An investigation of the metabolism of isoleucine to active Amyl alcohol in Saccharomyces cerevisiae

The metabolism of isoleucine to active amyl alcohol (2-methylbutanol) in yeast was examined by the use of (13)C nuclear magnetic resonance spectroscopy, combined gas chromatography-mass spectrometry, and a variety of mutants. From the identified metabolites a number of routes between isoleucine and active amyl alcohol seemed possible. All involved the initial decarboxylation of isoleucine to alpha-keto-beta-methylvalerate. The first, via branched chain alpha-ketoacid dehydrogenase to alpha-methylbutyryl-CoA, was eliminated because abolition of branched-chain alpha-ketoacid dehydrogenase in an lpd1 disruption mutant did not prevent the formation of active amyl alcohol. However, the lpd1 mutant still produced large amounts of alpha-methylbutyrate which initially seemed contradictory because it had been assumed that alpha-methylbutyrate was derived from alpha-methylbutyryl-CoA via acyl-CoA hydrolase. Subsequently it was observed that alpha-methylbutyrate arises from the non-enzymic oxidation of alpha-methylbutyraldehyde (the immediate decarboxylation product of alpha-keto-beta-methylvalerate). Mutant studies showed that one of the decarboxylases encoded by PDC1, PDC5, PDC6, YDL080c, or YDR380w must be present to allow yeast to utilize alpha-keto-beta-methylvalerate. Apparently, any one of this family of decarboxylases is sufficient to allow the catabolism of isoleucine to active amyl alcohol. This is the first demonstration of a role for the gene product of YDR380w, and it also shows that the decarboxylation steps for each alpha-keto acid in the catabolic pathways of leucine, valine, and isoleucine are accomplished in subtly different ways. In leucine catabolism, the enzyme encoded by YDL080c is solely responsible for the decarboxylation of alpha-ketoisocaproate, whereas in valine catabolism any one of the isozymes of pyruvate decarboxylase will decarboxylate alpha-ketoisovalerate.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

We have found that in the alcoholic fermentation of amino acids by yeast isobutyl alcohol is produced from alanine and n-propyl and active amyl alcohols are formed from α-amino-n-butyric acid or threonine contrary to the F. Ehrlich’s scheme. These results suggest the close relationship among the formation of these higher alcohols and biosynthesis of valine from alanine and biosynthesis of isoleucine from α-amino-n-butyric acid or threonine.

In this report, we studied the formation of n-propyl alcohol and active amyl alcohol from α-amino-n-butyric acid using washed yeast cells.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

It was found that washed yeast cells produced active amyl alcohol from aspartic acid, homoserine or α-hydroxy-aceto-n-butyric acid each of which was considered the intermediate in the synthetic pathway of active amyl alcohol and isoleucine. At the same time n-butanol and α-keto-β-methyl-n-valeric acid were detected in the fermented solutions, and α-keto-n-butyric acid was formed in the fermented solutions contained the former two compounds. These results seemed to support the reliability of this pathway. Acetylpropionyl and 2,3-pentanediol were formed from α-hydroxy-aceto-n-butyric acid.     

Study of Vitamin C ester synthesis by immobilized lipase from Candida sp.

Oleoyl ester of -ascorbic acid was synthesized by using immobilized lipases from Candida sp. A series of solvents, such as ethanol, tetrahydrofuran, pyridine, butanol, tertiary amyl alcohol (t-amyl alcohol), hexanol, octanol and hexane (log P from −0.24 to 3.5) were investigated for the reaction, and t-amyl alcohol was found to be the most suitable from the standpoint of the substrate concentration and the enzyme activity. And the equilibrium of the reaction was affected by the addition of the molecular sieves and the temperature. Reaction carried out at 55 °C and with 50 g/l of 4 Å molecular sieves is good for the enzyme to keep its activity and for making the equilibrium go to the product. The kinetic model was studied and the result showed that the reaction can be described by Ping-Pong mechanism. Parameters value of V_m and K_m′ were obtained. Last, the pure products of the reaction were attained and determined by IR spectra, mass spectrometry and ¹H NMR spectra.     

