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     

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.     

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.     

人工锌指蛋白介导调控的里氏木霉纤维素酶生产

里氏木霉Trichoderma reesei Rut-C30是目前研究最广泛的纤维素酶生产菌,选育高产纤维素酶的里氏木霉菌株有助于提高木质纤维素资源生物炼制的经济性。利用人工锌指蛋白文库转化T.reeseiRut-C30,筛选获得了两株高产纤维素酶的突变株T. reesei M1和M2,与出发菌株比较,突变株M1和M2滤纸酶活分别提高100%和53%,且M1突变株外泌蛋白量提高69%,M2内切纤维素酶活提高64%。实时定量PCR分析结果表明,与对照菌株相比,突变株M1和M2中主要纤维素酶基因转录均上调,但不同酶基因在两株菌中有不同的变化特征。此外,纤维素酶抑制转录因子基因ace1在两株突变株中都转录下调,而纤维素酶正调控转录因子基因xyr1仅在M1突变株中上调。以上结果表明,不同人工锌指蛋白对纤维素酶活性的影响具有多样性。对这些突变体中人工锌指蛋白靶基因进行深入分析,为进一步深入探究里氏木霉纤维素酶合成调控的机理,以及利用代谢工程选育更高效的产酶菌株提供了基础。     

Temperature Effects on Kinetic Parameters and Substrate Affinity of Cel7A Cellobiohydrolases

We measured hydrolytic rates of four purified cellulases in small increments of temperature (10–50 °C) and substrate loads (0–100 g/liter) and analyzed the data by a steady state kinetic model that accounts for the processive mechanism. We used wild type cellobiohydrolases (Cel7A) from mesophilic Hypocrea jecorina and thermophilic Rasamsonia emersonii and two variants of these enzymes designed to elucidate the role of the carbohydrate binding module (CBM). We consistently found that the maximal rate increased strongly with temperature, whereas the affinity for the insoluble substrate decreased, and as a result, the effect of temperature depended strongly on the substrate load. Thus, temperature had little or no effect on the hydrolytic rate in dilute substrate suspensions, whereas strong temperature activation (Q₁₀ values up to 2.6) was observed at saturating substrate loads. The CBM had a dual effect on the activity. On one hand, it diminished the tendency of heat-induced desorption, but on the other hand, it had a pronounced negative effect on the maximal rate, which was 2-fold larger in variants without CBM throughout the investigated temperature range. We conclude that although the CBM is beneficial for affinity it slows down the catalytic process. Cel7A from the thermophilic organism was moderately more activated by temperature than the mesophilic analog. This is in accord with general theories on enzyme temperature adaptation and possibly relevant information for the selection of technical cellulases.     

产纤维素酶黏细菌在生物转化的作用

目的:预处理对木质纤维素降解的影响.方法:从土壤中分离筛选到高纤维素酶活的黏细菌菌株So ce sh1008.该菌具有CMC酶活(CMCase)及微晶纤维素酶活性.研究NaOH联合黏细菌降解盐蒿、稻草、棉花秸秆和甘蔗渣四种木质纤维素的情况.结果:碱(2％ NaOH) -黏细菌处理的方法优于黏细菌-碱的方法,其中降解棉花秸秆降解效果最明显,以5.0g木质纤维素为原料,其最终干重损失达2.1g,溶液中总糖含量和还原糖含量均值分别为12.8 mg/mL和0.93 mg/mL.酵母菌发酵产乙醇的研究结果表明,最佳发酵时间为47h,碱-黏细菌甘蔗渣降解液发酵效果最好,乙醇产出达6.0％.结论:黏细菌联合2％ NaOH能有效降解甘蔗渣,提高乙醇产量.     

绿色木霉粗纤维素酶液水解壳聚糖的研究

利用自制绿色木霉粗纤维素酶液降解壳聚糖制备低聚壳聚糖.采用粘度法、乙酰丙酮法和还原糖浓度分析,研究了温度、pH值及反应时间等因素对壳聚糖水解程度和产物相对分子质量的影响,并采用质谱法对水解产物进行定性分析.结果表明,粗纤维素酶液水解壳聚糖作用的最适pH为5.0、最适反应温度为50 ℃、最适反应时间为12 h.粗纤维素酶...     

微生物资源库中产纤维素酶菌株的筛选及其酶学性质研究

利用平板碘液染色法对中国高校工业微生物资源与信息中心(CICIM-CU)资源库中保藏的625株细菌和放线菌进行筛选,从中筛出112株具有纤维素酶产生能力的菌株,建立了一个产纤维素酶微生物资源库.对筛选得到的纤维素酶产生菌株进行16S rDNA鉴定后发现,其中的Streptomyces tanashiensis、Paen...