Mode of action of pectic enzymes. II. Further purification of exopolygalacturonate lyase and pectinesterase from Clostridium multifermentans

Exopolygalacturonate lyase and pectinesterase from Clostridium multifermentans were purified 156-fold and 178-fold, respectively, by gel filtration chromatography on Sephadex G-200. The activities of both enzymes coincided in a single protein peak. Profiles of the two activities also coincided in diethylaminoethyl-cellulose chromatography and zonal centrifugation. These studies indicated that the esterase and the lyase were either complexed or similar molecular species. The former seems more probable because of the relatively high molecular weight. Both activities were most stable at pH 6.0. The esterase was inactivated rapidly at pH 5 or 7. Lyase preparations were freed of pectinesterase activity by heating for 30 min at 38 C and pH 7.0.     

Inducible thermoalkalophilic polygalacturonate lyase from Thermomonospora fusca.

A thermostable polygalacturonate lyase (PL; EC 4.2.2.2) was secreted by Thermomonospora fusca during stationary phase in pectin-mineral salts medium at 52 degrees C. Biosynthesis was induced by addition of pectic substances to cultures growing on glucose or cellulose but not cellobiose; the disaccharide repressed enzyme synthesis and triggered inactivation of enzyme previously secreted. The PL, purified to electrophoretic and serologic homogeneity, had a molecular size of 56 kilodaltons and an isoelectric point at pH 4.16. The amino acid composition closely resembled that of the major extracellular endoglucanases of the actinomycete. The enzyme had six cystine residues but no detectable sulfhydryl groups. It was inactivated by mild reducing agents and activated by oxygenation, indicating the necessity for disulfide bond maintenance. Temperature and pH optima for the PL reaction were 60 degrees C and 10.45, respectively. Calcium was essential for activity but not stability; calcium dependence curves were altered by low concentrations of toxic metals. The Km for pectin increased 30,000-fold as the percent esterification (methoxylation) of that substrate was increased from 0 to 60%. The size of the minimal susceptible site for PL attack on the pectin molecule was calculated as being equivalent to 10 unesterified residues, based on the correlation of Km values at various degrees of esterification with the percentage of cleavable bonds predicted by a random-number-generating computer program.     

The enzyme complex citramalate lyase from Clostridium tetanomorphum.

1. The enzyme citramalate from Clostridium tetanomorphum is not stable in crude extracts. However, the inactive enzyme can be reactivated by incubation with dithioerythritol followed by acetylation with acetic anhydride. Reactivation was also obtained with acetate, ATP, MgCl2 and acetate : SH-enzyme ligases (AMP) from C. tetanomorphum or Klebsiella aerogenes. 2. Incubation of the inactive enzyme with iodoacetate resulted in rapid loss of enzymic activity as determined by reactivation with acetic anhydride whereas the active enzyme was stable in the presence of iodoacetate. Using ido[2-(14)C]acetate the sites of carboxymethylation and acetylation where identified as cysteamine residues of the enzyme. The results demonstrate that the active enzyme contains acetyl thiolester residues which play the central role in the catalytic mechanism. 3. Citramalate lyase was purified by a procedure almost identical to that already described for citrate lyase from K. aerogenes. The molecular weight of citramalate lyase is equal to that of citrate lyase (Mr = 5.2--5.8 X 10(5)) as estimated by gel chromatography and sucrose gradient centrifugation. Polyacrylamide gel elctrophoresis of citramalate lyase in sodium dodecylsulfate yielded three polypeptide chains (Mr: alpha 5.3--5.6 X 10(4); beta 3.3--3.6 X 10(4); gamma 1.0--1.2 X 10(4)) in probably equal molar amounts. These data lead to a hexameric structure (alpha,beta,gamma)6 of the complete enzyme. 4. Pantothenate (5 mol/mol of enzyme) and the essential cysteamine residues were exclusively present in the gamma-chain, the acyl carrier protein of citramalate lyase. The acyl exchange and cleavage functions, probably catalysed by the alpha and beta-subunits, were measured with acyl-CoA derivatives which were able to substitute for the natural acyl carrier. 5. The results demonstrate that citramalate lyase is an enzyme complex with structure and functions closely resembling those of citrate lyase. Although the similarity between citramalate lyase and citrate lyases from various organisms suggests a close evolutionary relationship, these occur in very different, unrelated bacteria. A parallel situation found in the distribution of the nitrogenase system among procaryotes is discussed.     

Production and properties of polygalacturonate lyase by an alkalophilic microorganism Bacillus sp. RK9.

