Wickramasinghe Gammadde Hewa Ishan Maduka, Rathnayake Pilimathalawe Panditharathna Attanayake Mudiyanselage Samith Indika, Chandrasekharan Naduviladath Vishvanath, Weerasinghe Mahindagoda Siril Samantha, Wijesundera Ravindra Lakshman Chundananda, Wijesundera Wijepurage Sandhya Sulochana
Department of Chemistry, Faculty of Science, University of Colombo, Colombo, Sri Lanka.
Department of Plant Sciences, Faculty of Science, University of Colombo, Colombo, Sri Lanka.
BMC Microbiol. 2017 Jun 21;17(1):137. doi: 10.1186/s12866-017-1049-8.
Cellulose, a linear polymer of β 1-4, linked glucose, is the most abundant renewable fraction of plant biomass (lignocellulose). It is synergistically converted to glucose by endoglucanase (EG) cellobiohydrolase (CBH) and β-glucosidase (BGL) of the cellulase complex. BGL plays a major role in the conversion of randomly cleaved cellooligosaccharides into glucose. As it is well known, Saccharomyces cerevisiae can efficiently convert glucose into ethanol under anaerobic conditions. Therefore, S.cerevisiae was genetically modified with the objective of heterologous extracellular expression of the BGLI gene of Trichoderma virens making it capable of utilizing cellobiose to produce ethanol.
The cDNA and a genomic sequence of the BGLI gene of Trichoderma virens was cloned in the yeast expression vector pGAPZα and separately transformed to Saccharomyces cerevisiae. The size of the BGLI cDNA clone was 1363 bp and the genomic DNA clone contained an additional 76 bp single intron following the first exon. The gene was 90% similar to the DNA sequence and 99% similar to the deduced amino acid sequence of 1,4-β-D-glucosidase of T. atroviride (AC237343.1). The BGLI activity expressed by the recombinant genomic clone was 3.4 times greater (1.7 x 10 IU ml) than that observed for the cDNA clone (5 x 10 IU ml). Furthermore, the activity was similar to the activity of locally isolated Trichoderma virens (1.5 x 10 IU ml). The estimated size of the protein was 52 kDA. In fermentation studies, the maximum ethanol production by the genomic and the cDNA clones were 0.36 g and 0.06 g /g of cellobiose respectively. Molecular docking results indicated that the bare protein and cellobiose-protein complex behave in a similar manner with considerable stability in aqueous medium. The deduced binding site and the binding affinity of the constructed homology model appeared to be reasonable. Moreover, it was identified that the five hydrogen bonds formed between the amino acid residues of BGLI and cellobiose are mainly involved in the integrity of enzyme-substrate association.
The BGLI activity was remarkably higher in the genomic DNA clone compared to the cDNA clone. Cellobiose was successfully fermented into ethanol by the recombinant S.cerevisiae genomic DNA clone. It has the potential to be used in the industrial production of ethanol as it is capable of simultaneous saccharification and fermentation of cellobiose. Homology modeling, docking studies and molecular dynamics simulation studies will provide a realistic model for further studies in the modification of active site residues which could be followed by mutation studies to improve the catalytic action of BGLI.
纤维素是一种由β 1-4连接的葡萄糖组成的线性聚合物，是植物生物质（木质纤维素）中最丰富的可再生成分。它通过纤维素酶复合物中的内切葡聚糖酶（EG）、纤维二糖水解酶（CBH）和β-葡萄糖苷酶（BGL）协同转化为葡萄糖。BGL在将随机切割的纤维寡糖转化为葡萄糖的过程中起主要作用。众所周知，酿酒酵母在厌氧条件下能有效地将葡萄糖转化为乙醇。因此，对酿酒酵母进行了基因改造，目的是使绿色木霉的BGLI基因在细胞外异源表达，使其能够利用纤维二糖生产乙醇。
绿色木霉BGLI基因的cDNA和基因组序列被克隆到酵母表达载体pGAPZα中，并分别转化到酿酒酵母中。BGLI cDNA克隆的大小为1363 bp，基因组DNA克隆在第一个外显子之后还包含一个76 bp的单一内含子。该基因与深绿木霉1,4-β-D-葡萄糖苷酶（AC237343.1）的DNA序列相似度为90%，与推导的氨基酸序列相似度为99%。重组基因组克隆表达的BGLI活性比cDNA克隆（5×10 IU/ml）高3.4倍（1.7×10 IU/ml）。此外，该活性与本地分离的绿色木霉（1.5×10 IU/ml）的活性相似。估计该蛋白质的大小为52 kDa。在发酵研究中，基因组克隆和cDNA克隆的最大乙醇产量分别为0.36 g和0.06 g/克纤维二糖。分子对接结果表明，裸蛋白和纤维二糖-蛋白复合物在水性介质中表现出相似的行为，具有相当的稳定性。构建的同源模型的推导结合位点和结合亲和力似乎是合理的。此外，还确定了BGLI氨基酸残基与纤维二糖之间形成的五个氢键主要参与酶-底物结合的完整性。
与cDNA克隆相比，基因组DNA克隆中的BGLI活性显著更高。重组酿酒酵母基因组DNA克隆成功地将纤维二糖发酵成乙醇。由于它能够同时糖化和发酵纤维二糖，因此有潜力用于乙醇的工业生产。同源建模、对接研究和分子动力学模拟研究将为进一步研究活性位点残基的修饰提供一个现实的模型，随后可进行突变研究以改善BGLI的催化作用。
