Candida antarctica lipase B-catalyzed synthesis of poly(butylene succinate): shorter chain building blocks also work

Lipase catalysis was successfully employed to synthesize high molecular weight poly(butylene succinate) (PBS). Attempts to copolymerize succinic acid with 1,4-butanediol were unsuccessful due to phase separation of the reactants. To circumvent this problem, monophasic reaction mixtures were prepared from diethyl succinate and 1,4-butanediol. The reactions were studied in bulk as well as in solution. Of the organic solvents evaluated, diphenyl ether was preferred, giving higher molecular weight products. After 24 h in diphenyl ether, polymerizations at 60, 70, 80, and 90 degrees C yielded PBS with M(n) of 2000, 4000, 8000, and 7000, respectively. Further increase in reaction time to 72 h resulted in little or no further increase in M(n). However, increasing the reaction time produced PBS with extraordinarily low M(w)/M(n) due to the diffusion and reaction between low-molecular weight oligomers and chains that occurs at a greater frequency than interchain transesterification. Time-course studies and visual observation of polymerizations at 80 degrees C revealed PBS precipitates at 5 to 10 h, limiting the growth of chains. To maintain a monophasic reaction mixture, the polymerization temperature was increased from 80 to 95 degrees C after 21 h. The result was an increase in the PBS molecular weight to M(w) = 38 000 (M(w)/M(n) = 1.39). This work paves the way for the synthesis of PBS macromers and polymers that contain variable quantities of monomers with chemically sensitive moieties (e.g., silicone, epoxy, vinyl). Furthermore, this study established the feasibility of using lipase catalysis to prepare polyesters from alpha,omega-linear aliphatic diethyl ester/diol monomers with less than six carbons.     

Development of an efficient enzymatic production of gamma-D-glutamyl-L-tryptophan (SCV-07), a prospective medicine for tuberculosis, with bacterial gamma-glutamyltranspeptidase

Gamma-D-Glutamyl-L-tryptophan (SCV-07) is a prospective medicine for the treatment of tuberculosis, according to the phase two clinical trial. Because gamma-D-glutamyl-L-tryptophan has several reactive groups in its molecule, consists of D- and L-amino acids, and is connected by gamma-glutamyl linkage, its chemical synthesis is complicated. An efficient enzymatic method to synthesize gamma-D-glutamyl-L-tryptophan from D-glutamine and L-tryptophan employing bacterial gamma-glutamyltranspeptidase was developed. The optimum reaction conditions were 50 mM D-glutamine, 50 mM L-tryptophan, and 0.2 U ml(-1) gamma-glutamyltranspeptidase, pH 9-9.5, and incubation at 37 degrees C for 5 h. After a 5 h incubation, 33 mM gamma-D-glutamyl-L-tryptophan was obtained, the conversion rate being 66%. The product was purified by Dowex 1 x 8 column and was considered to be gamma-D-glutamyl-L-tryptophan.     

Enzymatic transesterifications of carbonates in water-restricted environments

We investigated the ability of several hydrolases to catalyze reactions with an abiotic water-insoluble substrate, carbonic acid diphenyl ester, also known as diphenyl carbonate (DPC). In single-phase water/organic systems, turnover numbers (TN) of greater than 2 x 10(4) min(-1)have been achieved for the hydrolysis of DPC. The K(m) values for the hydrolytic reaction were measured to be 200muM and 330muM for Candida cylindracea lipase and Porcine liver esterase, respectively. In addition to hydrolysis, we observed transesterification of carbonates with a wide variety of alcohol and phenol species. Transesterifications of DPC with bifunctional alcohols resulted in the synthesis of polycarbonates. We investigated the stability and transesterification activity of these enzymes in several water-restricted environments to limit competing hydrolysis reactions. We find that, with the removal of water, hydrolysis is reduced more than four orders of magnitude while transesterification is diminished only 10-fold (turnover numbers of 600 min(-1) in water-miscible systems to 60 min(-1) in water-restricted environments with pure Candida lipase). Stability of the Candida lipase in these water-restricted environments (half-life of longer than 3 days) is much greater than in water/organic single phase systems (5 h in 20% methanol). In addition, the Candida lipase displayed enantiomeric selectivity in transesterifications of DPC with racemic 2-butanol (greater than 80% ee).     

