L-核糖的生产研究进展

L-核糖是合成许多抗病毒药物的关键中间体,自然界和生物体中并不存在。L-核糖的制备方法有化学合成法和生物合成法。与化学合成法相比,生物合成法对环境更加有利。生物合成法是应用微生物及其酶以核糖醇或L-阿拉伯糖为原料生产L-核糖。综述了L-核糖的制备方法以及产品的分离纯化技术,并对L-核糖的研究现状和前景进行了展望。     

医药中间体L-核糖的生物制造研究进展

L-核糖(L-ribose)是自然界不存在的L-型稀有糖,L-核糖是多种核苷类似物药物的核心砌块,其衍生物2-脱氧-L-核糖也是昂贵的药物中间体;过去只能通过化学合成获得,近年来开始有酶法合成的报道。本文综述了L-核糖生物制备的三种关键酶-L-阿拉伯糖异构酶(L-AI)、甘露糖-6-磷酸异构酶(MPI)、以及L-核糖异构酶(L-RI)在制备L-核糖方面的研究进展,结合产品衍生物研发进行了总结,并展望了L-核糖在未来医药行业的技术需求。     

Efficient production of L-ribose with a recombinant Escherichia coli biocatalyst

A new synthetic platform with potential for the production of several rare sugars, with l-ribose as the model target, is described. The gene encoding the unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (garden celery) was synthetically constructed for optimal expression in Escherichia coli. This MDH enzyme catalyzes the interconversion of several polyols and their l-sugar counterparts, including the conversion of ribitol to l-ribose. Expression of recombinant MDH in the active form was successfully achieved, and one-step purification was demonstrated. Using the created recombinant E. coli strain as a whole-cell catalyst, the synthetic utility was demonstrated for production of l-ribose, and the system was improved using shaken flask experiments. It was determined that addition of 50 to 500 microM ZnCl(2) and addition of 5 g/liter glycerol both improved production. The final levels of conversion achieved were >70% at a concentration of 40 g/liter and >50% at a concentration of 100 g/liter. The best conditions determined were then scaled up to a 1-liter fermentation that resulted in 55% conversion of 100 g/liter ribitol in 72 h, for a volumetric productivity of 17.4 g liter(-1) day(-1). This system represents a significantly improved method for the large-scale production of l-ribose.     

Isolation and characterization of Escherichia coli mutants able to utilize the novel pentose L-ribose.

Wild-type strains of Escherichia coli were unable to utilize L-ribose for growth. However, L-ribose-positive mutants could be isolated from strains of E. coli K-12 which contained a ribitol operon. L-ribose-positive strains of E. coli, isolated after 15 to 20 days, had a growth rate of 0.22 generation per h on L-ribose. Growth on L-ribose was found to induce the enzymes of the L-arabinose and ribitol pathways, but only ribitol-negative mutants derived from strains originally L-ribose positive lost the ability to grow on L-ribose, showing that a functional ribitol pathway was required. One of the mutations permitting growth on L-ribose enabled the mutants to produce constitutively an NADPH-linked reductase which converted L-ribose to ribitol. L-ribose is not metabolized by an isomerization to L-ribulose, as would be predicted on the basis of other pentose pathways in enteric bacteria. Instead, L-ribose was metabolized by the reduction of L-ribose to ribitol, followed by the conversion to D-ribulose by enzymes of the ribitol pathway.     

Production of L-ribose from L-ribulose by a triple-site variant of mannose-6-phosphate isomerase from Geobacillus thermodenitrificans

A triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase from Geobacillus thermodenitrificans was obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (k(cat)/K(m)) for L-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co(2+). The triple-site variant produced 213 g/liter l-ribose from 300 g/liter L-ribulose for 60 min, with a volumetric productivity of 213 g liter(-1) h(-1), which was 4.5-fold higher than that of the wild-type enzyme. The k(cat)/K(m) and productivity of the triple-site variant were approximately 2-fold higher than those of the Thermus thermophilus R142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.     