Quinoprotein alcohol dehydrogenase from ethanol-grown Pseudomonas aeruginosa.

Cell-free extracts of Pseudomonas aeruginosa strains, grown on ethanol, showed dye-linked alcohol dehydrogenase activities. The enzyme responsible for this activity was purified to homogeneity. It appeared to contain two molecules of pyrroloquinoline quinone per enzyme molecule. In many respects, it resembled other quinoprotein alcohol dehydrogenases (EC 1.1.99.8), having a substrate specificity intermediate between that of methanol dehydrogenases and ethanol dehydrogenases in this group. On the other hand, it also showed dissimilarities: the enzyme was found to be a monomer (Mr 101 000), to need only one molecule of the suicide substrate cyclopropanol to become fully inactivated, and to have a different aromatic amino acid composition.     

Characterization of cinnamyl alcohol dehydrogenase of Helicobacter pylori. An aldehyde dismutating enzyme

Cinnamyl alcohol dehydrogenases (CAD; 1.1.1.195) catalyse the reversible conversion of p-hydroxycinnamaldehydes to their corresponding alcohols, leading to the biosynthesis of lignin in plants. Outside of plants their role is less defined. The gene for cinnamyl alcohol dehydrogenase from Helicobacter pylori (HpCAD) was cloned in Escherichia coli and the recombinant enzyme characterized for substrate specificity. The enzyme is a monomer of 42.5 kDa found predominantly in the cytosol of the bacterium. It is specific for NADP(H) as cofactor and has a broad substrate specificity for alcohol and aldehyde substrates. Its substrate specificity is similar to the well-characterized plant enzymes. High substrate inhibition was observed and a mechanism of competitive inhibition proposed. The enzyme was found to be capable of catalysing the dismutation of benzaldehyde to benzyl alcohol and benzoic acid. This dismutation reaction has not been shown previously for this class of alcohol dehydrogenase and provides the bacterium with a means of reducing aldehyde concentration within the cell.     

反胶束中单宁酶催化没食子酸烷基酯的生物合成

为了研究单宁酶在有机相中的催化性能,建立了AOT/异辛烷／水反胶束单宁酶催化没食子酸与脂肪醇酯合成反应体系。结果显示：反胶束单宁酶催化体系可成功催化合成C3-C5脂肪醇与没食子酸的酯合成反应。不同反应体系中由于不同脂肪醇的存在,单宁酶的动力学参数和紫外光谱存在差别。结果表明单宁酶对脂肪醇的专一性不强,根据Vmax/Km比值,丁醇与异丁醇是其最适底物,单宁酶催化没食子酸烷基酯合成的动力学符合米氏方程。反应体系中不同的脂肪醇导致了单宁酶构象的差别。     

Synthesis of short chain alkyl esters using cutinase from Burkholderia cepacia NRRL B2320

Short chain alkyl esters are well appreciated for fruity flavors they provide. These are mainly applied to the fruit-flavored products like jam, jelly, beverages, wine and dairy. Cutinase from Burkholderia cepacia NRRL B 2320 was found to be active in catalyzing the synthesis of alkyl esters in organic solvent. The optimal temperature range for the enzyme catalyzed synthesis was found to be from 35 °C to 40 °C. The maximum conversion (%) during synthesis of ester was obtained for butyric acid (C4) and valeric acid (C5) with butanol reflecting the specificity of the enzyme for short-chain length fatty acids. In case of alcohol specificity, butanol was found to be most preferred substrate by the enzyme and conversion (%) decreased with increasing carbon chain length of alcohol used in the esterification reaction. The kinetic analysis for the synthesis of butyl butyrate by varying concentration of one substrate at a time (butanol or butyric acid), showed that Ping–Pong Bi Bi model with acid inhibition and influence of initial water is most suitable model for the prediction of the reaction kinetics.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

We have found that some straight-chained α-amino acids are converted by yeast to the alcohols with correspondingly longer carbon chains in the alcoholic fermentation contrary to F. Ehrlich’s scheme, i.e., isobutyl alcohol from alanine and active amyl alcohol from α-amino-n-butyric acid or threonine.

