Controlling Autolysis During Flagella Insertion in Gram-Negative Bacteria.

The flagellum is an important macromolecular machine for many pathogenic bacteria. It is a hetero-oligomeric structure comprised of three major sub-structures: basal body, hook and thin helical filament. An important step during flagellum assembly is the localized and controlled degradation of the peptidoglycan sacculus to allow for the insertion of the rod as well as to facilitate anchoring for proper motor function. The peptidoglycan lysis events require specialized lytic enzymes, ß-N-acetylglucosaminidases and lytic transglycosylases, which differ in flagellated proteobacteria. Due to their autolytic activity, these enzymes need to be controlled in order to prevent cellular lysis. This review summarizes are current understanding of the peptidoglycan lysis events required for flagellum assembly and motility with a main focus on Gram-negative bacteria.

[Expression and characterization of ß-N-acetylglucosaminidases from Bacillus coagulans DSM1 for N-acetyl-ß-D glucosamine production].

ß-N-acetylglucosaminidases (NAGases) can convert natural substrates such as chitin or chitosan to N-acetyl-ß-D glucosamine (GlcNAc) monomer that is wildly used in medicine and agriculture. In this study, the BcNagZ gene from Bacillus coagulans DMS1 was cloned and expressed in Escherichia coli. The recombinant protein was secreted into the fermentation supernatant and the expression amount reached 0.76 mg/mL. The molecular mass of purified enzyme was 61.3 kDa, and the specific activity was 5.918 U/mg. The optimal temperature and pH of the BcNagZ were 75 °C and 5.5, respectively, and remained more than 85% residual activity after 30 min at 65 °C. The Mie constant Km was 0.23 mmol/L and the Vmax was 0.043 1 mmol/(L·min). The recombinant BcNagZ could hydrolyze colloidal chitin to obtain trace amounts of GlcNAc, and hydrolyze disaccharides to monosaccharide. Combining with the reported exochitinase AMcase, BcNagZ could produce GlcNAc from hydrolysis of colloidal chitin with a yield over 86.93%.

Structural and functional characterization of a glycoside hydrolase family 3 ß-N-acetylglucosaminidase from Paenibacillus sp. str. FPU-7.

Chitin, a ß-1,4-linked homopolysaccharide of N-acetyl-d-glucosamine (GlcNAc), is one of the most abundant biopolymers on Earth. Paenibacillus sp. str. FPU-7 produces several different chitinases and converts chitin into N,N'-diacetylchitobiose ((GlcNAc)2) in the culture medium. However, the mechanism by which the Paenibacillus species imports (GlcNAc)2 into the cytoplasm and divides it into the monomer GlcNAc remains unclear. The gene encoding Paenibacillus ß-N-acetyl-d-glucosaminidase (PsNagA) was identified in the Paenibacillus sp. str. FPU-7 genome using an expression cloning system. The deduced amino acid sequence of PsNagA suggests that the enzyme is a part of the glycoside hydrolase family 3 (GH3). Recombinant PsNagA was successfully overexpressed in Escherichia coli and purified to homogeneity. As assessed by gel permeation chromatography, the enzyme exists as a 57-kDa monomer. PsNagA specifically hydrolyses chitin oligosaccharides, (GlcNAc)2-4, 4-nitrophenyl N-acetyl ß-d-glucosamine (pNP-GlcNAc) and pNP-(GlcNAc)2-6, but has no detectable activity against 4-nitrophenyl ß-d-glucose, 4-nitrophenyl ß-d-galactosamine and colloidal chitin. In this study, we present a 1.9 Å crystal structure of PsNagA bound to GlcNAc. The crystal structure reveals structural features related to substrate recognition and the catalytic mechanism of PsNagA. This is the first study on the structural and functional characterization of a GH3 ß-N-acetyl-d-glucosaminidase from Paenibacillus sp.

A chitinolytic endochitinase and ß-N-acetylglucosaminidase-based system from Hevea latex in generating N-acetylglucosamine from chitin.

An endochitinase and ß-N-acetylglucosaminidase (NAGase) were purified and characterised from fresh rubber latex serum. These enzymes were used in a total enzyme-based system to produce pure N-acetylglucosamine (NAG) from chitin. The N-terminal amino acid sequences of both purified endochitinase (KEESRRRRHR) and NAGase (AAVDSDTLEI) lacked homology with other known chitinases, including hevamine from rubber latex lutoids. The apparent kinetic parameters, Km and Vmax, for the endochitinase using 4-MU-ß-(NAG)3 as a substrate were 99.73 µM and 29.49 pkat mg(-1), respectively. For NAGase, using 4-MU-ß-NAG as a substrate, the corresponding Km and Vmax values were 20.4 µM and 25.82 pkat mg(-1). When an enzyme incubation mixture containing a 1:1 (pkat/pkat) activity mixed ratio of endochitinase: NAGase was employed, the maximum yield of N-acetylglucosamine (NAG) obtained was 98% from ß-chitin and 20% from α-chitin. These yields were obtained after 4 days of hydrolysis of equal amounts of ß-chitin and α-chitin in the mixture. Thus, ß-chitin was the preferred substrate compared to α-chitin by a ratio of nearly five to one. Mass spectroscopic analysis, using electrospray ionisation mass spectrometry (ESI-MS), of the product obtained from ß-chitin after digestion (for 24h) depicted one distinct major molecular ion peak m/z 260.1, a small minor ion peak m/z 481.2, a potassium adduct of NAG and a potassium adduct of two NAG molecules. Furthermore, experiments to establish the commercial production of NAG using crude enzymes of Hevea latex serum are currently in progress.