Thermostable Artificial Enzyme Isolated by In Vitro Selection

Artificial enzymes hold the potential to catalyze valuable reactions not observed in nature. One approach to build artificial enzymes introduces mutations into an existing protein scaffold to enable a new catalytic activity. This process commonly results in a simultaneous reduction of protein stability as an undesired side effect. While protein stability can be increased through techniques like directed evolution, care needs to be taken that added stability, conversely, does not sacrifice the desired activity of the enzyme. Ideally, enzymatic activity and protein stability are engineered simultaneously to ensure that stable enzymes with the desired catalytic properties are isolated. Here, we present the use of the in vitro selection technique mRNA display to isolate enzymes with improved stability and activity in a single step. Starting with a library of artificial RNA ligase enzymes that were previously isolated at ambient temperature and were therefore mostly mesophilic, we selected for thermostable active enzyme variants by performing the selection step at 65°C. The most efficient enzyme, ligase 10C, was not only active at 65°C, but was also an order of magnitude more active at room temperature compared to related enzymes previously isolated at ambient temperature. Concurrently, the melting temperature of ligase 10C increased by 35 degrees compared to these related enzymes. While low stability and solubility of the previously selected enzymes prevented a structural characterization, the improved properties of the heat-stable ligase 10C finally allowed us to solve the three-dimensional structure by NMR. This artificial enzyme adopted an entirely novel fold that has not been seen in nature, which was published elsewhere. These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.     

Partial purification of saccharifying and cell wall-hydrolyzing enzymes from malt in waste from beer fermentation broth

A number of hydrolyzing enzymes that are secreted from malt during brewing, including cell wall-hydrolyzing, saccharide-hydrolyzing, protein-degrading, lipid-hydrolyzing, and polyphenol and thiol-hydrolyzing enzymes, are expected to exist in an active form in waste from beer fermentation broth (WBFB). In this study, the existence of these enzymes was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which enzyme extract was partially purified through a series of purification steps. The hydrolyzing enzyme activity was then measured under various conditions at each purification step using carboxymethyl cellulose as a substrate. The best hydrolyzing activities of partially purified enzymes were found at pH 4.5 and 50 °C in a citrate buffer system. The enzymes showed highest thermal stability at 30 °C when exposed for prolonged time. As the temperature increased gradually from 25 to 70 °C, yeast cells in the chemically defined medium with enzyme extract lost their cell wall and viability earlier than those without enzyme extract. Cell wall degradation and the release of cell matrix into the culture media at elevated temperature (45–70 °C) in the presence of enzyme extract were monitored through microscopic pictures. Saccharification enzymes from malt were relatively more active in the original WBFB than supernatant and diluted sediments. The presence of hydrolyzing enzymes from malt in WBFB is expected to play a role in bioethanol production using simultaneous saccharification and fermentation without the need for additional enzymes, nutrients, or microbial cells via a cell-free enzyme system.     

The potential of enzyme recycling during the hydrolysis of a mixed softwood feedstock

Despite recent improvement in cellulase enzymes properties, the high cost associated with the hydrolysis step remains a major impediment to the commercialization of full-scale lignocellulose-to-ethanol bioconversion process. As part of a research effort to develop a commercial process for bioconversion of softwood residues, we have examined the potential for recycling enzymes during the hydrolysis of mixed softwood substrate pretreated by organosolv process. We have used response surface methodology to determine the optimal temperature, pH, ionic strength, and surfactant (Tween 80) concentration for maximizing the recovery of bound protein and enzyme activity from the residual substrates after hydrolysis. Data analysis showed that the temperature, pH and surfactant concentration were the major factors governing enzyme desorption from residual substrate. The optimized conditions were temperature 44.4 °C, pH 5.3 and 0.5% Tween 80. The optimal conditions significantly increased the hydrolysis yield by 25% after three rounds of hydrolysis. This bound enzyme desorption combining with free enzyme re-adsorption is a potential method to recover cellulase enzymes and reduce the cost of enzymatic hydrolysis.     

Regeneration of reduced-denatured seminal ribonuclease: Effect of modification at cysteines 31 and 32

The temperature dependences of glutathione-facilitated regeneration of ribonuclease A and seminal ribonuclease are quite different although the two proteins are homologous. This difference in the two enzymes appears to result from the presence of two additional half-cystine residues in seminal ribonuclease. When these two cysteines are alkylated with either a neutral or positively charged blocking agent, the regeneration process becomes seemingly temperature insensitive. On the other hand, negatively charged agents are less effective in restoring normal regeneration kinetics. The modifications also render the protein more stable against thermal inactivation, a process which presumably contributes to the unusual temperature dependence of regeneration. These data reveal the potential importance of peripheral groups in the regeneration and stability of proteins. A model is proposed to explain these observations.     