A two-step acid-catalyzed process for the production of biodiesel from rice bran oil

A study was undertaken to examine the effect of temperature, moisture and storage time on the accumulation of free fatty acid in the rice bran. Rice bran stored at room temperature showed that most triacylglyceride was hydrolyzed and free fatty acid (FFA) content was raised up to 76% in six months. A two-step acid-catalyzed methanolysis process was employed for the efficient conversion of rice bran oil into fatty acid methyl ester (FAME). The first step was carried out at 60 degrees C. Depending on the initial FFA content of oil, 55-90% FAME content in the reaction product was obtained. More than 98% FFA and less than 35% of TG were reacted in 2 h. The organic phase of the first step reaction product was used as the substrate for a second acid-catalyzed methanolysis at 100 degrees C. By this two-step methanolysis reaction, more than 98% FAME in the product can be obtained in less than 8 h. Distillation of reaction product gave 99.8% FAME (biodiesel) with recovery of more than 96%. The residue contains enriched nutraceuticals such as gamma-oryzanol (16-18%), mixture of phytosterol, tocol and steryl ester (19-21%).     

Systematic synthesis of purine 8,5'-imino and substituted imino cyclonucleosides

To achieve a systematic synthesis of purine 8,5'-imino and substituted imino cyclonucleosides, 2',3'-O-isopropylidene-purinenucleosides substituted with a methylamino (4a,b), benzyl-amino (4c,d,g and h) and allylamino group (4e,f,i and j) at the C8 were synthesized. With these substrates in hand, extensive 8,5'-cyclization reactions were carried out using diphenyl carbonate/Et3N (Method A), N,N'-carbonyldiimidazole (Method B) and the Mitsunobu reaction (Method C) to give 8,5'-substituted imino cyclonucleosides (5a,c,d,e,f and g). The yields of cyclization by Method C are generally higher than by the other two methods. 5a, b,c,d,e,f,g and h were deprotected to the corresponding mother compounds 8 through one or two steps. In guanosine series, a new cyclic system comprising an 8,5'-carbamate ester bridge (6a-c) has been introduced.     

Enhanced cell-free protein synthesis using a S30 extract from Escherichia coli grown rapidly at 42 degrees C in an amino acid enriched medium

Growths of Escherichia coli strain A19 were investigated in a 5-L fermentor at 37 and 42 degrees C either in Pratt's medium (a standard medium for cell-free protein synthesis using its S30 extract) or in a casamino acids supplemented Pratt's medium (aa-enriched medium). Specific growth rates in Pratt's medium at 37 and 42 degrees C were 0.77 and 0.46 h(-1), respectively, whereas those in the aa-enriched medium at 37 and 42 degrees C were 0.87 and 1.49 h(-1), respectively. The extent of cell-free chloramphenicol acetyltransferase (CAT) synthesis was compared at 37 degrees C incubation (from a plasmid pK7-CAT) for S30 extracts prepared from the cells cultured in the aa-enriched medium at 37 or 42 degrees C. A 40% increase in CAT synthesis occurred when the 42 degrees C/S30 extract was used as compared with 37 degrees C/S30 extract. CAT and both the light and heavy chains (Lc and Hc) of the Fab fragment of an antibody 6D9 were synthesized at 37 degrees C in the cell-free synthesis in the presence of [(14)C]Leu. Their reaction mixtures were subjected to SDS-PAGE autoradiographic analysis. It was found that most of the synthesized proteins were in the soluble fraction when 42 degrees C/S30 extract was used, suggesting that the 42 degrees C/S30 extract contained greater amounts of various protein folding factors. A dialysis membrane minibioreactor with a reaction volume ca. 0.5 mL was handmade by the authors. The advantages of the minibioreactor are a simple configuration, a low manufacturing cost, and the capability of the dialysis membrane replacement. Increased CAT synthesis was also observed for continuous exchange cell-free (CECF) protein synthesis at 37 degrees C when the 42 degrees C/S30 extract was used in the minibioreactor. Some plausible reasons to give higher protein synthesis activity of the 42 degrees C/S30 extract are discussed.     

