Covalent immobilization of the multienzyme enniatin synthetase

Summary The multienzyme enniatin synthetase was covalently immobilized to N-hydroxysuccinimide activated agarose. The stability of the immobilized enzyme at 25°C was enhanced compared to the soluble enzyme. Immobilization experiments also indicated that the enniatins are synthesized by a single molecule and thus do not require interactions of several enzyme molecules.     

Synthesis of the penicillin precursor, δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV), by an immobilized enzyme preparation

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase activity has been partially-purified from cell-free extracts of Streptomyces clavuligerus by ammonium sulfate precipitation. The salt precipitated enzyme was immobilized on an anion exchange resin and synthesis of ACV was observed by exposing the immobilized enzyme preparation to a reaction mixture containing l--aminoadipic acid, l-valine and l-cysteine in the presence of appropriate cofactors. Reaction mixtures containing l--aminobutyric acid(aB) in place of l-valine synthesized the ACV analog ACaB. Immobilized ACV synthetase can be reused, and after six cycles of reaction, 28.9% of original activity remains.     

Constitutive Expression of Enniatin Synthetase during Fermentative Growth of Fusarium scirpi

The production of enniatins by Fusarium scirpi during fermentative growth in submerged cultures was measured. The fungus produced the antibiotic during mycelial growth, but not during the stationary phase of cultivation. By contrast, enniatin synthetase, the enzyme responsible for enniatin synthesis, was present during growth, during the stationary phase, and even in spores. Similarly, the enniatin synthetase mRNA was present at every stage of the cultivation of the fungus. Therefore, this multifunctional peptide synthetase is a constitutive enzyme, the expression of which is not regulated by any specific mechanism. The findings stand in contrast to the common assumption that production of secondary metabolites underlies regulatory control, leading to separation of the trophophase and the idiophase.     

Mechanism of depsipeptide formation catalyzed by enniatin synthetase

Covalently bound intermediates of enniatin B synthesis could be isolated from enniatin synthetase by treatment with performic acid. By comparison with products of mild alkaline cleavage of authentic enniatin B they could be identified as the dipeptide D-2-hydroxyisovaleryl-N-methylvaline and the corresponding tetrapeptide. Synthesis of enniatins apparently proceeds via condensation of dipeptides. This was confirmed by the use of the substrate analogue isovaleric acid, which has shown to be a strong inhibitor for enniatin synthesis by formation of N-isovaleryl-N-methyl valine.     

Enniatin Production by Fusarium Strains and Its Effect on Potato Tuber Tissue

Several Fusarium strains produce the cyclohexadepsipeptide enniatin, a host-nonspecific phytotoxin. Enniatins are synthesized by the 347-kDa multifunctional enzyme enniatin synthetase. In the present study, 36 Fusarium strains derived from a wide range of host plants were characterized with respect to enniatin production in different media. Thirteen of these strains produced enniatins on one or more of these media. To determine whether enniatin production affected virulence, an assay on potato tuber tissue was performed. Seven enniatin-producing and 16 nonproducing strains induced necrosis of potato tuber tissue, so that enniatin synthesis is not essential for the infection of potato tuber tissue. The application of a mixture of enniatins to slices of potato tuber, however, caused necrosis of the tissue. Therefore, enniatin production by the enniatin-synthesizing strains may affect their pathogenicity. The enniatin synthetase gene (esyn1) of Fusarium scirpi ETH 1536 was used as a probe to determine if similar sequences were present in the strains examined. In Southern blot analyses, DNA sequences hybridizing with the esyn1 probe were present in all but two of the strains examined. In some cases, enniatin-nonproducing strains had the same hybridization pattern as enniatin producers.     