Influence of Process Parameter on Campylobacter spp. Counts on Poultry Meat in a Slaughterhouse Environment

Campylobacter spp. are the most important food-borne pathogens in broilers. Exposure of the consumer can be influenced by the reduction of contaminated broiler meat at various steps along the production line. This study was performed at a poultry slaughterhouse in Germany. Steps within the slaughter process were defined by the slaughterhouse quality control for potential Campylobacter reduction. Their impact was tested for two process variations. The first process variation was the increase of the temperature of the scalding water from 53.0 to 53.9 °C. The second step was the application of an additional outside sprayer which was placed after plucking. The increase of the scalding water temperature was the most effective measure (>2 log reduction), but resulted in defects to the broiler skin. This would limit marketing of fresh broiler meat with skin. The additional water spray after plucking had no additional effect. In fact, numbers of Campylobacter were lower before introduction of the sprayer. In conclusion, modifications of the processing technology have to be evaluated carefully, but can have additional effects for Campylobacter reduction.     

Biphasic Kinetic Behavior of E. coli WrbA, an FMN-Dependent NAD(P)H:Quinone Oxidoreductase

The E. coli protein WrbA is an FMN-dependent NAD(P)H:quinone oxidoreductase that has been implicated in oxidative defense. Three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds. The present work was undertaken to evaluate these suggestions and to characterize the kinetic behavior of WrbA. Steady-state kinetics results reveal that WrbA conforms to a ping-pong mechanism with respect to the constancy of the apparent Vmax to Km ratio with substrate concentration. However, the competitive/non-competitive patterns of product inhibition, though consistent with the general class of bi-substrate reactions, do not exclude a minor contribution from additional forms of the enzyme. NMR results support the presence of additional enzyme forms. Docking and energy calculations find that electron-transfer-competent binding sites for NADH and benzoquinone present severe steric overlap, consistent with the ping-pong mechanism. Unexpectedly, plots of initial velocity as a function of either NADH or benzoquinone concentration present one or two Michaelis-Menten phases depending on the temperature at which the enzyme is held prior to assay. The effect of temperature is reversible, suggesting an intramolecular conformational process. WrbA shares these and other details of its kinetic behavior with mammalian DT-diaphorase, an FAD-dependent NAD(P)H:quinone oxidoreductase. An extensive literature review reveals several other enzymes with two-plateau kinetic plots, but in no case has a molecular explanation been elucidated. Preliminary sedimentation velocity analysis of WrbA indicates a large shift in size of the multimer with temperature, suggesting that subunit assembly coupled to substrate binding may underlie the two-plateau behavior. An additional aim of this report is to bring under wider attention the apparently widespread phenomenon of two-plateau Michaelis-Menten plots.     

Citric acid cycle biomimic on a carbon electrode

The citric acid cycle is one of the main metabolic pathways living cells utilize to completely oxidize biofuels to carbon dioxide and water. The overall goal of this research is to mimic the citric acid cycle at the carbon surface of an electrode in order to achieve complete oxidation of ethanol at a bioanode to increase biofuel cell energy density. In order to mimic this process, dehydrogenase enzymes (known to be the electron or energy producing enzymes of the citric acid cycle) are immobilized in cascades at an electrode surface along with non-energy producing enzymes necessary for the cycle to progress. Six enzymatic schemes were investigated each containing an additional dehydrogenase enzyme involved in the complete oxidation of ethanol. An increase in current density is observed along with an increase in power density with each additional dehydrogenase immobilized on an electrode, reflecting increased electron production at the bioanode with deeper oxidation of the ethanol biofuel. By mimicking the complete citric acid cycle on a carbon electrode, power density was increased 8.71-fold compared to a single enzyme (alcohol dehydrogenase)-based ethanol/air biofuel cell.     

Influence of high temperature and ethanol on thermostable lignocellulolytic enzymes

Lignocellulolytic enzymes are among the most costly part in production of bioethanol. Therefore, recycling of enzymes is interesting as a concept for reduction of process costs. However, stability of the enzymes during the process is critical. In this work, focus has been on investigating the influence of temperature and ethanol on enzyme activity and stability in the distillation step, where most enzymes are inactivated due to high temperatures. Two enzyme mixtures, a mesophilic and a thermostable mixture, were exposed to typical process conditions [temperatures from 55 to 65 °C and up to 5 % ethanol (w/v)] followed by specific enzyme activity analyses and SDS-PAGE. The thermostable and mesophilic mixture remained active at up to 65 and 55 °C, respectively. When the enzyme mixtures reached their maximum temperature limit, ethanol had a remarkable influence on enzyme activity, e.g., the more ethanol, the faster the inactivation. The reason could be the hydrophobic interaction of ethanol on the tertiary structure of the enzyme protein. The thermostable mixture was more tolerant to temperature and ethanol and could therefore be a potential candidate for recycling after distillation.     

