Huang Yuhong, Busk Peter Kamp, Grell Morten Nedergaard, Zhao Hai, Lange Lene
Section for Sustainable Biotechnology, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University Copenhagen, DK-2450 Copenhagen SV, Denmark; Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan 610041, China; University of the Chinese Academy of Sciences, Beijing 100049, China.
Section for Sustainable Biotechnology, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University Copenhagen, DK-2450 Copenhagen SV, Denmark.
Enzyme Microb Technol. 2014 Dec;67:47-52. doi: 10.1016/j.enzmictec.2014.09.002. Epub 2014 Sep 16.
Mucor circinelloides produces plant cell wall degrading enzymes that allow it to grow on complex polysaccharides. Although the genome of M. circinelloides has been sequenced, only few plant cell wall degrading enzymes are annotated in this species. We applied peptide pattern recognition, which is a non-alignment based method for sequence analysis to map conserved sequences in glycoside hydrolase families. The conserved sequences were used to identify similar genes in the M. circinelloides genome. We found 12 different novel genes encoding members of the GH3, GH5, GH9, GH16, GH38, GH47 and GH125 families in M. circinelloides. One of the two GH3-encoding genes was predicted to encode a β-glucosidase (EC 3.2.1.21). We expressed this gene in Pichia pastoris KM71H and found that the purified recombinant protein had relative high β-glucosidase activity (1.73U/mg) at pH5 and 50°C. The Km and Vmax with p-nitrophenyl-β-d-glucopyranoside as substrate was 0.20mM and 2.41U/mg, respectively. The enzyme was not inhibited by glucose and retained 84% activity at glucose concentrations up to 140mM. Although zygomycetes are not considered to be important degraders of lignocellulosic biomass in nature, the present finding of an active β-glucosidase in M. circinelloides demonstrates that enzymes from this group of fungi have a potential for cellulose degradation.
卷枝毛霉能产生植物细胞壁降解酶，使其能够在复杂多糖上生长。尽管卷枝毛霉的基因组已被测序，但该物种中只有少数植物细胞壁降解酶得到注释。我们应用了肽模式识别，这是一种基于非比对的序列分析方法，用于绘制糖苷水解酶家族中的保守序列。这些保守序列被用于在卷枝毛霉基因组中鉴定相似基因。我们在卷枝毛霉中发现了12个不同的新基因，它们编码GH3、GH5、GH9、GH16、GH38、GH47和GH125家族的成员。两个编码GH3的基因之一被预测编码一种β-葡萄糖苷酶（EC 3.2.1.21）。我们在毕赤酵母KM71H中表达了该基因，发现纯化后的重组蛋白在pH5和50°C时具有相对较高的β-葡萄糖苷酶活性（1.73U/mg）。以对硝基苯基-β-D-吡喃葡萄糖苷为底物时，其Km和Vmax分别为0.20mM和2.41U/mg。该酶不受葡萄糖抑制，在葡萄糖浓度高达140mM时仍保留84%的活性。尽管接合菌在自然界中不被认为是木质纤维素生物质的重要降解者，但目前在卷枝毛霉中发现的一种活性β-葡萄糖苷酶表明，这组真菌的酶具有纤维素降解的潜力。