Synthesis of soluble heat shock proteins in seminal root tissues of some cultivated and wild wheat genotypes

Effect of heat stress on the synthesis of soluble heat shock proteins (HSPs) and the regrowth in seminal roots of three cultivated and three wild wheat genotypes was examined. In regrowth experiments, 2-d-old etiolated seedlings were exposed to 23 (control), 32, 35, 37 and 38 degrees C for 24 h, and 35 and 37 degrees C (24 h) followed by 50 degrees C (1 h). The lengths of the seminal roots generally decreased significantly at the end of 48 and 72 h recovery growth periods at 35, 37 and 38 degrees C temperature treatments compared with control. Genotypic variability was significant level at all temperature treatments for the seminal root length. Also, genotypic differences for the number of seminal roots were determined among the wheat cultivars and between the wild wheat species and the wheat cultivars at all temperature treatments; but genotypic differences among wild wheat species were only detected at 37-->50 degrees C treatment. Acquired thermotolerance for the seminal root length is over 50% at 37-->50 degrees C treatment. The genotypic variability of soluble heat shock proteins in seminal root tissues were analyzed by two-dimensional electrophoresis (2-DE). Total number of low molecular weight (LMW) HSPs was more than intermediate-(IMW) and high- (HMW) HSPs at high temperature treatments. The most of LMW HSPs which were generally of acidic character ranged between 14.2-30.7 kDa. The genotypes had both common (43 HSP spots between at least two genotypes and 23 HSP spots between 37 and 37-->50 degrees C) and genotype-specific (72 HSP spots) LMW HSPs.     

Enzymatic Regioselective Alkoxycarbonylation of Nucleosides and Its Utility in Nucleoside Derivative Synthesis

The regioselective enzymatic alkoxycarbonylation of nucleosides is described for α-, xylo-, anhydro-, and arabino-nucleosides to obtain Cbz-derivatives. The utility of these compounds and of the related vinyl carbonates of 2‘-deoxynucleosides is shown by the synthesis of 3‘-O-acetates of α-and xylo-thymidine and the synthesis of some nucleoside carbamates, respectively.     

Lipase-catalyzed synthesis of precursor dipeptides of RGD in aqueous water-miscible organic solvents

PPL-catalyzed synthesis of the precursor dipeptides of RGD as a cellular adhesion factor, Benzyl-Arg-Gly-NH2 and CBZ-Gly-Asp-NH2, was conducted in water-organic cosolvents systems. Five water-miscible organic solvents, which have some advantage over the water-immiscible organic solvent systems or the anhydrous organic solvent systems used often in protease-catalyzed synthesis of a peptide bond, were tested. The reaction condition of PPL-catalyzed synthesis of the dipeptides was optimized by examining the main factors affecting the product yield. The optimal reaction condition for the synthesis of Benzyl-Arg-Gly-NH2 was set up as pH 8.0, 15 degrees C in 40% MeOH for 10 h with the maximum yield of 73.6%. The optimum condition for the synthesis of CBZ-Gly-Asp-NH2 was pH 7.0, 15 degrees C in 50% MeOH for 10h with the maximum yield of 67.0%.     

Two-step freezing of cattle blastocysts with dimethylsulfoxyde (DMSO) or glycerol

Sixty five cattle blastocysts were frozen by the so-called two-step freezing method: The samples were seeded at -7 degrees C and then directly brought at -30 degrees C for 30 minutes before being taken into liquid nitrogen. Results in terms of survival rates at thawing and after short term cultures were compared to two controlled linear cooling rate procedures (i.e. 0.3 degrees C/min and 1.3 degrees C/min). The results demonstrate that: 1) two-step freezing yielded approximately the same survival rate as the two others techniques and 2) Glycerol yielded better survival rates than DMSO treatments (56 vs 31% after 24 hours in culture).     

In vitro amplification of DNA fragments greater than 10 kb.

The synthesis of a 10.9-kb DNA fragment from a bacteriophage lambda template was used in the search for conditions to extend the range for the polymerase chain reaction (PCR). Using the same primer sequences and conditions (denaturation at 94 degrees C, 1 min; annealing at 57 degrees C, 1 min; polymerization at 70 degrees C, 20 to 30 min) as published by W. Rychlik, W. J. Spencer, and R. E. Rhoads [(1990) Nucleic Acids Res. 18, 6409-6412], unsatisfactory results were obtained with AmpliTaq and native Taq polymerase (poor reproducibility, low product yield, nonspecific products), whereas Tub polymerase completely failed to amplify this fragment. Only after changes in the following parameters were reliable results obtained but only with Tub polymerase: A two-step PCR procedure with primer annealing and extension at 65 degrees C followed by DNA denaturation at 94 degrees C for 1.5 min was performed. The DNA fragment desired was specifically amplified when the enzyme concentration was reduced to 0.4 U/50 microliters and extension times as low as 4 to 12 min with an optimum at 8 min were used. A prolongation to 20 min or more resulted in an accumulation of unspecific products with a concomitant reduction in the yield of the fragment. Under the conditions described above it was also possible to amplify a DNA fragment even significantly longer (15.6 kb).     