Characterization of recombinant L-ribose isomerase acquired from Cryobacterium sp. N21 with potential application in L-ribulose production

l-Ribose isomerase (lRI) is an enzyme that can catalyze the reversible isomerization between l-ribose and l-ribulose. It can also perform the conversion between many aldoses into their corresponding ketoses. l-RI was produced from Cryobacterium sp. N21 (CrL-RIse), and l-ribose was utilized as a substrate. The recombinant l-RI gene was cloned and overexpressed from Cryobacterium sp. N21. The purification of CrL-RIse was performed by metal-affinity chromatography. The enzyme displayed a corresponding band with an approximate size of 35 kDa on the SDS-PAGE analysis. The protein for this gene contains 266 amino acids with an expected molecular weight (M_w) of 29.6 kDa. The measured M_w of CrL-RIse calculated by HPLC was 125 kDa. CrL-RIse was extremely active in glycine buffer at 35 °C, pH 9.0, showing a specific activity of 54.96 U mg⁻¹. CrL-RIse displayed no major increase in activity with metal ions, excluding Mn²⁺. The estimated Km, K_cat, K_cat/Km and V_max values of CrL-RIse were 37.8 mM, 10,416 min⁻¹, 275.43 min⁻¹ mM⁻¹, and 250 U mg⁻¹, respectively. The rate of l-ribulose production was 31 % (6.24, 12.11, and 20.89 g L⁻¹) at equilibrium by utilizing 20, 40, and 70 g L⁻¹ of the substrate, respectively. The results indicated that CrL-RIse has the capability to manufacture l-ribulose from l-ribose.     

Cloning and characterization of a novel gene encoding L-ribose isomerase from Acinetobacter sp. strain DL-28 in Escherichia coli.

The gene encoding a novel L-ribose isomerase (L-RI) from Acinetobacter sp. was cloned into Escherichia coli and nucleotide sequence was determined. The gene corresponded to an open reading frame of 747 bp that codes for a deduced protein of 249 amino acids, which showed no amino acid sequence similarity with any other sugar isomerases. After expression of the gene in E. coli using pUC118 the recombinant L-RI was purified to homogeneity using different chromatographic methods. The overall enzymatic properties of the purified recombinant L-RI were the same as those of the authentic L-RI. To our knowledge, this is the first time report concerning the L-RI gene.     

Characterization of a mannose-6-phosphate isomerase from Thermus thermophilus and increased L-ribose production by its R142N mutant

An uncharacterized gene from Thermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme for L-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu(2+). Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of L-ribulose to L-ribose, a potential starting material for many L-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase in L-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (k(cat)/K(m)) for L-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. The k(cat)/K(m) of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans mannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reported k(cat)/K(m). The R142N mutant enzyme produced 213 g/liter L-ribose from 300 g/liter L-ribulose for 2 h, with a volumetric productivity of 107 g liter(-1) h(-1), which was 1.5-fold higher than that of the wild-type enzyme.     

Stochastic boundary molecular dynamics simulation of L-ribose in the active site of Actinoplanes missouriensis xylose isomerase and its Val135Asn mutant with improved reaction rate

We used molecular dynamics simulations to study how a non-natural substrate, L-ribose, interacts with the active site of Actinoplanes missouriensis xylose isomerase. The simulations showed that L-ribose does not stay liganded in the active site in the same way as D-xylose, in which the oxygens O2 and O4 are liganded to the metal M1. The oxygen O4 of L-ribose moved away from the metal M1 to an upside down position. Furthermore, the distances of the carbons C1 and C2 of L-ribose to the catalytic metal M2 were higher than in the case of D-xylose. These findings explain the extremely low reaction rate of xylose isomerase with L-ribose. The mutation V135N close to the C5-OH of the substrate increased the reaction efficiency 2- to 4-fold with L-ribose. V135N did not affect the reaction with D-xylose and L-arabinose, whereas the reaction with D-glucose was impaired, probably due to a hydrogen bond between Asn-135 and the substrate. When L-ribose was the substrate, Asn-135 formed a hydrogen bond to Glu-181. As a consequence, O4 of L-ribose stayed liganded to the metal M1 in the V135N mutant in molecular dynamics simulations. This explains the decreased K(m) of the V135N mutant with L-ribose.     

