Engineering Escherichia coli cell Factories for continuous 5'-cytidine monophosphate production via biofilm-anchored dual-enzyme cascade catalysis.

The pathway of synthesis of cytidylic and uridylic acids has been tentatively established in certain microbial and animal cells according to recent reviews (14, 35, 28). The sequence of reactions is probably as follows: CO, + ATP + NH3 > carbamyl -H.,O aspartate phosphate --> carbamyl aspartate > di-2 H PRPP3 hydroorotic acid orotic acid > orotidine-5'-phosphate -> UMP + CO2. In yeast, UMP was further converted to UTP by kinases requiring ATP (23). Amination of uridine nucleotides to form cytidine nucleotides involved participation of glutamine in mammalian cells (18, 34) and NH3 in Escherichia coli (21). Neither uracil or uridine, cytosine or cytidine, are normal intermediates in synthesis of uridylic and cytidylic acids. The only pyrimidine involved is orotic acid, according to this pathway. In higher plants, uracil, uridine, and cytidine are incorporated into RNA (2, 8, 15), although incorporation of cytosine has apparently not been reported. Anabolism of -uracil may occur either by addition of ribose from ribose-1-phosphate to produce uridine (11) or by conversion directly to UMP upon reaction with PRPP (11, 13). Uridine was converted to UMP in the presence of ATP and a uridine kinase (11). The ,enzymes responsible for cytidine utilization have ap_parently not been extensively studied. Considerably less is known of the pathway of pyrimidine nucleotide synthesis in higher plants. In a preliminary communication Kapoor and Waygood (19) reported in wheat embryos the presence of the enzyme converting orotic acid to OMP, orotidine-5'phosphate pyrophosphorylase, and of dihydroortic de-hydrogenase, which forms orotate from dihydroorotate. In a similar brief report, King and Wang (20) showed that C14 from both uracil and aspartic acid labelled pyrimidines of wheat leaves. Neumann and Jones (25) rccently studied aspartic acid transcarbamylase, and demonstrated several of its properties. Buchowicz and Reifer (7) found orotic acid to be converted to UMP, CMP, uridine, and uracil in wheat seedling leaves. Carbamyl aspartate also labelled these compounds and, in addition, orotic acid (30). This work suggests a pathway of nucleotide synthesis similar to that in other organisms. However, the latter workers reported that the conversion of orotic acid to UMP involved uridine as an intermediate product (9), and were unable to detect any OMP in their wheat leaves. Buchowicz (8) also reported that uracil could be incorporated directly into the nucleotides of RNA in wheat seedling homogenates without first labelling UMP and CMP. A direct binding of uracil by a polynucleotide acceptor was suggested to explain this surprising result. The present experiments were undertaken to provide more evidence as to some of the reactions involved in synthesis of uridylic and cytidylic acids in higher plants and to extend the results to plants other than wheat. Quantitative comparisons of the rate of incorporation of certain precursors into RNA were made and several metabolites were identified, including OMP.

An Escherichia coli strain expressing three recombinant enzymes, i.e., cytidine 5'-monophosphate (CMP) kinase, sialic acid aldolase and cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase, was utilized as a biocatalyst for the production of CMP-NeuAc. Both recombinant E. coli extract and whole cells catalyzed the production of CMP-NeuAc from CMP (20 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetylphosphate (60 mM), resulting in 90% conversion yield based on initial CMP concentration used. It was confirmed that endogenous acetate kinase can catalyze not only the ATP regeneration in the conversion of CMP to CDP but also the conversion of CDP to CTP. On the other hand, endogenous pyruvate kinase and polyphosphate kinase could not regenerate ATP efficiently. The addition of exogenous acetate kinase to the reaction mixture containing the cell extract increased the conversion rate of CMP to CMP-NeuAc by about 1.5-fold, but the addition of exogenous inorganic pyrophosphatase had no influence on the reaction. This E. coli strain could also be employed as an enzyme source for in situ regeneration of CMP-NeuAc in a sialyltransferase catalyzed reaction. About 90% conversion yield of alpha2,3-sialyl-N-acetyllactosamine was obtained from N-acetyllactosamine (20 mM), CMP (2 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetyl phosphate (80 mM) using the recombinant E. coli extract and alpha2,3-sialyltransferase.