Thermostable endoglucanases in the liquefaction of hydrothermally pretreated wheat straw

Background  

Thermostable enzymes have several benefits in lignocellulose processing. In particular, they potentially allow the use of increased substrate concentrations (because the substrate viscosity decreases as the temperature increases), resulting in improved product yields and reduced capital and processing costs. A short pre-hydrolysis step at an elevated temperature using thermostable enzymes aimed at rapid liquefaction of the feedstock is seen as an attractive way to overcome the technical problems (such as poor mixing and mass transfer properties) connected with high initial solid loadings in the lignocellulose to ethanol process.     

Gamma-irradiated chymotrypsin-like proteins. II. Connection between inactivation and structural changes.

The radiation yeilds of unfolding (Gconf) determined by the method of tryptophan fluorescence coincide with the radiation yields of proteolytic inactivation (Gin) for chymotrypsin-like (CT-like) enzymes on irradiation in air, both in solution and in the dry state with futher dissolution at pH7. It can be supposed that the unfolding is the main process determining the proteolytic gamma-inactivation of CT-like enzymes. It was also shown that the transition of chymotrypsin and trypsin gamma-irradiated at acid pH to neutral pH is an additional action, leading to unfolding of part of the molecules.     

Reconstitution of the DNA base excision—repair pathway

Background: The base excision–repair pathway is the major cellular defence mechanism against spontaneous DNA damage. The enzymes involved have been highly conserved during evolution. Base excision–repair has been reproduced previously with crude cell-free extracts of bacterial or human origin. To further our understanding of base excision–repair, we have attempted to reconstitute the pathway in vitro using purified enzymes.Results We report here the successful reconstitution of the base excision–repair pathway with five purified enzymes from Escherichia coli: uracil-DNA glycosylase, a representative of the DNA glycosylases that remove various lesions from DNA; the AP endonuclease IV that specifically cleaves at abasic sites; RecJ protein which excises a 5′ terminal deoxyribose-phosphate residue; DNA polymerase I; and DNA ligase. The reaction proceeds with high efficiency in the absence of additional factors in the reconstituted system. Four of the enzymes are absolutely required for completion of the repair reaction. An unusual feature we have discovered is that the pathway branches after enzymatic incision at an abasic DNA site. RecJ protein is required for the major reaction, which involves replacement of only a single nucleotide at the damaged site; in its absence, an alternative pathway is observed, with generation of longer repair patches by the 5′ nuclease function of DNA polymerase I.Conclusion Repair of uracil in DNA is achieved by a very short-patch excision–repair process involving five different enzymes. No additional protein factors seem to be required. There is a minor, back-up pathway that uses replication factors to generate longer repair patches.     

Structural comparison of tRNA m1A58 methyltransferases revealed different molecular strategies to maintain their oligomeric architecture under extreme conditions

Background

tRNA m¹A58 methyltransferases (TrmI) catalyze the transfer of a methyl group from S-adenosyl-L-methionine to nitrogen 1 of adenine 58 in the T-loop of tRNAs from all three domains of life. The m¹A58 modification has been shown to be essential for cell growth in yeast and for adaptation to high temperatures in thermophilic organisms. These enzymes were shown to be active as tetramers. The crystal structures of five TrmIs from hyperthermophilic archaea and thermophilic or mesophilic bacteria have previously been determined, the optimal growth temperature of these organisms ranging from 37°C to 100°C. All TrmIs are assembled as tetramers formed by dimers of tightly assembled dimers.

Results

In this study, we present a comparative structural analysis of these TrmIs, which highlights factors that allow them to function over a large range of temperature. The monomers of the five enzymes are structurally highly similar, but the inter-monomer contacts differ strongly. Our analysis shows that bacterial enzymes from thermophilic organisms display additional intermolecular ionic interactions across the dimer interfaces, whereas hyperthermophilic enzymes present additional hydrophobic contacts. Moreover, as an alternative to two bidentate ionic interactions that stabilize the tetrameric interface in all other TrmI proteins, the tetramer of the archaeal P. abyssi enzyme is strengthened by four intersubunit disulfide bridges.

Conclusions

The availability of crystal structures of TrmIs from mesophilic, thermophilic or hyperthermophilic organisms allows a detailed analysis of the architecture of this protein family. Our structural comparisons provide insight into the different molecular strategies used to achieve the tetrameric organization in order to maintain the enzyme activity under extreme conditions.     

