Haq Ikram Ul, Tahir Syed Fahad, Aftab Muhammad Nauman, Akram Fatima, Nawaz Ali, Mukhtar Hamid
Institute of Industrial Biotechnology, Government College University, Lahore 54000, Pakistan.
Protein Pept Lett. 2018;25(11):1003-1014. doi: 10.2174/0929866525666181108101824.
Cellulose, being the most abundant biopolymer found in nature, can be utilized for bioethanol production to cater the future energy needs. Due to increased usage of fossil fuel it has been predicted that fossil fuel reserves may be depleted by year 2050. These concerns need serious attention and focus should be diverted to renewable fuels that are based on natural resources. Cellulases including exoglucanase (cellobiohydrolases) are the key enzymes that are produced by cellulolytic micro-organisms for the biodegradation of natural resource (cellulose) into fermentable reducing sugars. Many members of genus Clostridium possess supramolecular structures known as cellulosomes which contain various cellulases. Cellulase are composed of catalytic subunits that include endoglucanase, β-glucosidase and cellobiohydrolases which concurrently can catalyse and subsequently convert cellulose into glucose and other sugars. After the action of cellulases, the sugars can be conveniently converted into bioethanol.
In the present study, characterization of a thermostable cellobiohydrolase enzyme from Thermotoga petrophila was carried out. The main purpose of this study is the utilization of thermostable cellobiohydrolase along with other cellulases in the process of saccharification of the cellulosic biomass to produce fermentable sugars that could in turn be converted into bioethanol which is the fuel of the future.
In this article, we propose a framework for achieving our a forementioned object. We started with the cloning of thermophilic cellobiohydrolase gene in mesophilic hosts to ease enzyme production. After cloning of cellobiohydrolase gene, submerged fermentation was performed for intracellular enzyme production. Microbial pellet obtained after centrifugation was sonicated and subjected to ammonium sulphate precipitation. The fraction obtained was purified to isoelectric homogeneity through ion exchange chromatography. Finally SDS analysis of purified cellobiohydrolase was carried out alongwith its characterization, kinetics and thermodynamics studies.
Purification fold of 4.05 was obtained along with enzyme activity and specific activity of 11.5 U ml-1 min-1 and 66.5 U mg-1, respectively. The molecular mass of purified recombinant enzyme was 37 kDa as calculated by means of SDS-PAGE analysis. The enzyme showed 50% residual activity at 90°C and also at a wide pH range of 4-10. The enzyme retained its activity in the presence of most of the metal ions except Fe+2, Hg+2 and Pb+2. EDTA has an inhibitory effect on the function of the enzyme. The catalytic activity of the enzyme was maintained in the presence of the organic solvents. The enzyme had a Km and Vmax of 4.6 mM and 25.64±1.87 µM min-1 for PNP-β- D-cellobioside under optimal conditions.
The present study demonstrated that cellobiohydrolase produced from Thermotoga petrophila can be employed in many industries like paper and pulp and food processing. Most recent application of the cellobiohydrolases is their utilization in the production of bioethanol.
纤维素是自然界中最丰富的生物聚合物，可用于生产生物乙醇以满足未来的能源需求。由于化石燃料使用量的增加，预计到2050年化石燃料储备可能会耗尽。这些问题需要认真关注，应将重点转向基于自然资源的可再生燃料。包括外切葡聚糖酶（纤维二糖水解酶）在内的纤维素酶是纤维素分解微生物产生的关键酶，用于将自然资源（纤维素）生物降解为可发酵的还原糖。梭菌属的许多成员拥有称为纤维小体的超分子结构，其中包含各种纤维素酶。纤维素酶由催化亚基组成，包括内切葡聚糖酶、β-葡萄糖苷酶和纤维二糖水解酶，它们可以同时催化并随后将纤维素转化为葡萄糖和其他糖类。在纤维素酶作用后，糖类可方便地转化为生物乙醇。
在本研究中，对嗜热栖热菌的一种耐热纤维二糖水解酶进行了表征。本研究的主要目的是在纤维素生物质糖化过程中利用耐热纤维二糖水解酶和其他纤维素酶来生产可发酵糖，这些糖进而可转化为未来的燃料生物乙醇。
在本文中，我们提出了一个实现上述目标的框架。我们首先在嗜温宿主中克隆嗜热纤维二糖水解酶基因以简化酶的生产。克隆纤维二糖水解酶基因后，进行深层发酵以生产细胞内酶。离心后获得的微生物沉淀进行超声处理并进行硫酸铵沉淀。通过离子交换色谱将获得的级分纯化至等电均一性。最后对纯化的纤维二糖水解酶进行SDS分析以及其表征、动力学和热力学研究。
获得了4.05的纯化倍数，酶活性和比活性分别为11.5 U ml-1 min-1和66.5 U mg-1。通过SDS-PAGE分析计算，纯化的重组酶的分子量为37 kDa。该酶在90°C以及4 - 10的宽pH范围内显示出50%的残余活性。除Fe+2、Hg+2和Pb+2外，该酶在大多数金属离子存在下仍保持其活性。EDTA对该酶的功能有抑制作用。在有机溶剂存在下该酶的催化活性得以维持。在最佳条件下，该酶对对硝基苯基-β-D-纤维二糖苷的Km和Vmax分别为4.6 mM和25.64±1.87 μM min-1。
本研究表明，嗜热栖热菌产生的纤维二糖水解酶可用于许多行业，如造纸和制浆以及食品加工。纤维二糖水解酶的最新应用是它们在生物乙醇生产中的利用。
