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.
摘要：
通过定向进化改造了一种来自巴伦博尔氏芽孢杆菌的几丁质酶（PbChi70），以提高其对粉末几丁质的水解效率。通过两轮筛选，获得了最大比活性为73.21U/mg的突变体（mPbChi70），这是迄今为止报道的最高值。将突变基因进一步转化到黑曲霉FBL-B（ΔglaA）中，该黑曲霉可以分泌高水平的内源性β-N-乙酰氨基葡萄糖苷酶（GlcNAcase），构建了双酶表达系统。通过高细胞密度发酵，在5-L发酵罐中获得最高的几丁质酶活性为61.33U/mL，GlcNAcase活性为353.1U/mL。几丁质降解酶混合物直接用于从粉末几丁质中生物转化GlcNAc，最高转化率高达71.9%(w/w)，GlcNAc纯度≥95%(w/w)。本研究为几丁质材料的高效生物转化提供了一种优良的几丁质酶和双酶混合体系。