Cytidine 5′-monophosphate(CMP) was widely applied in thefood and pharmaceutical industries. Currently, CMP is mainly producedby enzyme catalysis. However, the starting materials for enzyme catalysiswere relatively expensive. Therefore, seeking a low-cost productionprocess for CMP was attractive. In this study, Escherichiacoli (E. coli) wassystematically modified to produce CMP. First, a the cytidine-producingstrain was constructed by deleting cdd, rihA, rihB, and rihC. Second, the genesinvolved in the pyrimidine precursor competing pathway and negativeregulation were deleted to increase cyti dine biosynthesis. Third,the deletion of the genes that caused the loss of CMP phosphataseactivity led to the accumulation of CMP, and the overexpression ofthe rate-limiting step genes and feedback inhibition resistance genesgreatly increased the yield of CMP. The yield of CMP was further increasedto 1013.6 mg/L by blocking CMP phosphorylation. Ultimately, the yieldof CMP reached 15.3 g/L in a 50 L bioreactor. Overall, the engineered E. coli with a high yield of CMP was successfullyconstructed and showed the potential for industrial production.

Citric acid is widely used in the food, chemical and pharmaceutical industries. Aspergillus niger is the workhorse used for citric acid production in industry. A canonical citrate biosynthesis that occurred in mitochondria was well established; however, some research suggested that the cytosolic citrate biosynthesis pathway may play a role in this chemical production. Here, the roles of cytosolic phosphoketolase (PK), acetate kinase (ACK) and acetyl-CoA synthetase (ACS) in citrate biosynthesis were investigated by gene deletion and complementation in A. niger. The results indicated that PK, ACK and ACS were important for cytosolic acetyl-CoA accumulation and had significant effects on citric acid biosynthesis. Subsequently, the functions of variant PKs and phosphotransacetylase (PTA) were evaluated, and their efficiencies were determined. Finally, an efficient PK-PTA pathway was reconstructed in A. niger S469 with Ca-PK from Clostridium acetobutylicum and Ts-PTA from Thermoanaerobacterium saccharolyticum. The resultant strain showed an increase of 96.4% and 88% in the citrate titer and yield, respectively, compared with the parent strain in the bioreactor fermentation. These findings indicate that the cytosolic citrate biosynthesis pathway is important for citric acid biosynthesis, and increasing the cytosolic acetyl-CoA level can significantly enhance citric acid production.

Surface display of carbonic anhydrase on Escherichia coli for CO2 capture and mineralization

Nucleoside triphosphates and deoxynucleoside triphosphates are important biochemical molecules. In this study, recombinant Escherichia coli that could display nucleotide kinases (INP-N-NMKases) and acetate kinase (INP-N-ACKase) on the cell surface were constructed by fusing an enzyme (NMKase/ACKase) to the N-terminus of ice nucleation protein (INP-N). By using intact recombinant bacteria cells as a catalyst coupled with an ACKase-catalyzed adenosine-5'-triphosphate (ATP) regeneration system, nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) could be synthesized efficiently. In a reaction system with 5mmol/l substrate, the conversion rates of cytidine-5'-triphosphate (CTP) and deoxycytidine-5'-triphosphate (dCTP) were 96% and 93%, respectively, the conversion rate of ATP and deoxyadenosine-5'-triphosphate (dATP) was 96%, the conversion rate of deoxythymidine-5'-triphosphate (dTTP) was 91%, and the conversion rate of uridine-5'-triphosphate (UTP) was 80%. There was no obvious degradation. At 37°C, the stability of the surface-displayed fusion protein, especially in the presence of the substrate, was significantly improved. Each whole cell could be reused more than 8 times.

Immobilization and stabilization of α-galactosidase on Sepabeads EC-EA and EC-HA

In the fast-developing fields of pharmaceutical research and industry, the implementation of Raman spectroscopy and related technologies has been very well received due to the combination of chemical selectivity and the option for non-invasive analysis of samples. This chapter explores established and potential applications of Raman spectroscopy, confocal Raman microscopy and related techniques from the early stages of drug development research up to the implementation of these techniques in process analytical technology (PAT) concepts for large-scale production in the pharmaceutical industry. Within this chapter, the implementation of Raman spectroscopy in the process of selection and optimisation of active pharmaceutical ingredients (APIs) and investigation of the interaction with excipients is described. Going beyond the scope of early drug development, the reader is introduced to the use of Raman techniques for the characterization of complex drug delivery systems, highlighting the technical requirements and describing the analysis of qualitative and quantitative composition as well as spatial component distribution within these pharmaceutical systems. Further, the reader is introduced to the application of Raman techniques for performance testing of drug delivery systems addressing drug release kinetics and interactions with biological systems ranging from single cells up to complex tissues. In the last part of this chapter, the advantages and recent developments of integrating Raman technologies into PAT processes for solid drug delivery systems and biologically derived pharmaceutics are discussed, demonstrating the impact of the technique on current quality control standards in industrial production and providing good prospects for future developments in the field of quality control at the terminal part of the supply chain and various other fields like individualized medicine. On the way from the active drug molecule (API) in the research laboratory to the marketed medicine in the pharmacy, therapeutic efficacy of the active molecule and safety of the final medicine for the patient are of utmost importance. For each step, strict regulatory requirements apply which demand for suitable analytical techniques to acquire robust data to understand and control design, manufacturing and industrial large-scale production of medicines. In this context, Raman spectroscopy has come to the fore due to the combination of chemical selectivity and the option for non-invasive analysis of samples. Following the technical advancements in Raman equipment and analysis software, Raman spectroscopy and microscopy proofed to be valuable methods with versatile applications in pharmaceutical research and industry, starting from the analysis of single drug molecules as well as complex multi-component formulations up to automatized quality control during industrial production.