Immobilization and characterization of a thermostable β-galactosidase from a thermophilic anaerobe on a porous ceramic support

A thermostable -galactosidase (EC 3.2.1.23) from a thermophilic anaerobe, strain NA10, was purified from the crude extract of the Escherichia coli transformant harboring the lacN gene. The purified enzyme was physically and covalently immobilized to a porous ceramic support, SM-10. Among the supports tested, the highest residual activity after 3 h incubation at 70° C was obtained when the enzyme was covalently immobilized to silanized SM-10 with 3-[2-(2-amino-ethylaminoethylamino)propyl]trimethoxysilane. The amount of the enzyme immobilized was about 60 mg/g of this support. The enzymatic properties were almost the same as those of the free enzyme. The half-life of this immobilized enzyme was estimated to be approximately 450 h at the pasteurization temperature (65° C).On leave from Aichi Institute of Technology, Yakusa-cho, Toyota 470-03, Japan Correspondence to: T. Saito     

Genetic Changes of Regulatory Mechanisms Occurred in Leucine and Valine Producing Mutants Derived from Brevibacterium lactofermentum 2256

The regulatory mechanisms in branched-chain amino acid synthesis were compared between 2-thiazolealanine (2-TA) resistant l-leucine and l-valine producing mutants and the 2-TA sensitive original strains of Brevibacterium lactofermentum 2256.

In the original strains, sensitive to 2-TA, α-isopropylmalate (IPM) synthetase, the initial enzyme specific for l-leucine synthesis, is sensitive to feedback inhibition and to repression by l-leucine, and α-acetohydroxy acid (AHA) synthetase, the common initial enzyme for synthesis of l-isoleucine, l-valine as well as l-leucine, is sensitive to feedback inhibition by each one of these amino acids, and to repression by them all. In strain No. 218, a typical l-leucine producer resistant to 2-TA, IPM synthetase was found to be markedly desensitized and derepressed, and AHA synthetase remained unaltered. On the contrary, in strain No. 333, l-valine producer resistant to 2-TA, AHA synthetase was found to be desensitized and partially derepressed, and IPM synthetase remained unaltered.

The genetic alteration of these regulatory mechanisms was discussed in connection with the accumulation pattern of amino acids.     

Biosynthesis of PF1022A and related cyclooctadepsipeptides

PF1022A belongs to a recently identified class of N-methylated cyclooctadepsipeptides (CODPs) with strong anthelmintic properties. Described here is the cell-free synthesis of this CODP and related structures, as well as the purification and enzymatic characterization of the responsible synthetase. For PF1022A synthesis extracts of Mycelia sterilia were incubated with the precursors L-leucine, D-lactate, D-phenyllactate, and S-adenosyl-L-methionine in the presence of ATP and MgCl(2). A 350-kDa depsipeptide synthetase, PFSYN, responsible for PF1022A synthesis was purified to electrophoretic homogeneity. Like other peptide synthetases, PFSYN follows a thiotemplate mechanism in which the substrates are activated as thioesters via adenylation. N-Methylation of the substrate L-leucine takes place after covalent binding prior to peptide bond formation. The enzyme is capable of synthesizing all known natural cyclooctadepsipeptides of the PF1022 type (A, B, C, and D) differing in the content of D-lactate and D-phenyllactate. In addition to PF1022 types A, B, C, and D, the in vitro incubations produced PF1022F (a CODP consisting of D-lactate and N-methyl-L-leucine), as well as di-, tetra-, and hexa-PF1022 homologs. PFSYN strongly resembles the well documented enniatin synthetase in size and mechanism. Our results suggest that PFSYN, like enniatin synthetase, is an enzyme with two peptide synthetase domains and forms CODP by repeated condensation of dipeptidol building blocks. Due to the low specificity of the d-hydroxy acid binding site, D-lactate or D-phenyllactate can be incorporated into the dipeptidols depending on the concentration of these substrates in the reaction mixture.     

Synthesis of electroconductive polyaniline using immobilized laccase

A new method for synthesis of the conductive complex between polyaniline (PANI) and poly(2-acrylamido-2-methyl-1-propanosulfonic acid) (PAMPS) was proposed; in this method, the immobilized laccase from the basidiomycete Trametes hirsuta is used as a biocatalyst for aniline oxidative polymerization. The conditions for laccase immobilization on CM cellulose by bifunctional Woodward’s reagent were optimized. The catalytic properties of immobilized and native laccases were compared. The immobilized laccase appeared an efficient catalyst for the oxidative radical polymerization of aniline on polysulfonic acid matrix at 4°C. It was demonstrated that the immobilized enzyme could be repeatedly used for enzymatic synthesis of this polymer. Several spectral characteristics of the PANI/PAMPS complexes synthesized at various pH values were studied. The conductance of PANI specimens produced using immobilized laccase as a catalyst was 13 mS/cm.     

