatidylglycerol and diphosphatidylglycerol as polar lipids; menaquinones of the MK-10 series from MK-10(H(0)) to MK-10(H(8)); and iso/anteiso-branched and 10-methyl-branched fatty acids, the principal fatty acids being anteiso-17 : 0 and tuberculostearic acid. Nocardiopsis lucentensis and Nocardiopsis alba are the phylogenetic neighbours of strain SNG49(T), respectively showing 98.8 and 98.7 % 16S rRNA gene sequence similarity; however, moderate DNA-DNA reassociation values between these two species and strain SNG49(T) (44 and 60 %, respectively) showed that strain SNG49(T) could be clearly separated from them. These data, together with distinct physiological traits, led to the conclusion that this isolate represents a novel species within the genus Nocardiopsis, for which the name Nocardiopsis aegyptia is proposed. The type strain is SNG49(T) (=DSM 44442(T)=NRRL B-24244(T)).

Isolation and enumeration of marine actinomycetes from seawater and sediments in Alexandria.

Actinomycetes were isolated from near-shore marine sediments and water at four different sites in Alexandria. Statistical analysis revealed that variation in temperature, pH, and dissolved phosphate were of insignificant values, but that variation in total nitrogen and organic matter were significant. The treatment of sediments and water samples by heat resulted in a selective reduction of the nonactinomycetal heterotrophic microflora. Four selective culture media were used for counting actinomycetes in marine water and sediments. The starch nitrate medium favored the growth of these microorganisms. The diversity and counts of actinomycetes varied with the seasonal variation, and the highest counts were detected in dry warm seasons. The numbers of this bacterial group in sediments exceeded by far their numbers in seawater. A positive correlation was found between population size and location. Actinomycetes were found in the highest numbers in the upper layers (0-20 cm depth). In a few cases, the counts of actinomycetes showed bimodal maxima 0-20 and 60-100 cm deep. Sediments were the best source of marine actinomycetes, and their distribution varied depending on the depth from which samples were collected. The ratio of actinomycetes to the total microflora ranged from 0.48 to 2.29, depending on location.