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

Abstract

BackgroundN-acetyl-β-D-glucosamine (GlcNAc) is widely used as a valuable pharmacological agent and a functional food additive. The traditional chemical process for GlcNAc production has some problems such as high production cost, low yield, and acidic pollution. Hence, to identify a novel chitinase that is suitable for bioconversion of chitin to GlcNAc is of great value.ResultsA novel chitinase gene (PbChi74) from Paenibacillus barengoltzii was cloned and heterologously expressed in Escherichia coli as an intracellular soluble protein. The gene has an open reading frame (ORF) of 2,163 bp encoding 720 amino acids. The recombinant chitinase (PbChi74) was purified to apparent homogeneity with a purification fold of 2.2 and a recovery yield of 57.9%. The molecular mass of the purified enzyme was estimated to be 74.6 kDa and 74.3 kDa by SDS-PAGE and gel filtration, respectively. PbChi74 displayed an acidic pH optimum of 4.5 and a temperature optimum of 65°C. The enzyme showed high activity toward colloidal chitin, glycol chitin, N-acetyl chitooligosaccharides, and p-nitrophenyl N-acetyl β-glucosaminide. PbChi74 hydrolyzed colloidal chitin to yield N-acetyl chitobiose [(GlcNAc)2] at the initial stage, which was further converted to its monomer N-acetyl glucosamine (GlcNAc), suggesting that it is an exochitinase with β-N-acetylglucosaminidase activity. The purified PbChi74 coupled with RmNAG (β-N-acetylglucosaminidase from Rhizomucor miehei) was used to convert colloidal chitin to GlcNAc, and GlcNAc was the sole end product at a concentration of 27.8 mg mL-1 with a conversion yield of 92.6%. These results suggest that PbChi74 may have great potential in chitin conversion.ConclusionsThe excellent thermostability and hydrolytic properties may give the exochitinase great potential in GlcNAc production from chitin. This is the first report on an exochitinase with N-acetyl-β-D-glucosaminidase activity from Paenibacillus species.

Purification and biochemical characterization of novel acidic chitinase from Paenicibacillus barengoltzii

“…2), which is obviously lower than those of the two chitinases (PbChi70, 70.6 kDa; PbChi74, 74 kDa) from the same strain reported in our previous studies [4,5]. [31]).…”

“…PbChi74 is an exochitinase with molecular mass of 74 kDa. It was most active at pH 4.5 and 65 °C, respectively, and suitable for GlcNAc production [5]. Here, for the first time, we described production, purification and characterization of novel acidic endochitinase from Paenibacillus barengoltzii.…”

“…Paenicibacillus barengoltzii CAU904 is a thermophilic marine bacterium which has been reported to produce different types of chitinases in our previous studies, including an endochitinase suitable for (GlcNAc)2 production [4] and an acidic and thermostable exochitinase (PbChi74) suitable for GlcNAc production [5]. Here, we report on production, purification and characterization of another novel acidic endochitinase from Paenicibacillus barengoltzii.…”

“…Aliquots withdrawn at different time intervals were immediately boiled for 10 min to terminate the enzyme activity, and the hydrolysis products were then analyzed by thin layer chromatography (TLC). TLC analysis was performed according to the method of Fu et al [5]. The hydrolysis products from colloidal chitin at the initial stage were quantitatively determined by HPLC equipped with an evaporative light-scattering detector (Waters, USA) using acetonitrile/water (75:25, v/v) as the mobile phase at a flow rate of 1 mL/min.…”

“…They have received a great deal of attention in recent years due to their biotechnological applications in different fields [3]. They can be used in the production of N-acetyl GOSs [4] and GlcNAc [5] in food or pharmaceutical industry [2], bioconversion of chitin materials to ethanol in energy industry [6], production of single cell protein in feed industry [7] and bio-controlling of fungal phytopathogens in agriculture [8,9].…”