Studies on purification of 1,3-propanediol by molecular distillation

The separation of 1,3-propanediol using molecular distillation has been studied. The effects of operating temperature and feed flow rate through a sequential distillation strategy were investigated. The optimal experimental temperature was at 70°C for separating 1,3-propanediol and the by-product 2,3-butanediol. Meanwhile, the volume flow rate was 10 mL/min. As a result, the recovery of 1,3-propanediol and 2,3-butanediol were 87.6 and 87.5%, respectively. Furthermore, the integrated separation characteristic of 1,3-propanediol was evaluated through macrolevel and micro-level models. The separation factors of 1,3-propanediol versus 2,3-butanediol and glycerol were 0.11 and 1.07, respectively, affirming that the separation of 1,3-propanediol by molecular distillation was feasible.     

Fructose production by immobilizedArthrobacter cells

Summary ImmobilizedArthrobacter cells (NRRL-B-3728) were used for continuous isomerization of glucose to fructose in a bioreactor system. The system utilized stationary phase (55h) cells (2.2×10⁹ CFU/ml saline) immobilized onto K-carrageenan (3% w/v) beads [cells were heated at 65°C for 10 min to inactivate endogenous proteolytic enzymes]. Immobilized-cell preparations were hardened using three different glutaraldehyde systems. Glutaraldehyde (0.2 M) treated-immobilized cells (pH 7.0, 5°C for 30 min) exhibited good gel strength and high glucose isomerase activities. Maximal bioreactor isomerization of 44% was achieved when a buffered feedstock containing 40% glucose was fed into the column (60°C) at a flow rate of 0.2 ml/min. The biological half-life of glucose isomerase activities in this system was 400 h. Scanning electron microscopy revealed large numbers of cells distributed within the beads. A thin layer surrounding the beads following glutaraldehyde treatment was mainly due to cross-linking reactions between cell proteins and glutaraldehyde. This layer prevented leaking of cells during continuous isomerization reaction.     

Fructose transport by Escherichia coli

The utilization of fructose by Escherichia coli involves, as first step, the uptake of the sugar, normally via the phosphoenolpyruvate-dependent phosphotransferase system (PTS). This fructose-specific PTS differs in several ways from that effecting the uptake of other sugars that also possess the 3,4,5-D-arabino-hexose configuration: these differences are discussed. Mutants that lack the genes ptsI and ptsH, which specify components of the PTS common to most PT-sugars, can mutate further to regain the ability to utilize fructose when this is present in relatively high concentration (i.e. greater than 2 mM) in the medium. Some of the properties of this unusual uptake system is discussed.     

Fructose phosphorylation by zymomonas mobilis glucokinase

Zymomonas mobilis ATCC 53431, a fructokinase negative mutant is unable to utilize fructose as a sole carbon source for growth. At high fructose concentrations, however, fructose was converted to ethanol. The fructose uptake displayed MICHAELIS -MENTEN kinetics with an apparent Km of 185 mM fructose. Purified glucokinase from ATCC 53431 and the wild strain ATCC 29191 both exhibited fructose phosphorylating activity at high fructose concentrations with an apparent Km value of 222 mM fructose. Glucokinase substrate specificity was found not be absolute, as previously reported.     

Partial purification and determination of molecular weights of glyoxalases by filtration on dextran gel

Glyoxalase I and glyoxalase II (EC. 4.4.1.5 and EC 3.1.2.6) were separated by gel filtration on Sephadex G-75 and G-100. This simple procedure permitted also the partial purification of glyoxalase II. The purification coefficient in a single run from supernatant from beef liver was about 1 : 30 compared with 1 : 15 after the fifth step of purification with classical methods.     

Sorption of mono- and sesquiterpenes by natural polysaccharides

Sorption of terpenoids (essential oil components) from aqueous solutions by six types of native food starches was studied by capillary gas chromatography. Sorption of volatile substances did not depend on amylose content in starch and specific surface of its granules. The degree of sorption was maximum (86%) for corn starch containing 25-28% amylose and decreased in the following order: tapioca starch (77%) > potato starch (74%) > wheat starch (70%) > high-amylose corn starch (58%) > amylopectin corn starch (57%). Amylopectin corn starch differed from other starches in the mechanism of sorption and selectivity to compounds with various functional groups.     

Semisynthesis and purification of homogeneous plasmenylcholine molecular species.

Methods for the efficient use of limiting amounts of fatty acid probes in the synthesis of individual molecular species of plasmenylcholine have been developed. Plasmenylcholine molecular species were synthesized through acylation of homogeneous 1-O-(Z)-hexadec-1'-enyl-sn-glycero-3-phosphocholine utilizing fatty acid anhydrides generated in situ from combined pools of reactant and recycled fatty acids by repeated addition of small amounts (10 mol%) of N,N'-dicyclohexylcarbodiimide. The efficient generation of reactive anhydrides was accomplished through minimizing irreversible formation of N-acyl urea adducts by maintaining a persistent molar excess of fatty acid (with respect to carbodiimide) during the entire reaction time course. The synthesis of multiple different sn-2 labeled plasmenylcholine probes for utilization in fluorescence, ESR, or 2H NMR spectroscopy as well as isotopically labeled plasmenylcholines for metabolic studies has been achieved in good yield (40-50% of theoretical yield based on fatty acid) by these methods. Rapid and effective purification methods utilizing high-performance liquid chromatography were developed for both large- and small-scale purifications of individual reaction mixtures which collectively resulted in the isolation of homogeneous plasmenylcholine molecular species in high yield from limiting amounts of fatty acid probes.     