Asparaginase conjugated magnetic nanoparticles used for reducing acrylamide formation in food model system

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.

The stabilization effect of ‘bilayer encagement’ on enzymes from mesophilic organisms and their ‘thermophilic’ counterparts was compared. Lactate dehydrogenases from pig heart and from a thermophilic bacterium (Clostridium thermohydrosulfuricum), respectively, showed stabilization factors of 4·5 (at 47°C) and 12·8 (at 70°C), respectively. For ‘thermophilic’ acetate kinase no stabilization effect of encagement was observed. Lactate dehydrogenase and acetate kinase from Clostridium thermohydrosulfuricum were immobilized to controlled porous glass and tested for their long-term stability. The ‘thermophilic’ enzymes showed by far a longer half-life as compared with the corresponding enzymes from pig heart and Escherichia coli, respectively, the half-life time of the flow injection system response with thermophilic lactate dehydrogenase at 35°C attaining 250 h (mesophilic enzyme 89 h), and with thermophilic acetate kinase 79 h (mesophilic enzyme 24 h). © 1997 SCI.

The rapid development and the economic growth of oil palm industry have exacerbated environmental pollution since it results in large quantity of waste products produced from the oil extraction process. In many cases, however, low catalytic efficiency and instability of enzymes were considered as barriers for the development of large-scale operations and applications. In this study, hydrolysis of oil palm empty fruit bunch to reducing sugar was achieved using cellulase enzyme immobilized on multi-wall carbon nanotubes. The hydrolysis evaluation was performed using free cellulase enzyme. The amount of reducing sugar concentration released during the reaction was determined using dinitrosalicylic acid (DNS) assay. Using the immobilized form of the enzyme, it was found that the reducing sugar concentration was increased and the enzymatic hydrolysis of oil palm empty fruit bunch was enhanced greatly over long time of hydrolysis. Key words: Oil palm empty fruit bunch, enzymatic hydrolysis, cellulase enzyme, immobilized cellulase enzyme

The demand for synthetic flavor ester is high, especially in the food, beverage, and cosmetic and pharmaceutical industries. It is derived from the reaction between a short-chain fatty acid and alcohol. Lipases from Antarctic bacteria have gained huge interest in the industry due to its ability react at low temperatures. The use of immobilization enzymes is one of the methods that can improve the stability of the enzyme. The current work encompasses the low temperature enzymatic synthesis of ethyl hexanoate by direct esterification of ethanol with hexanoic acid in a toluene and solvent-free system. The effects of various reaction parameters such as the organic solvent, temperature, time, substrate, substrate ratio and concentration, enzyme concentration on ethyl hexanoate synthesis were tested. Several matrices were used for immobilization and comparisons of the efficiency of immobilized enzyme with free enzyme in the synthesis of flavor ester were conducted. Ester production was optimally synthesized at 20 °C in both systems— immobilized and free enzyme. A 69% ester conversion rate was achieved after a two-hour incubation in toluene, compared to 47% in a solvent-free system for free enzyme. Immobilized AMS8 lipase showed a higher conversion of ester in toluene with respect to free-solvents, from 80% to 59%, respectively. Immobilized enzymes showed enhancement to the stability of the enzyme in the presence of the organic solvent. The development of AMS8 lipase as an immobilized biocatalyst demonstrates great potential as a cost-effective enzyme for biocatalysis and biotransformation in the food industry.

A global tendency for products considered environmentally sustainable, and ecologically obtained led the industry related to personal care formulations to fund the research and the development of personal care/cosmetics containing ingredients from natural resources. Furthermore, consumers are aware of environmental and sustainability issueans, thus not harming the environment represents a key consideration when developing a new cosmetic ingredient. In this study we review some examples of active ingredients or raw materials used in cosmetics/personal care/biomedical products that are coming from either through biotechnological systems, or as byproducts of several industries. A skin formulation containing biosynthetic actives, prepared by us and the study regarding its dermocosmetic properties are also described. The need for the standardization processes, the safety assessment tools, the improvement of the exploitation methods of these renewable sources in order the production to be ecologically and economically better are also discussed.

Functional expression of mammalian NADPH–cytochrome P450 oxidoreductase on the cell surface of Escherichia coli