Identification of Glyceryl MonoRicinoleate (GMR) isomers using RP-HPLC and regio-specificity of lipases.

In the present study, we report a reverse-phase high-performance liquid chromatography (RP-HPLC) method for separation of the regio-isomers of Glyceryl MonoRicinoleate (GMR) identified using position specificity of lipases. The approaches explored to identify these regio-isomers include LC-mass spectrometry, UV spectroscopy, and selective hydrolysis with lipases. A distinct UV absorption spectrum and λmax values for each isomer were noted, and mass spectral analysis further revealed their molecular weight. Lastly, the purified regio-isomers were subjected to hydrolysis with two distinctive regio-specific lipases to identified as sn-2 and sn-1(3) GMR. The current methodology of using analytic tool and enzyme specificity provides a useful platform for identifying regio-isomers for structured lipid synthesis.

Engineering Geobacillus thermoglucosidasius for direct utilisation of holocellulose from wheat straw.

BACKGROUND: A consolidated bioprocessing (CBP), where lignocellulose is converted into the desired product(s) in a single fermentative step without the addition of expensive degradative enzymes, represents the ideal solution of renewable routes to chemicals and fuels. Members of the genus Geobacillus are able to grow at elevated temperatures and are able to utilise a wide range of oligosaccharides derived from lignocellulose. This makes them ideally suited to the development of CBP. RESULTS: In this study, we engineered Geobacillus thermoglucosidasius NCIMB 11955 to utilise lignocellulosic biomass, in the form of nitric acid/ammonia treated wheat straw to which expensive hydrolytic enzymes had not been added. Two different strains, BZ9 and BZ10, were generated by integrating the cglT (ß-1,4-glucosidase) gene from Thermoanaerobacter brockii into the genome, and localising genes encoding different cellulolytic enzymes on autonomous plasmids. The plasmid of strain BZ10 carried a synthetic cellulosomal operon comprising the celA (Endoglucanase A) gene from Clostridium thermocellum and cel6B (Exoglucanase) from Thermobifida fusca; whereas, strain BZ9 contained a plasmid encoding the celA (multidomain cellulase) gene from Caldicellulosiruptor bescii. All of the genes were successfully expressed, and their encoded products secreted in a functionally active form, as evidenced by their detection in culture supernatants by Western blotting and enzymatic assay. In the case of the C. bescii CelA enzyme, this is one of the first times that the heterologous production of this multi-functional enzyme has been achieved in a heterologous host. Both strains (BZ9 and BZ10) exhibited improved growth on pre-treated wheat straw, achieving a higher final OD600 and producing greater numbers of viable cells. To demonstrate that cellulosic ethanol can be produced directly from lignocellulosic biomass by a single organism, we established our consortium of hydrolytic enzymes in a previously engineered ethanologenic G. thermoglucosidasius strain, LS242. We observed approximately twofold and 1.6-fold increase in ethanol production in the recombinant G. thermoglucosidasius equivalent to BZ9 and BZ10, respectively, compared to G. thermoglucosidasius LS242 strain at 24 h of growth. CONCLUSION: We engineered G. thermoglucosidasius to utilise a real-world lignocellulosic biomass substrate and demonstrated that cellulosic ethanol can be produced directly from lignocellulosic biomass in one step. Direct conversion of biomass into desired products represents a new paradigm for CBP, offering the potential for carbon neutral, cost-effective production of sustainable chemicals and fuels.

Specific fusion of ß-1,4-endoglucanase and ß-1,4-glucosidase enhances cellulolytic activity and helps in channeling of intermediates.

Identification and design of new cellulolytic enzymes with higher catalytic efficiency are a key factor in reducing the production cost of lignocellulosic bioalcohol. We report here identification of a novel ß-glucosidase (Gluc1C) from Paenibacillus sp. strain MTCC 5639 and construction of bifunctional chimeric proteins based on Gluc1C and Endo5A, a ß-1,4-endoglucanase isolated from MTCC 5639 earlier. The 448-amino-acid-long Gluc1C contained a GH superfamily 1 domain and hydrolyzed cellodextrin up to a five-sugar chain length, with highest efficiency toward cellobiose. Addition of Gluc1C improved the ability of Endo5A to release the reducing sugars from carboxymethyl cellulose. We therefore constructed six bifunctional chimeric proteins based on Endo5A and Gluc1C varying in the positions and sizes of linkers. One of the constructs, EG5, consisting of Endo5A-(G(4)S)(3)-Gluc1C, demonstrated 3.2- and 2-fold higher molar specific activities for ß-glucosidase and endoglucanase, respectively, than Gluc1C and Endo5A alone. EG5 also showed 2-fold higher catalytic efficiency than individual recombinant enzymes. The thermal denaturation monitored by circular dichroism (CD) spectroscopy demonstrated that the fusion of Gluc1C with Endo5A resulted in increased thermostability of both domains by 5°C and 9°C, respectively. Comparative hydrolysis experiments done on alkali-treated rice straw and CMC indicated 2-fold higher release of product by EG5 than that by the physical mixture of Endo5A and Gluc1C, providing a rationale for channeling of intermediates. Addition of EG5 to a commercial enzyme preparation significantly enhanced release of reducing sugars from pretreated biomass, indicating its commercial applicability.

One step flow-through adsorptive purification of tubulin from tissue homogenate.

Tubulin, a potential target for anti-cancer drugs, has been purified in one step and obtained as flow-through fraction directly from an extract of a mammalian brain tissue by adsorption chromatography on H-CELBEADS, an indigenously developed rigid, superporous cross-linked cellulose based weakly hydrophobic adsorbent. The fibrous polymerized tubulin mass passed through the H-CELBEADS bed while the associated proteins were separated by adsorption. The final tubulin preparation was obtained free from other proteins as seen on SDS-PAGE. Purified tubulin was obtained in a yield of about 29 mg/100 g brain, and its bioactivity, evaluated through its ability to bind colchicine, was found to be preserved.