Expression of secreted His-tagged S-adenosylmethionine synthetase in the methylotrophic yeast Pichia pastoris and its characterization, one-step purification, and immobilization

S-Adenosylmethionine synthetase (SAM synthetase) catalyzes the synthesis of S-adenosylmethionine (SAM), which plays an important role in cellular functions such as methylation, sulfuration, and polyamine synthesis. To develop a simple and effective way to enzymatically synthesize and produce SAM, a soluble form of SAM synthetase encoded by SAM2 from Saccharomyces cerevisiae was successfully produced at high level ( approximately 200 mg/L) by the recombinant methylotrophic yeast Pichia pastoris. The secreted His6-tagged SAM synthetase was purified in a single chromatography step with a yield of approximately 82% for the total activity. The specific activity of the purified synthetase was 23.84 U/mg. The recombinant SAM synthetase could be a kind of allosteric enzyme with negative regulation. The enzyme functioned optimally at a temperature of 35 degrees C and pH 8.5. The stability of the recombinant synthetase and the effectiveness of different factors in preventing the enzyme from inactivation were also studied. Additional experiments were performed in which the recombinant SAM synthetase was purified and immobilized in one step using immobilized metal-chelate affinity chromatography. The immobilized synthetase was found to be 40.4% of the free enzyme activity in catalyzing the synthesis of SAM from dl-Met and ATP.     

Enzymatic propyl gallate synthesis in solvent-free system: Optimization by response surface methodology

The ability of a non-commercial immobilized Staphylococcus xylosus lipase to catalyze the esterification of propanol with gallic acid was investigated and the antioxidant as well as the antimicrobial activities of the ester formed were evaluated. The response surface methodology, based on a three variables Box–Behnken design (reaction temperature, enzyme amount and 1-propanol/gallic acid molar ratio), was used to optimize the experimental conditions of propylgallate synthesis. The maximum conversion yield (90% ±3.5) was obtained by using 400 IU of immobilized lipase and a propanol/gallic acid at a molar ratio of 160 at 52 °C. The obtained ester was characterized by spectroscopic methods, NMR and FTIR. The antioxidant activity of propyl gallate was evaluated and compared to the synthetic classical antioxidants, BHA and ascorbic acid, taken as references. In addition, the antimicrobial activity of the propyl gallate was tested against S. xylosus, Escherchia coli and Staphylococcus aureus using disc diffusion and macrodilution methods. Our results show that the synthesized propyl gallate ester presents a higher antioxidant and antimicrobial power than the parent gallic acid as well as the synthetic classical antioxidants.     

Fatty acid synthetase from pigeon red blood cells

• 1.1. Fatty acid synthetase has been purified 200-fold from pigeon erythrocytes.
• 2.2. The enzyme gave 2 major staining bands on disc gel electrophoresis corresponding to the complex and dissociated forms of the enzyme.
• 3.3. Sucrose density gradient centrifugation of the enzyme showed only one sedimenting peak and high performance liquid chromatography also showed only 1 major light absorbing peak.
• 4.4. The molecular weight of the enzyme was estimated to be 300,000–330,000 and the enzyme is comprised of 2 subunits of similar molecular weights.
• 5.5. The red blood cell fatty acid synthetase was found to be immunochemically nonidentical with the liver fatty acid synthetase.

     

The control of glutamine synthetase level in Lemna minor L.

Summary The specific activity of glutamine synthetase (E.C. 6.3.1.2) of Lemna minor L. is markedly reduced when either ammonium ions or glutamine are present in the growth medium. Combinations of 5 mM ammonia and 5 mM glutamic acid or 5 mM ammonia and 5 mM glutamine as nitrogen source, lead to a 4–5 fold reduction of the maximum activity measurable on 5 mM -aminobutyric acid. Analyses of the soluble pool of nitrogen indicate that the reduction in enzyme level is associated with an increase in the pool of glutamine. There is an inverse correlation between the apparent rate of synthesis of glutamine synthetase and the intracellular concentration of glutamine, and this relationship suggests that the glutamine synthetase of Lemna minor is subject to end product repression by the endogenous pool of glutamine.     