Homogeneous esterification of cellulose in the lithium chloride-N,N-dimethylacetamide solvent system: effect of temperature and catalyst

Commercial rayon grade cellulose was dissolved in the lithium chloride-N,N-dimethylacetamide (LiCl-DMAc) solvent system and esterified with acetic anhydride using p-toluenesulfonyl chloride (p-TsCl) and pyridine as catalysts. The reaction temperature was varied from 28 to 70 degrees C and the time of reaction from 2 to 24 h. Full substitution took place at 60 and 70 degrees C at respective reaction times of 10 and 8 h for p-TsCl, and 10 and 6 h for pyridine. Esterification of cellulose followed a second-order reaction path. The rate constants at different reaction temperatures and the activation energy for the reaction are reported. Mechanisms for these reactions using the two catalysts are also suggested. The degrees of substitution (DS) of the esters prepared using both catalysts show that pyridine is a better catalyst than p-TsCl. Molecular weights of the esters, determined viscosimetrically, show that some degradation in the cellulose chain occurred at a reaction temperature of 70 degrees C. Hence, the optimum temperature for esterification appears to be 50-60 degrees C at 10 h reaction time to obtain full degree of acetyl substitution.     

Thermal factors influencing detection of Vibrio vulnificus using real-time PCR

Five thermal factors, including initial denaturation temperature, cycling denaturation temperature, annealing temperature, extension temperature and the temperature at which the intensity of the fluorescent signal is read, were evaluated for their effects on the detection of Vibrio vulnificus via real-time PCR. Fluorescent signal detection after extension was set between the Tm value of the primer-dimers (79 degrees C) and that of the PCR target amplicons (84 degrees C). This effectively eliminated the overestimation of the yield of PCR amplicons due to the presence of primer-dimers which otherwise led to erroneously lower Ct values (1.91+/-0.22 cycles lower). The annealing and extension steps were combined to convert a three-step PCR to a two-step PCR. This consisted of initial denaturation at 95 degrees C for 3 min, cycling denaturation at 94 degrees C for 15 s and a combined annealing and extension step at 60 degrees C for 5 s in each PCR cycle. One genomic target per real-time PCR reaction was detected with the simplified two-step PCR.     

Kinetic modeling of the enzymatic hydrolysis of pretreated cellulose

The production of sugars by the enzymatic hydrolysis of cellulose is a two-step process that includes conversion of the intermediate cellobiose to glucose by beta-glucosidase. The hydrolysis was followed by analyzing the two sugar products (cellobiose and glucose). The enzyme showed maximum activity at pH 4.8. Thermal deactivation was significant at temperatures above 45 degrees C. At 50 degrees C (optimum temperature) thermal deactivation was found to follow first-order kinetics. Several models were tested by modeling the kinetics of the reaction. Their parameter values were determined by numerical optimization, including temperature dependence. The best fitting model was a competitive product inhibition for the two reactions in the operational range.     

Biodiesel production from crude Jatropha curcas L. seed oil with a high content of free fatty acids

A technique to produce biodiesel from crude Jatropha curcas seed oil (CJCO) having high free fatty acids (15%FFA) has been developed. The high FFA level of JCJO was reduced to less than 1% by a two-step pretreatment process. The first step was carried out with 0.60 w/w methanol-to-oil ratio in the presence of 1% w/w H(2)SO(4) as an acid catalyst in 1-h reaction at 50 degrees C. After the reaction, the mixture was allowed to settle for 2h and the methanol-water mixture separated at the top layer was removed. The second step was transesterified using 0.24 w/w methanol to oil and 1.4% w/w NaOH to oil as alkaline catalyst to produce biodiesel at 65 degrees C. The final yield for methyl esters of fatty acids was achieved ca. 90% in 2 h.     