Separation of mono- and diglycerides by gas--liquid chromatography

The parameters affecting the separation and quantification of trimethylsilyl ethers of mono- and diglycerides have been investigated by gas-liquid chromatography with QF-1 and SE-30 as stationary phases and a flame ionization detector. Results have been compared with those obtained earlier for triglycerides. The isothermal characteristics of a range of trimethylsilyl ethers of mono- and diglycerides on both stationary phases showed that log retention volume was directly proportional to carbon number and inversely proportional to absolute temperature. However, glyceride derivatives with lower carbon numbers deviated from these relationships. By using various rates of programmed temperature rise, we have determined the elution temperatures (Kelvin scale) of the mono- and diglyceride trimethylsilyl ethers relative to that of glycerol trilaurate. The "carbon equivalent of a trimethylsilyl group" is defined and shown to be useful in comparing the chromatographic properties of different glyceride classes. Weight and molar correction factors have been obtained and used to analyze diglycerides derived from egg and bovine brain lecithins.     

Synergistic Inhibition of Fructose 1,6-Bisphosphatase by Fructose 2,6-Bisphosphate and Adenosine Monophosphate in Round Spermatids of Rats

The effects of adenosine monophosphate (AMP) and fructose 2, 6-bisphosphate (fruc-2, 6-P₂) on the key-enzyme of gluconeogenesis, fructose 1, 6-bisphosphatase (fruc-P₂ase; D-fructose 1, 6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) in spermatid extract from rat testes were studied. The fruc-P₂ase activity in the spermatids of rats was suppressed by AMP and fruc-2, 6-P₂. The inhibition of fruc-2, 6-P₂ was much stronger at low than at high substrate concentrations, and enhanced synergistically with AMP. The substrate saturation curve was changed by fruc-2, 6-P₂ hyperbolic to sigmoidal. Furthermore, the concentration of AMP that decreased the activity to 50% was much lower in the presence than in the absence of fruc-2, 6-P₂. These results indicate the possibility that gluconeogenesis in spermatids of rats is controlled by AMP and fruc-2, 6-P₂.     

Rapid extraction and purification of environmental DNA for molecular cloning applications and molecular diversity studies

A rapid method for the extraction and purification of DNA from environmental samples for molecular cloning applications was developed. The indigenous cells from plant debris, organic materials, sediments, and soils were lysed directly by using DAS-I^Z solution and the nucleic acids were precipitated with isopropanol. A simple purification step using DAS-II^Z solution without binding matrix produced highly pure, colorless and undegraded DNA with molecular weight of more than 20 kb. The superiority of this method was tested for wide applications in molecular cloning, i.e., construction of genomic library by using Lambda DASH^(R)II Vector and Gigapack^(R)III XL, plasmid library, cloning of gene encoding protease, and molecular microbial diversity analysis. An additional advantage of this method is that only 0.1 g of sample is required, if analysis of many samples in short time should be done. To extract large amounts of environmental DNA for molecular cloning lasts only 30 min and to purify it less than 1 h.     

Immunoaffinity purification and identification of the molecular chaperone calnexin.

We have developed a method for the immunoaffinity purification of calnexin, an endoplasmic reticulum molecular chaperone, and analyzed the molecular weight of purified calnexin using matrix-assisted laser adsorption ionization time of flight mass spectrometry (MALDI TOF-MS). Calnexin was thereby found to have a molecular weight of 66.1 x 10(3), which is nearly identical to the molecular weight estimated from the protein sequence.     

Affinity purification and characterization of the Escherichia coli molecular chaperones

The molecular chaperones are a group of proteins that are effective in vitro and in vivo folding aids and show a well-documented affinity for proteins lacking tertiary structure. The molecular chaperones were induced from lon(-) Escherichia coli mutants, affinity purified with an immobilized beta-casein column, and assayed for refolding activity with thermally and chemically denatured carbonic anhydrase B (CAB). Chaperones were induced with three treatments: heat shock at 39 degrees C, heat shock 42 degrees C, and alcohol shock with 3% ethanol (v/v). Lysates were applied to an immobilized beta-casein (30 mg/g beads) column. After removing nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water or 1 mM Mg-ATP. The cold water and Mg-ATP eluates were analyzed by SDS-PAGE. Western analysis identified five E. coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES. The purity of eluted chaperones was 58% with cold water and 100% with Mg-ATP. Refolding denatured CAB in the presence of Mg-ATP resulted in a 97% recovery of heat-denatured CAB and a 68% recovery of chemically denatured CAB. The use of affinity matrices for the chaperone purification which are effective as in vitro folding aids will be presented.