In this report, we confirmed this fact in the alcoholic fermentation of many aliphatic amino acids by 2 yeast strains using gas chromatography. Moreover, n-propyl alcohol was proved to come from α-amino-n-butyric acid or threonine. Small quantities of n-propyl, isobutyl, active amyl and isoamyl alcohols were found in all the fermented solutions. There was some difference in the composition of higher alcohols of the alcoholic solutions fermented by different yeasts.     

非水相脂肪酶催化合成L-抗坏血酸棕榈酸酯的研究Ⅰ

对催化合成Ｌ－捩民血酸棕榈酸本反应的脂肪酶（ＮＯＶＯ４３５、ＭＭＬ、ＬＩＰＯＬＡＳＥ、ＰＰＩ）和反应介质进行比较,得出最佳酶为ＮＯＶＯ４３５,最佳介质为椒戊醇,同时对影响合成Ｌ－抗坏血酸棕榈酸酯反应的初速度的因素（转速、温度、水分含量、酶氏物浓度）进行了探讨,确定了最适反应１条件,转速为２００ｔ／ｍｉｎ,温度为５５℃,水分含量为０,酶浓度为１２．５％。     

3 beta-Hydroxysteroid dehydrogenase of Ruminococcus sp. from human intestinal bacteria

Ruminococcus sp. PO1-3 obtained from human intestinal flora is able to reduce dehydrocholate as well as 3-ketoglycyrrhetinate. From this bacterium dehydrocholate- and 3-ketoglycyrrhetinate-reducing activities were purified one thousand-fold together with 3-ketocholanate-reducing and 3-beta-hydroxyglycyrrhetinate (glycyrrhetic acid) oxidizing activities by means of Matrex Red A, Sephadex G-200 and Octyl-Sepharose column chromatography. The purified enzyme catalyzed the reduction of dehydrocholic acid to 3 beta-hydroxy-7,12-diketocholanic acid and of 3-ketocholanic acid to 3 beta-hydroxycholanic acid. Studies on substrate specificity revealed that the enzyme had absolute specificity for the beta-configuration of a hydroxyl group at the 3 position of bile acid and steroids having no double bond in the A/B ring. This enzyme was neither beta-hydroxysteroid dehydrogenase [EC 1.1.1.51] nor 3 beta-hydroxy-delta 5-steroid dehydrogenase [EC 1.1.1.145], but a novel type of enzyme, defined as 3 beta-hydroxysteroid dehydrogenase.     

Enzyme instability and proteolysis during the purification of an alcohol dehydrogenase from Drosophila melanogaster.

The alcohol dehydrogenase of the Drosophila melanogaster adhUF allele (alloenzyme with ultra-fast electrophoretic mobility) was unstable in crude or partially purified preparations. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that inactivation was porbably due to proteolytic degradation, and new method of purification of the enzyme was developed. After three steps, namely salmine sulphate precipitation, hydroxyapatite chromatography and Sephadex G-100 gel filtration, a 10-fold purified preparation was obtained. The enzyme produced was relatively stable compared with alcohol dehydrogenase purified by other methods, and was shown to be proteinase-free. The enzyme had a subunit mol.wt. of 24000 and had a single thiol residue per subunit available for titration with 5,5'-dithiobis-(2-nitrobenzoic acid). The amino acid composition and C-terminal amino acid sequence of the enzyme were determined. The substrate specificity of this alcohol dehydrogenase was also characterized. These results are discussed in relation to experiments on the evolutionary significance of thermostability at the adh locus.     

On the Formation of Higher Alcohols in the Fermentation by Yeasts

When sugar mixed with certain nitrogenous compounds other than leucine is fermented by yeasts, a small quantity of amyl alcohol is always obtained. We have examined this mechanism and concluded that amyl alcohol is produced from leucine which is caused from the decomposition of yeast protein. The decomposition products of yeast protein also contain valine, but no trace of iso-butyl alcohol was detected after fermentation.     

