Khan Ibrar, Qayyum Sadia, Ahmed Shehzad, Niaz Zeeshan, Fatima Nighat, Chi Zhen-Ming
UNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Qingdao 266003, China; Department of Microbiology, Hazara University, 21300 Mansehra, Pakistan.
Department of Microbiology, Hazara University, 21300 Mansehra, Pakistan.
Gene. 2016 Nov 5;592(2):291-302. doi: 10.1016/j.gene.2016.07.032. Epub 2016 Jul 15.
The PVGOX gene (accession number: KT452630) was isolated from genomic DNA of the marine fungi Penicillium viticola F1 by Genome Walking and their expression analysis was done by Fluorescent RT-PCR. An open reading frame of 1806bp encoding a 601 amino acid protein (isoelectric point: 5.01) with a calculated molecular weight of 65,535.4 was characterized. The deduced protein showed 75%, 71%, 69% and 64% identity to those deduced from the glucose oxidase (GOX) genes from different fungal strains including; Talaromyces variabilis, Beauveria bassiana, Aspergillus terreus, and Aspergillus niger, respectively. The promoter of the gene (intronless) had two TATA boxes around the base pair number -88 and -94 and as well as a CAAT box at -100. However, the terminator of the PVGOX gene does not contain any polyadenylation site (AATAAA). The protein deduced from the PVGOX gene had a signal peptide containing 17 amino acids, three cysteine residues and six potential N-linked glycosylation sites, among them, -N-K-T-Y- at 41 amino acid, -N-R-S-L- at 113 amino acid, -N-G-T-I- at 192 amino acid, -N-T-T-A at 215 amino acid, -N-F-T-E at 373 amino acid and -N-V-T-A- at 408 amino acid were the most possible N-glycosylation sites. Furthermore, the relative transcription level of the PVGOX gene was also stimulated in the presence of 4% (w/v) of calcium carbonate and 0.5 % (v/v) of CSL in the production medium compared with that of the PVGOX gene when the fungal strain F1 was grown in the absence of calcium carbonate and CSL in the production medium, suggesting that under the optimal conditions, the expression of the PVGOX gene responsible for gluconic acid biosynthesis was enhanced, leading to increased gluconic acid production. Therefore, the highly glycosylated oxidase enzyme produced by P. viticola F1 strain might be a good producer in the fermentation process for the industrial level production of gluconic acid.
通过基因组步移技术从海洋真菌葡萄穗青霉F1的基因组DNA中分离出PVGOX基因（登录号：KT452630），并通过荧光逆转录聚合酶链式反应对其进行表达分析。鉴定出一个1806bp的开放阅读框，编码一个601个氨基酸的蛋白质（等电点：5.01），计算分子量为65,535.4。推导的蛋白质与来自不同真菌菌株（包括多变拟青霉、球孢白僵菌、土曲霉和黑曲霉）的葡萄糖氧化酶（GOX）基因推导的蛋白质分别具有75%、71%、69%和64%的同一性。该基因的启动子（无内含子）在碱基对-88和-94附近有两个TATA框，在-100处还有一个CAAT框。然而，PVGOX基因的终止子不包含任何多聚腺苷酸化位点（AATAAA）。从PVGOX基因推导的蛋白质有一个包含17个氨基酸的信号肽、三个半胱氨酸残基和六个潜在的N-糖基化位点，其中，第41个氨基酸处的-N-K-T-Y-、第113个氨基酸处的-N-R-S-L-、第192个氨基酸处的-N-G-T-I-、第215个氨基酸处的-N-T-T-A、第373个氨基酸处的-N-F-T-E和第408个氨基酸处的-N-V-T-A-是最可能的N-糖基化位点。此外，与在生产培养基中不添加碳酸钙和CSL培养真菌菌株F1时PVGOX基因的情况相比，在生产培养基中存在4%（w/v）碳酸钙和0.5%（v/v）CSL时，PVGOX基因的相对转录水平也受到刺激，这表明在最佳条件下，负责葡萄糖酸生物合成的PVGOX基因的表达增强，导致葡萄糖酸产量增加。因此，葡萄穗青霉F1菌株产生的高度糖基化氧化酶可能是工业水平生产葡萄糖酸发酵过程中的良好生产者。
