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首页> 外文期刊>Applied and Environmental Microbiology >Correction of the beta-mannanase domain of the celC pseudogene from Caldocellulosiruptor saccharolyticus and activity of the gene product on kraft pulp.
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Correction of the beta-mannanase domain of the celC pseudogene from Caldocellulosiruptor saccharolyticus and activity of the gene product on kraft pulp.

机译：卡尔杜氏纤维素分解酵母糖酶celC假基因的β-甘露聚糖酶结构域的校正和牛皮纸浆上基因产物的活性。

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The celA, manA, and celB genes from Caldocellulosiruptor saccharolyticus compose a cellulase-hemicellulase gene cluster and are arranged on a 12-kb C. saccharolyticus genomic fragment of the recombinant lambda bacteriophage NZP lambda 2. The beginning of a fourth open reading frame (celC) which was homologous to the C. saccharolyticus manA and celA genes was located at the 3' end of the 12-kb NZP lambda 2 genomic fragment. Genome-walking PCR was used to isolate DNA fragments downstream of the C. saccharolyticus celB gene, and the entire nucleotide sequence of celC was obtained. From the preliminary nucleotide sequence, celC appeared to encode yet another multidomain bifunctional enzyme (CelC) consisting of an N-terminal endo-1,4-beta-D-glucanase domain (75% similar to CelA domain 1), two central cellulose-binding domains, and a C-terminal endo-1,4-beta-D-mannanase domain (98% similar to ManA domain 1). However, upon completion of the celC sequencing, two -1 frameshifts were identified in the region encoding the putative CelC mannanase domain. The isolated CelC mannanase domain exhibited no beta-mannanase activity, which supported this observation. Recombinant PCR was used to correct the celC frameshifts by inserting the appropriate nucleotides into the gene. The repaired celC fragment containing the base insertions (manB) expressed strong beta-mannanase activity on soluble mannan substrates and showed significant activity on kraft pulp as judged by the release of reducing sugars.
机译：产自卡尔杜氏纤维素分解酵母糖酶的celA，manA和celB基因组成一个纤维素酶-半纤维素酶基因簇，并排列在重组λ噬菌体NZPλ2的12 kb糖解梭菌基因组片段上。第四个开放阅读框（celC）的开始与解糖梭菌的manA和celA基因同源的）位于12kbNZPλ2基因组片段的3'末端。使用基因组步行PCR分离解糖梭菌celB基因下游的DNA片段，并获得了celC的完整核苷酸序列。从初步的核苷酸序列来看，celC似乎编码了另一种多结构域双功能酶（CelC），该酶由一个N末端1,4-beta-D-葡聚糖酶结构域（与CelA结构域1相似，为75％），两个中央纤维素-结合结构域和一个C末端1,4-beta-D-甘露聚糖酶C末端结构域（98％与ManA结构域1相似）。但是，完成celC测序后，在编码推定的CelC甘露聚糖酶结构域的区域中发现了两个-1移码。分离的CelC甘露聚糖酶结构域没有显示β-甘露聚糖酶活性，这支持了这一观察结果。通过将适当的核苷酸插入基因，重组PCR被用于校正celC移码。通过还原糖的释放判断，含有碱基插入片段（manB）的已修复celC片段在可溶性甘露聚糖底物上表达了很强的β-甘露聚糖酶活性，并对牛皮纸浆显示出显着的活性。

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