Development of a selection system for the detection of L-ribose isomerase expressing mutants of <Emphasis Type="Italic">Escherichia coli</Emphasis>

L-Arabinose isomerase (E.C. 5.3.1.14) catalyzes the reversible isomerization between L-arabinose and L-ribulose and is highly selective towards L-arabinose. By using a directed evolution approach, enzyme variants with altered substrate specificity were created and screened in this research. More specifically, the screening was directed towards the identification of isomerase mutants with L-ribose isomerizing activity. Random mutagenesis was performed on the Escherichia coli L-arabinose isomerase gene (araA) by error-prone polymerase chain reaction to construct a mutant library. To enable screening of this library, a selection host was first constructed in which the mutant genes were transformed. In this selection host, the genes encoding for L-ribulokinase and L-ribulose-5-phosphate-4-epimerase were brought to constitutive expression and the gene encoding for the native L-arabinose isomerase was knocked out. L-Ribulokinase and L-ribulose-5-phosphate-4-epimerase are necessary to ensure the channeling of the formed product, L-ribulose, to the pentose phosphate pathway. Hence, the mutant clones could be screened on a minimal medium with L-ribose as the sole carbon source. Through the screening, two first-generation mutants were isolated, which expressed a small amount of L-ribose isomerase activity.     

Synthesis of ethyl (1S,2R,3S,4S,5S)-2,3-O-(isopropylidene)-4-hydroxy-bicyclo[3.1.0]hexane-carboxylate from L-ribose: a versatile chiral synthon for preparation of adenosine and P2 receptor ligands

Substitution of the ribose moiety of various nucleosides and nucleotides with the (N)-methanocarba ring system increases the potency and selectivity as ligands at certain subtypes of adenosine and P2 receptors. We have prepared a key intermediate in the synthesis of these derivatives, ethyl (1S,2R,3S,4S,5S)-2,3-O-(isopropylidene)-4-hydroxybicyclo[3.1.0]hexane-carboxylate (15), starting from L-ribose (8) as a readily available, enantiopure building block. L-ribose was converted to the corresponding 5'-iodo derivative (9), which was cleaved reductively with Zn. Improvements were made in subsequent steps corresponding to a published route to biologically important (N)-methanocarba 5'-uronamido nucleosides, and new steps were added to prepare related 5'-nucleotides.     

Investigation of the mechanism of temperature sensitivity of the electroreceptors of ampullae of lorenzini

In experiments on Black Sea skates (Raja clavata), the potential of the receptor epithelium of the ampullae of Lorenzini and spike activity of single nerve fibers connected to them were investigated during electrical and temperature stimulation. Usually the potential within the canal was between 0 and –2 mV, and the input resistance of the ampulla 250–400 k. Heating of the region of the receptor epithelium was accompanied by a negative wave of potential, an increase in input resistance, and inhibition of spike activity. With worsening of the animal's condition the transepithelial potential became positive (up to +10 mV) but the input resistance of the ampulla during stimulation with a positive current was nonlinear in some cases: a regenerative spike of positive polarity appeared in the channel. During heating, the spike response was sometimes reversed in sign. It is suggested that fluctuations of the transepithelial potential and spike responses to temperature stimulation reflect changes in the potential difference on the basal membrane of the receptor cells, which is described by a relationship of the Nernst's or Goldman's equation type.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. I. M. Sechenov, Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Pacific Institute of Oceanology, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 67–74, January–February, 1980.     

Principles of organization and evolution of systems of regulation of functions

Evolution of living organisms is closely connected with evolution of structure of the system of regulations and its mechanisms. The functional ground of regulations is chemical signalization. As early as in unicellular organisms there is a set of signal mechanisms providing their life activity and orientation in space and time. Subsequent evolution of ways of chemical signalization followed the way of development of delivery pathways of chemical signal and development of mechanisms of its regulation. The mechanism of chemical regulation of the signal interaction is discussed by the example of the specialized system of transduction of signal from neuron to neuron, of effect of hormone on the epithelial cell and modulation of this effect. These mechanisms are considered as the most important ways of the fine and precise adaptation of chemical signalization underlying functioning of physiological systems and organs of the living organism