Purification and regulation of glutamine synthetase in a collagenolytic Vibrio alginolyticus strain

Glutamine synthetase (EC 6.3.1.2) has been purified from a collagenolytic Vibrio alginolyticus strain. The apparent molecular weight of the glutamine synthetase subunit was approximately 62,000. This indicates a particle weight for the undissociated enzyme of 744,000, assuming the enzyme is the typical dodecamer. The glutamine synthetase enzyme had a sedimentation coefficient of 25.9 S and seems to be regulated by a denylylation and deadenylylation. The pH profiles assayed by the -glutamyltransferase method were similar for NH₄-shocked and unshocked cell extracts and isoactivity point was not obtained from these eurves. The optimum pH for purified and crude cell extracts was 7.9. Cell-free glutamine synthetase was inhibited by some amino acids and AMP. The transferase activity of glutamine synthetase from mid-exponential phase cells varied greatly depending on the sources of nitrogen or carbon in the growth medium. Glutamine synthetase level was regulated by nitrogen catabolite repression by (NH₄)₂SO₄ and glutamine, but cells grown, in the presence of proline, leucine, isoleucine, tryptophan, histidine, glutamic acid, glycine and arginine had enhanced levels of transferase activity. Glutamine synthetase was not subject to glucose, sucrose, fructose, glycerol or maltose catabolite repression and these sugars had the opposite effect and markedly enhanced glutamine synthetase activity.Abbreviations GS glutamine synthetase - SMM succinate minimal medium - ASMM ammonium/succinate minimal medium - GT -glutamyl transferase - SVP snake venom phosphodiesterase     

Synthesis and proteolytic activation of chitin synthetase in Phycomyces blakesleeanus Burgeff

The chitin synthetase of Phycomyces blakesleeanus mycelium is a particulate enzyme sedimenting mostly at 1000xg. The activity in crude extracts or cellular fractions can be increased more than tenfold by mild trypsin treatment. Plotting the reaction velocity versus UDP-N-acetylglucosamine concentration yields a sigmoidal curve. N-acetylglucosamine, which greatly stimulates the enzyme, changes the kinetics to an almost normal hyperbolic relationship.The enzyme is nearly absent in dormant spores and is synthesized de novo in germinating spores (from 4 h germination on). Trypsin treatment of extracts from germinating spores to assay the synthesis of the proenzyme did not reveal an earlier synthesis of the zymogen, which therefore might have some activity of its own.Abbreviations Used UDP-GlcNAc Uridinediphosphate-N-acetylglucosamine - GlcNAc N-acetylglucosamine - Chitin synthetase UDP-2-acetylamino-deoxyglucosyltransferase (EC 2.4.1.16)     

The influence of environmental and incubation temperature on fatty acid synthetase from flounder (Platichthys flesus L.) liver

• 1.1. Fatty acid synthetase from liver of cold and warm adapted flounder and rabbit was purified to homogenity and compared.
• 2.2. The mol. wt of the cold and warm flounder enzyme was estimated to be about 457,000.
• 3.3. The kinetic properties were found to be similar for warm and cold adapted flounder liver enzyme and not different from the rabbit liver enzyme when measured at 5, 10, 15, 20 and 37°C.
• 4.4. Palmitic acid was the main product of both the flounder and rabbit enzyme, but significant amounts of butyric acid were also synthesized. The product composition did not change for any of the enzymes tested when the incubation temperature was changed.
• 5.5. It was concluded that fatty acid synthetase from flounder liver is similar to mammalian fatty acid synthetase with regard to molecular weight and kinetic properties.

     

Coordinate regulation of acetyl coenzyme A carboxylase and fatty acid synthetase in liver cells of the developing chick in vivo and in culture.