Immobilization of α-amylase and amyloglucosidase onto ion-exchange resin beads and hydrolysis of natural starch at high concentration

α-Amylase was immobilized on Dowex MAC-3 with 88 % yield and amyloglucosidase on Amberlite IRA-400 ion-exchange resin beads with 54 % yield by adsorption process. Immobilized enzymes were characterized to measure the kinetic parameters and optimal operational parameters. Optimum substrate concentration and temperature were higher for immobilized enzymes. The thermal stability of the enzymes enhanced after the immobilization. Immobilized enzymes were used in the hydrolysis of the natural starch at high concentration (35 % w/v). The time required for liquefaction of starch to 10 dextrose equivalent (DE) and saccharification of liquefied starch to 96 DE increased. Immobilized enzymes showed the potential for use in starch hydrolysis as done in industry.     

Dynamic model for CHO cell engineering

Industrial CHO cell fed-batch processes have progressed significantly over the past decade, with recombinant protein titer consistently reaching the gram per liter level. Such improvements have largely resulted from separate advances in process and cell line development. Model-based selection of targets for metabolic engineering in CHO cells is confounded by the dynamic nature of the fed-batch process. In this work, we use a dynamic model of CHO cell metabolism to simultaneously identify both process and cell modifications that improve antibody production. Model simulations explored ca. 9200 combinations of process variables (shift temperature, shift day, seed density, and harvest day) and knockdowns (8 metabolic enzymes). A comprehensive examination of a simulated solution space showed that optimal gene knockdown clearly depends on the process parameters such as temperature shift day, shift temperature, and seed density. Knockdown of enzymes related to lactate production were the most beneficial; however, depending on the process conditions, modulating such enzymes yielded varying productivities, ranging from a reduction in final titer to greater than 2-fold improvement.     

Enzymatic synthesis of carbonate monomers and polycarbonates

Diphenyl carbonate is an attractive monomer for copolymerization with Bisphenol-A to produce the strong, high melting polycarbonate, Bisphenol-A Polycarbonate. Diphenyl carbonate is an ideal candidate for this polymerization as the phenols constitute good leaving groups during polymerization. Industrially, diphenyl carbonate is produced via the phosgenation of a phenolic sodium salt. Using phosgene creates additional safety hazards as well as concerns in treating or disposing of the reaction by-products. The enzymatic synthesis of diphenyl carbonate via alcoholysis of dimethyl carbonate by phenol is presented. While the process is environmentally benign and eliminates the considerable safety issues related to the use of phosgene, phenol is a poor nucleophile and conversion to diphenyl carbonate is limited. Enzyme catalyzed condensation polymerization of carbonate monomers and diols is a more feasible and direct enzymatic route to polycarbonate. We describe an AA-BB condensation polymerization to make polycarbonates using enzymes at ambient conditions. Molecular weights of up to 8, 500 MW are achieved. Unlike the industrial polymerization, this process is performed without the use of acid catalysts, significant energy input, or high temperature or pressure.     

Role of additional substrates in DDT degradation by cultures of Pseudomonas aeruginosa

The activity of enzymes was compared during Pseudomonas aeruginosa 640x growth on compounds activating DDT to a different degree and on acetate which did not stimulate this process. The activity of dehydrogenases generating reduced cofactors necessary for DDT dechlorination was found to be much higher on effective additional substrates than on compounds which either had no effect on DDT degradation or stimulated this process only slightly. The activity of enzymes generating the reduced cofactors was shown to correlate with the extent of DDT degradation by this culture.     

冬小麦籽粒淀粉合成相关酶活性的日变化

蔗糖向淀粉的转化是决定小麦籽粒产量的重要因素.田间条件下研究了两个小麦(Triticum aestivum L.)品种"鲁麦22"和"鲁麦14"籽粒淀粉合成相关酶:蔗糖合酶(sucrose synthase,SS)、腺苷二磷酸葡萄糖焦磷酸化酶(ADP-glucose pyrophosphorylase,ADPGPPase)、可溶性淀粉合酶(soluble starch synthase,SSS)、束缚态淀粉合酶(starch granule-bound synthase,GBSS)的活性以及籽粒ATP含量的日变化.结果表明,上述酶活性呈现明显的昼夜变化特征,酶活性一般在白天较低,而在夜间呈现较高活性,而籽粒ATP含量趋势相反.相关分析表明,白天较低的酶活性可能与气温超过其适宜温度有关.对籽粒淀粉合成相关酶活性日变化的可能因子进行了讨论.     

A fast,continuous enzyme-based pretreatment process concept for cotton containing textiles

A fast integrated enzyme-based pretreatment process concept for cotton containing textiles has been developed for operation in the continuous mode. The total processing time for the desizing and scouring operation is 3–10 minutes for fabrics with a weight of 120–300 g/m². Essential elements in the process are the high starting temperature, the presence of surfactant, application of vacuum technology and a robust rinsing process afterwards to remove the degraded pectin together with hydrophobic compounds. This rinsing procedure is realized with water containing chelator and surfactant and at high operation temperature beyond 80°C. A mixture of enzymes is used, consisting of a temperature stable α-amylase and a pectate lyase, both operating under alkaline conditions.