Time course and magnitude of synthesis of heat-shock proteins in congeneric marine snails (Genus tegula) from different tidal heights

The time course and magnitude of the heat-shock response in relation to severity of thermal stress are important, yet poorly understood, aspects of thermotolerance. We examined patterns of protein synthesis in congeneric marine snails (genus Tegula) that occur at different heights along the subtidal to intertidal gradient after a thermal exposure (30 degrees C for 2.5 h, followed by 50 h recovery at 13 degrees C) that induced the heat-shock response. We monitored the kinetics and magnitudes of protein synthesis by quantifying incorporation of 35S-labeled methionine and cysteine into newly synthesized proteins and observed synthesis of putative heat-shock proteins (hsp's) of size classes 90, 77, 70, and 38 kDa. In the low- to mid-intertidal species, Tegula funebralis, whose body temperature frequently exceeds 30 degrees C during emersion, synthesis of hsp's commenced immediately after heat stress, reached maximal levels 1-3 h into recovery, and returned to prestress levels by 6 h, except for hsp90 (14 h). In contrast, in the low-intertidal to subtidal species, Tegula brunnea, for which 2.5 h at 30 degrees C represents a near lethal heat stress, synthesis of hsp's commenced 2-14 h after heat stress; reached maximal levels after 15-30 h, which exceeded magnitudes of synthesis in T. funebralis; and returned to prestress levels in the case of hsp90 (50 h) and hsp77 (30 h) but not in the case of hsp70 and hsp38. Exposures to 30 degrees C under aerial (emersion) and aquatic (immersion) conditions resulted in differences in hsp synthesis in T. brunnea but not in T. funebralis. The different time courses and magnitudes of hsp synthesis in these congeners suggest that the vertical limits of their distributions may be set in part by thermal stress.     

Reaction kinetics and modeling of the enzyme-catalyzed production of lactosucrose using beta-fructofuranosidase from Arthrobacter sp. K-1

Lactosucrose synthesis from sucrose and lactose was carried out by using beta-fructofuranosidase from Arthrobacter sp. K-1. The transfructosylation mechanism was found to be of an ordered bi-bi type in which sucrose was bound first to the enzyme and lactosucrose was released last. Hydrolysis side-reaction experiments indicated that the reactions were uncompetitively inhibited by glucose and lactose, while no inhibition by fructose was apparent. The overall reaction rates were formulated. The reaction rate constants, equilibrium constant, and dissociation and Michaelis constants were determined at 35 degrees C and 50 degrees C by fitting the experimental concentration changes with the calculated values by a nonlinear least-square method. The average relative derivation for the concentrations was 9.67%. The kinetic parameters were also calculated for 43 degrees C and 60 degrees C by assuming the Arrhenius law, and the course of reaction was predicted. The obtained reaction rate equations well represented the concentration changes during the experiment at all temperatures.     

Overcoming limiting side reactions associated with an NHS-activated precursor of polymethacrylamide-based polymers

Combinatorial polymer libraries have recently gained popularity for the development of novel materials for a variety of biomedical applications including non-viral gene delivery systems and biodegradable polymers for tissue engineering. To streamline the nontrivial task of library synthesis, activated ester homopolymers have been used to serve as a backbone to which primary amine-containing functional groups (NH2-FGs) can be covalently bound at varying ratios. Polymethacryloxysuccinimide (poly(MAOS)) is one such homopolymer that was previously reported to be an attractive precursor for polymeric drug and gene delivery systems. The reported functionalization protocols entailed conjugating the precursor with 2 equiv of the NH2-FG at a reaction concentration of 25 mg poly(MAOS)/150 microL DMSO for either 5 h at 50 degrees C or 16 h at 25 degrees C. More recently, both protocols were revealed to be associated with ring-opening and glutarimide-forming side reactions that compromise the utility of the homopolymer. Using 1-dimensional and 2-dimensional NMR spectroscopy techniques, we have characterized the side product distributions that result from conjugations performed at 50 degrees C/5 h and 25 degrees C/16 h. Moreover, by systematically altering the equivalents of the NH2-FGs, polymer concentration, reaction time, and reaction temperature, we have established a protocol that overcomes these side reactions. Using a final reaction protocol of 5 equiv of the NH2-FG at a reaction concentration of 25 mg poly(MAOS)/600 microL DMSO for 24 h at 75 degrees C, we have obtained functionalized polymers with minimal side products. This protocol is applicable for polymers ranging from 5000 to 50,000 g/mol, compatible with a variety of functional groups, and amenable to conjugating combinations of functional groups.