The activities of hepatic acetyl-CoA carboxylase and fatty acid synthetase undergo two distinct types of development in the perinatal chick. The first increase begins prior to hatching, continues after hatching in the starved chick, and is independent of feeding. The second increase is caused by feeding and is reversed by starvation (A. G. Goodridge (1973) J. Biol. Chem.248, 1932–1938). We have purified these enzymes to homogeneity and raised antibodies to them in rabbits. Using immunochemical techniques we have established that the activity changes in both types of development were a function of changes in the concentrations of enzyme proteins. All activity changes were accompanied by similar changes in the relative rates of synthesis of the two enzymes. Regulation of the activities of acetyl-CoA carboxylase and fatty acid synthetase was further characterized in liver cells from 19-day-old embryos maintained in culture in a chemically defined medium. After 3 days in culture in the absence of hormones, the activities of the enzymes increased significantly with respect to the activities of the freshly prepared cells. Addition of either insulin or triiodothyronine alone caused additional small increases. Insulin plus triiodothyronine caused 8- and 15-fold increases in acetyl-CoA carboxylase and fatty acid synthetase, respectively, relative to cells incubated without hormones. In the presence of insulin alone glucagon had no effect on the activity of either enzyme. In the presence of insulin plus triiodothyronine, glucagon inhibited the increase in enzyme activities by about 75%. The results of quantitative immunoprecipitin tests indicated that activity changes caused by the various hormones were functions of changes in the concentrations of the enzyme proteins. The effects of the hormones on enzyme activities were accompanied by comparable or larger changes in the relative rates of synthesis of the enzymes. Under a wide variety of experimental conditions, both in vivo and in culture, the relative rates of synthesis of acetyl-CoA carboxylase and fatty acid synthetase are regulated coordinately. Under some of these conditions, synthesis of malic enzyme also is regulated coordinately with the syntheses of acetyl-CoA carboxylase and fatty acid synthetase. The common intracellular mechanisms underlying the coordinate control remain to be elucidated.     

Synthesis of antioxidant propyl gallate using tannase from Aspergillus niger van Teighem in nonaqueous media

Tannase from Aspergillus niger van Teighem has been used for synthesis of food additive antioxidant propyl gallate by direct transesterification of tannic acid. The optimized yield of 86% was obtained by using simultaneously pH tuned enzyme, immobilized on Celite and using the right amount of water in the non aqueous media.     

The effect of prolonged fasting on total lipid synthesis and enzyme activities in the liver of the European eel (Anguilla anguilla)

• 1.1. The extent of fatty acid synthesis from [1-¹⁴C]acetate in liver slices was reduced 6-fold when eels were fasted for 1–7 weeks and 20-fold when fasted for 39 weeks; thereafter hepatic lipogenesis seemed to remain constant for up to 95 weeks of fasting.
• 2.2. After a 1–3 week fast some hepatic enzyme activities were reduced (acetyl-CoA carboxylase decreased 2-fold and fatty acid synthetase declined 5-fold), while others remained unchanged (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, α-glycerol phosphate dehydrogenase as well as malic enzyme and ATP-citrate lyase).
• 3.3. The optimum temperature for measuring both total lipid synthesis and lipogenic enzyme activity in eel liver was found to be 30°C.

     

Stabilization of an intracellular Mucor circinelloides lipase for application in non-aqueous media

Different methods for stabilization of Mucor circinelloides lipase, facilitating its application in organic solvents were tested. Lipase was either isolated from the mycelium and immobilized on solid carriers (derivatives of cellulose, diatomaceous earth, modified porous glass) or immobilized in situ in the mycelium pellets and stabilized. The immobilized enzyme preparations were used for synthesis of sucrose, glucose, butyl and propyl oleates and caprylates, carried out in petroleum and di-n-pentyl ethers. Immobilized preparations of either crude or purified lipase isolated from the mycelium were at least 4–6 times less effective in sucrose esters synthesis than mycelium-bound lipase preparations. Lipase preparation with the highest synthetic activity was obtained by cross-linking of M. circinelloides mycelium pellets with glutardialdehyde (operational stability in sucrose caprylate synthesis was 94% after 4 runs (24 h each), and caprylic acid conversion was 91–85%). The best method for production of mechanically durable biocatalyst, which efficiently catalyzed sucrose esters synthesis, was found to be entrapment of the mycelium-bound lipase in polyvinyl pyrrolidone-containing chitosan beads solidified with hexametapolyphosphate.