Evaluation and optimization of immobilized lipase for esterification of fatty acid and monohydric alcohol

Commercial available lipases viz. Lipozyme™, Novozyme-735 and Candida antartica lipase-B (CAL-B) were immobilized on seven different supports by simple adsorption process. The importance of suitable enzyme–support combination in esterification of lauric acid and iso-propanol was validated experimentally. Effect of long chain fatty acids (C4–C18) and small chain monohydric alcohols (C1–C6) on specific activities of different immobilized lipases were evaluated. Lauric acid (C12) was found to be the most preferred fatty acid and t-amyl alcohol (C5) being the best alcohol. CAL-B adsorbed on Lewatit was the most efficient immobilized enzyme for esterification reaction. Selectivity constant for lauric acid (3.4) was the highest among all fatty acids tested, whereas there was not much difference in selectivity between different alcohols. Furthermore, increase in fatty acid unsaturation leads to decrease catalytic efficiency of immobilized CAL-B. The optimum conditions for t-amyllaurate synthesis were as follows: lauric acid—0.5 M, t-amyl alcohol—0.3 M and amount of immobilized enzyme—150 mg. Finally, CAL-B adsorbed on Lewatit was reused for three consecutive cycles.     

Purification and specificity of prolyl dipeptidase from bovine kidney.

Prolyl dipeptidase (iminodipeptidase, L-prolyl-amino acid hydrolase, EC 3.4.13.8) was purified 180-fold from bovine kidney. The enzyme which was obtained in a 10% yield was completely separated from a number of known kidney peptidases including an enzyme of very similar substrate specificity, proline aminopeptidase (L-prolyl-peptide hydrolase, EC 3.4.11.5). The specific activity of the enzyme with L-prolylglycine as substrate is 1600 units of activity per mg protein. Optimum activity of the enzyme is at pH 8.75 and the molecular weight on gel filtration was estimated to be 100 000. The isoelectric point of the enzyme is pH 4.25. Studies of substrate specificity showed that the enzyme preferentially hydrolyzes dipeptides and dipeptidyl amides with L-proline or hydroxy-L-proline at the N-terminus. Longer chain substrates with N-terminal proline were not hydrolyzed.     

The action of Vibrio cholerae and Corynebacterium diphtheriae neuraminidases on the Sendai virus receptor of human erythrocytes.

Studies of the Sendai virus haemagglutinin receptor on the human erythrocyte surface have confirmed that it involves 2 leads to 3 linked sialic acid. Because the primary specificity of Vibrio cholerae neuraminidase is for this linkage, it is able to compete with the virus for the receptor, to which it adsorbs strongly at low temperatures. Corynebacterium diphtheriae neuraminidase, whose principal specificity is for a sialic acid linkage other than 2 leads to 3, does not easily remove Sendai virus receptors, nor does it adsorb to the erythrocyte surface. A new definition of the term "receptor-destroying enzyme" is given which takes both enzyme and virus specificity into account, and a modified assay method is suggested in order to overcome the problems due to enzyme adsorption.     

