N-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. A 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. The 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.

Butyl butyrate has been considered as a promising fuel source because it is a kind of natural ester which can be converted from renewable and sustainable lignocellulosic biomass. Compared with the conventional chemical methods for butyl butyrate production, the enzymatic approach has been demonstrated to be more attractive, mainly owing to the mild reaction conditions, high specificity, low energy consumption, and environmental friendliness. Cutinases play an important role in the butyl butyrate production process. However, the production level of cutinases is still relatively low. Thus, to identify novel cutinases suitable for butyl butyrate synthesis and enhance their yields is of great value in biofuel industry. A novel cutinase gene (McCut) was cloned from a thermophilic fungus Malbranchea cinnamomea and expressed in Pichia pastoris. The highest cutinase activity of 12, 536 U/mL was achieved in 5-L fermentor, which is by far the highest production for a cutinase. McCut was optimally active at pH 8.0 and 45 °C. It exhibited excellent stability within the pH range of 3.0–10.5 and up to 75 °C. The cutinase displayed broad substrate specificity with the highest activity towards p-nitrophenyl butyrate and tributyrin. It was capable of hydrolyzing cutin, polycaprolactone, and poly(butylene succinate). Moreover, McCut efficiently synthesized butyl butyrate with a maximum esterification efficiency of 96.9% at 4 h. The overall structure of McCut was resolved as a typical α/β-hydrolase fold. The structural differences between McCut and Aspergillus oryzae cutinase in groove and loop provide valuable information for redesign of McCut. These excellent features make it useful in biosynthesis and biodegradation fields. A novel cutinase from M. cinnamomea was identified and characterized for the first time. High-level expression by P. pastoris is by far the highest for a cutinase. The enzyme exhibited excellent stability and high esterification efficiency for butyl butyrate production, which may make it a good candidate in biofuel and chemical industries.