Development of a two-enzyme system in Aspergillus niger for efficient production of N-acetyl-β-D-glucosamine from powdery chitin

Abstract

A chitinase (PbChi70) from Paenibacillus barengoltzii was engineered by directed evolution to enhance its hydrolysis efficiency towards powder chitin. Through two rounds of screening, a mutant (mPbChi70) with a maximum specific activity of 73.21 U/mg was obtained, which is by far the highest value ever reported. The mutant gene was further transformed into Aspergillus niger FBL-B (ΔglaA) which could secrete high level of endogenously β-N-acetylglucosaminidase (GlcNAcase), thus a two-enzyme expression system was constructed. The highest chitinase activity of 61.33 U/mL with GlcNAcase activity of 353.1 U/mL was obtained in a 5-L fermentor by high-cell density fermentation. The chitin-degrading enzyme cocktail was used for the bioconversion of GlcNAc from powder chitin directly, and the highest conversion ratio reached high up to 71.9 % (w/w) with GlcNAc purity ≥95 % (w/w). This study may provide an excellent chitinase as well as a double enzyme cocktail system for efficient biological conversion of chitin materials.

Keywords: Chitin degradation; Chitinase; Directed evolution; N-acetyl-D-glucosamine; Two-enzyme system; β-N-acetylglucosaminidase.