Microbial degradation of polyethers

This paper summarizes studies on microbial degradation of polyethers. Polyethers are aerobically metabolized through common mechanisms (oxidation of terminal alcohol groups followed by terminal ether cleavage), well-characterized examples being found with polyethylene glycol (PEG). First the polymer is oxidized to carboxylated PEG by alcohol and aldehyde dehydrogenases and then the terminal ether bond is cleaved to yield the depolymerized PEG by one glycol unit. Most probably PEG is anaerobically metabolized through one step which is catalyzed by PEG acetaldehyde lyase, analogous to diol dehydratase. Whether aerobically or anaerobically, the free OH group is necessary for metabolization of PEG. PEG with a molecular weight of up to 20,000 was metabolized either in the periplasmic space (Pseudomonas stutzeri and sphingomonads) or in the cytoplasm (anaerobic bacteria), which suggests the transport of large PEG through the outer and inner membranes of Gram-negative bacterial cells. Membrane-bound PEG dehydrogenase (PEG-DH) with high activity towards PEG 6,000 and 20,000 was purified from PEG-utilizing sphingomonads. Sequencing of PEG-DH revealed that the enzyme belongs to the group of GMC flavoproteins, FAD being the cofactor for the enzyme. On the other hand, alcohol dehydrogenases purified from other bacteria that cannot grow on PEG oxidized PEG. Cytoplasmic NAD-dependent alcohol dehydrogenases with high specificity towards ether-alcohol compound, either crude or purified, showed appreciable activity towards PEG 400 or 600. Liver alcohol dehydrogenase (equine) also oxidized PEG homologs, which might cause fatal toxic syndrome in vivo by carboxylating PEG together with aldehyde dehydrogenase when PEG was absorbed. An ether bond-cleaving enzyme was detected in PEG-utilizing bacteria and purified as diglycolic acid (DGA) dehydrogenase from a PEG-utilizing consortium. The enzyme oxidized glycolic acid, glyoxylic acid, as well as PEG-carboxylic acid and DGA. Similarly, dehydrogenation on polypropylene glycol (PPG) and polytetramethylene glycol (PTMG) was suggested with cell-free extracts of PPG and PTMG-utilizing bacteria, respectively. PPG commercially available is atactic and includes many structural (primary and secondary alcohol groups) and optical (derived from pendant methyl groups on the carbon backbone) isomers. Whether PPG dehydrogenase (PPG-DH) has wide stereo- and enantioselective substrate specificity towards PPG isomers or not must await further purification. Preliminary research on PPG-DH revealed that the enzyme was inducibly formed by PPG in the periplasmic, membrane and cytoplasm fractions of a PPG-utilizing bacterium Stenotrophomonas maltophilia. This finding indicated the intracellular metabolism of PPG is the same as that of PEG. Besides metabolization of polyethers, a biological Fenton mechanism was proposed for degradation of PEG, which was caused by extracellular oxidants produced by a brown-rot fungus in the presence of a reductant and Fe3+, although the metabolism of fragmented PEG has not yet been well elucidated.     

Purification and characterization of an acidic amino acid specific endopeptidase of Streptomyces griseus obtained from a commercial preparation (Pronase)

A protease was purified 163-fold from Pronase, a commercial product from culture filtrate of Streptomyces griseus, by a series of column chromatographies on CM-Toyopearl (Fractogel), Sephadex G-50, hydroxyapatite, and Z-Gly-D-Phe-AH-Sepharose 4B using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gel isoelectric focusing. Studies on the substrate specificity with peptide p-nitroanilides revealed that this protease preferentially hydrolyzed peptide bonds on the carbonyl-terminal side of either glutamic acid or aspartic acid. It was most active at pH 8.8 for the hydrolysis of Boc-Ala-Ala-Pro-Glu-pNA. The molecular weight of the protease was estimated to be 20,000 by gel filtration on Sepharose 6B using 6 M guanidine hydrochloride as an eluent, and 22,000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the enzyme was 8.4. The enzyme was inactivated by diisopropyl phosphofluoridate (DFP) but not by p-chloromercuribenzoate (PCMB) or EDTA.     

Stabilities and rates in the laccase/TEMPO-catalyzed oxidation of alcohols

The influence of alcohol, 4-acetylamino,2,2,6,6'-tetramethylpiperidinyloxy (4-acetylamino-TEMPO) and laccase (from Trametes versicolor, TvL) concentration in the aerobic oxidation of furfuryl alcohol was investigated. Studies show that the K _m for 4-acetylamino-TEMPO is around 6.3 mM (V _max=0.18 mM min^-1) using 6.6 U mL^-1 of laccase and a furfuryl alcohol concentration of 140 mM. Under these optimized conditions, the reaction rate is still dependent on the concentration of enzyme in solution. Laccase can be reused, with a residual activity of around 25%. An important conclusion is that laccase is not stable in the presence of oxoammonium salts, presumably due to degradation via oxidation of essential amino acid residues or the glycosyl moieties on the periphery of the enzyme.     

Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities.