Substrate docking and molecular dynamic simulation for prediction of fungal enzymes from Trichoderma species-assisted extraction of nanocellulose from oil palm leaves

Abstract

Fungi of the Trichoderma species are valued industrial enzymes in support of the ‘zero-waste’ technology to convert agro-industrial biomass into valuable products, i.e. nanocellulose (NC). In this study, an in silico approach using substrate docking and molecular dynamic (MD) simulation was used to predict the order of which the multilayers of cellulosic polymers, i.e. lignin, hemicellulose and cellulose in oil palm leaves (OPL) are degraded by fungal enzymes, endocellulase and exocellulase. The study aimed to establish the catalytic tendencies of the enzymes to optimally degrade the cellulosic components of OPL for high yield production of NC. Energy minimized endocellulase and exocellulase models revealed satisfactory scores of PROCHECK (90.0% and 91.2%), Verify3D (97.23% and 98.85%) and ERRAT (95.24% and 91.00%) assessments. Active site prediction by blind docking, COACH meta-server and multiple sequence alignment indicated the catalytic triads for endocellulase and exocellulase were Ser116–His205–Glu249 and Ser382–Arg124–Asp385, respectively. Binding energy of endocellulase docked with hemicellulose (?6.0 ? kcal mol^?1) was the most favourable followed by lignin (?5.6 ? kcal mol^?1) and cellulose (?4.4 ? kcal mol^?1). Exocellulase, contrarily, bonded favorably with lignin (?8.7 ? kcal mol^?1), closely followed by cellulose (?8.5 ? kcal mol^?1) and hemicellulose (?8.4 ? kcal mol^?1). MDs simulations showed that interactions of complexes, endocellulase–hemicellulose and the exocellulase–cellulose being the most stable. Thus, the findings of the study successfully identified the specific actions of sugar-acting enzymes for NC production.

Communicated by Ramaswamy H. Sarma     

Note Floristiche Valdarnesi

Abstract

The involvement of cellulase (endo-b-l,4-glucanase, EC 3.2.1.4) in a number of different cell separation events which occur in higher plants has been well established. Besides their significance for the plant growth and differentiation, these events can be economically important since they also comprise softening of fleshy fruits and abscission of fruits, flowers and leaves. In higher plants cellulase is present in a number of different biochemical isoforms which are encoded by different genes. This finding is in accordance with the wide range of physiological events which require the intervention of cellulase activity, and whose peculiarities and amplitudes can be quite various.     

Convenient and Efficient Syntheses of Oligodeoxyribonucleotides Containing O 6-(Carboxymethyl)Guanine and O 6-(4-Oxo-4-(3-Pyridyl)Butyl)Guanine

O ⁶-(carboxymethyl)guanine (O ⁶-CMG) and O ⁶-(4-oxo-4-(3-pyridyl)butyl)guanine (O ⁶-pobG) are toxic lesions formed in DNA following exposure to alkylating agents. O ⁶-CMG results from exposure to nitrosated glycine or nitrosated bile acid conjugates and may be associated with diets rich in red meat. O ⁶-pobG lesions are derived from alkylating agents found in tobacco smoke. Efficient syntheses of oligodeoxyribonucleotides (ODNs) containing O ⁶-CMG and O ⁶-pobG are described that involve nucleophilic displacement by the appropriate alcohol on a common synthetic ODN containing the reactive base 2-amino-6-methylsulfonylpurine. ODNs containing O ⁶-pobG and O ⁶-CMG were found to be good substrates for the S. pombe alkyltransferase-like protein Atl1.

[Supplemental materials are available for this article. Go to the publisher's online edition of Nucleosides, Nucleotides & Nucleic Acids to view the free supplemental file.]     

Effects of adsorption properties and mechanical agitation of two detergent cellulases towards cotton cellulose

Abstract

The impacts of two hybrid cloned commercial cellulases designed for detergency on cotton fibres were compared. HiCel45 has a family 45 catalytic domain and a fungal cellulose binding module (CBM) from the fungus Humicola insolens. BaCel5 has a family 5 catalytic domain and a fungal CBM from Bacillus spp. BaCel5 bound irreversibly to cellulose under the buffer conditions tested while HiCel45 was found to bind reversibly to cellulose because it showed low adsorption. BaCel5 seems to yield more activity towards cotton than HiCel45 under mild stirring conditions, but under strong mechanical agitation both enzymes produce similar amount of sugars. HiCel45 had a more progressive production of residual reducing ends on the fabric than BaCel5. These studies seem to indicate that HiCel45 is a more cooperative enzyme with detergent processes where high mechanical agitation is needed.     

兼性厌氧纤维素降解菌的筛选和产酶研究

从我国天津地区堆肥中分离筛选出一株兼性厌氧的纤维素分解细菌D1,初步鉴定为纤维单孢菌(Cellulomonas sp.)。产酶最适碳源为葡萄糖,氮源为复合蛋白胨,酶的最适作用温度和pH值是50℃和6．4,在70℃以下和pH在5．2～8．4里稳定。     

酶法提取杜仲叶中绿原酸工艺研究

采用纤维素酶法提取杜仲叶中的主要药效成分———苯丙素类的绿原酸(CHA),通过单因素试验、正交试验和方差分析确定了纤维素酶法提取杜仲叶中绿原酸的最佳操作条件。结果表明,加入550 U/g纤维素酶0.50%,pH 4.5,温度40℃,提取率最高可达到51.84 mg/g。     

黄瓜膨胀素的重组表达及活性分析

目的:提高纤维素的酶水解效率和开发高效的纤维素酶水解过程。方法:采用RT-PCR方法从黄瓜胚轴细胞中分离了膨胀素S1的cDNA,并使之与毕赤酵母表达质粒pPICZ(A连接,形成重组质粒pPICZ(A-exs1。通过电转化方法,用质粒pPICZ(A-exs1转化巴氏毕赤酵母GS115,得到重组菌株P.pastoris-exs1。在该重组菌株中,膨胀素的基因通过同源重组整合在毕赤酵母的染色体上,并处于毕赤酵母甲醇氧化酶启动子的下游。重组菌株P.pastoris-exs1在甲醇诱导下可合成并分泌膨胀素。结果:培养上清液没有纤维素酶活性,但具有破坏滤纸纤维素结晶结构的能力。培养上清液与里氏木霉纤维素酶等量混合后,可使纤维素酶的滤纸酶活力提高50%。结论:采用巴氏毕赤酵母GS115重组成功表达了黄瓜膨胀素,其表达产物可以促进纤维素酶对滤纸的水解。     

红色荧光蛋白在丝状真菌里氏木霉中的表达

目的:建立红色荧光蛋白在里氏木霉中的表达方法,为深入研究里氏木霉中纤维素酶的合成机理打下基础。方法:采用PCR方法分离了里氏木霉纤维二糖水解酶Ⅰ(CBHⅠ)的启动子(Pcbh1)和终止序列(Tcbh1),将这两个片段与红色荧光蛋白(DsRed)的基因连接,得到Pcbh1-DsRed-Tcbh1表达盒。用此表达盒和质粒pAN7-1对里氏木霉QM9414的原生质体进行共转化,并用含100μg/ml潮霉素B的选择性平板进行筛选。结果:经筛选得到20个抗性转化子,在乳糖的诱导下有5个转化子可以表达红色荧光蛋白。对插入片段进行了扩增和序列测定,结果表明DsRed通过同源重组整合到了转化子的基因组DNA上,并处于cbh1启动子的下游。结论:通过cbh1启动子可以实现红色荧光蛋白在里氏木霉细胞内的稳定表达。     

我国秸秆生物转化燃料酒精研究现状

重点介绍了我国自主技术开发的秸杆预处理、产酶菌的诱变育种、纤维素酶活性提高技术、酶系组分的现代生物技术应用、酿酒酵母工程菌的构建和发酵工艺、酒精脱水等新技术应用,展望了秸杆生物转化酒精的可能的工业化途径。