Bacillus sp. RK9 was isolated from soil and produced a constitutive polygalacturonate lyase. Production of the enzyme required the presence of complex nitrogen (peptone and yeast extract). Highest activity was obtained with an initial pH of 9.7. The organism was alkalophilic. No growth occurred below pH 7.5. The enzyme was purified by salt precipitation and diethylaminoethyl (DEAE) cellulose ion-exchange chromatography. The pH optimum for activity was 10.0 in 0.01 M glycine-NaOH buffer. Calcium alone, of divalent cations, activated the enzyme by 2.9-fold. Complete inhibition of enzyme activity was achieved by 1 mM ethylenediaminetetraacetic acid (EDTA). Hydrolysis of substrate occurred in a random fashion and the enzyme was 50% more active towards acid soluble pectic acid (ASPA) than towards sodium polypectate.     

Isolation and characterization of an extracellular glycosylated protein complex from Clostridium thermosaccharolyticum with pectin methylesterase and polygalacturonate hydrolase activity.

An extracellular protein complex was isolated from the supernatant of a pectin-limited continuous culture of Clostridium thermosaccharolyticum Haren. The complex possessed both pectin methylesterase (EC 3.1.1.11) and exo-poly-alpha-galacturonate hydrolase (EC 3.2.1.82) activity and produced digalacturonate from the nonreducing end of the pectin chain. The protein consisted of 230- and 25-kDa subunits. The large subunit contained 10% (wt/wt) sugars (N-acetylgalactosamine and galactose). Under physiological conditions both activities acted in a coordinated manner: the ratio between methanol and digalacturonate released during degradation was constant and equal to the degree of esterification of the pectin used. Prolonged incubation of the enzyme with pectin led to a nondialyzable fraction that was enriched in neutral sugars, such as arabinose, rhamnose, and galactose; the high rhamnose/galacturonic acid ratio was indicative of hairy region-like structures. The smallest substrate utilized by the hydrolase was a tetragalacturonate. Vmax with oligogalacturonates increased with increasing chain length. The Km and Vmax for the polygalacturonate hydrolase with citrus pectate as a substrate were 0.8 g liter-1 and 180 mumol min-1 mg of protein-1, respectively. The Km and Vmax for the esterase with citrus pectin as a substrate were 1.2 g liter-1 and 440 mumol min-1 mg of protein-1, respectively. The temperature optima for the hydrolase and esterase were 70 and 60 degrees C, respectively. Both enzyme activities were stable for more than 1 h at 70 degrees C. The exo-polygalacturonate hydrolase of Clostridium thermosulfurogenes was partially purified while the methylesterase was also copurified.     

Cellular and environmental factors affecting the synthesis of polygalacturonate lyase byBacillus subtilis

Summary Cellular and environmental factors affecting the synthesis of polygalacturonate lyase in batch and chemostat cultures ofBacillus subtilis were investigated. The lyase was produced constitutively during growth on a wide range of carbon sources in a defined minimal medium and in medium containing complex organic carbon and nitrogen sources. The highest activity was obtained during batch growth in minimal medium containing glucose and ammonium sulphate. Over 99% of the activity was present extracellularly in the supernatant medium at all stages of the batch growth cycle. Two distinct differential rates of synthesis were observed during exponential growth. The lyase was unable to attack pectin rapidly unless pectin methyl-esterase was also present. Pectin was a poor substrate for growth and polygalacturonate lyase induction because the organism did not produce pectin methyl-esterase. In continuous-flow chemostat cultures with glucose medium, polygalacturonate lyase activity declined to a very low level owing to the selection of non-productive mutant strains. Loss of activity did not occur when polypectate was the carbon source. Steady-state specific polygalacturonate lyase activity in polypectate medium was relatively independent of dilution rate in the range 0.04 to 0.36/h. When polypectate was supplied in excess of the growth requirement lyase activity was 5 times higher than during polypectate-limited growth.     

Lowering induction temperature for enhanced production of polygalacturonate lyase in recombinant Pichia pastoris

Various effects of temperature on heterologous alkaline polygalacturonate lyase produced in recombinant Pichia pastoris were investigated. The results indicated that PGL activity could be improved significantly by decreasing the cultivation temperature. It was reached 931 U/mL with temperature lowered to 22 °C at the beginning of induction phase, which were 2.1-fold and 2.9-fold increase compared to that at 30 and 26 °C. The mechanisms behind the temperature effect on recombinant PGL production may be ascribed to poor cell viability, decrease of intracellular adenosine phosphate levels, of AOX activity but increase of extracellular proteases activities. Our study demonstrated that cultivation at lower temperatures resulted in higher cell viability, significant improvement of PGL stability and an increase intracellular AOX activity, but a lower activity of released host proteases which possibly caused the degradation of recombinant PGL. In addition, the evidence of higher intracellular adenosine phosphate levels but lower energy charge level was provided at a lower temperature induction.     

添加保护剂促进碱性果胶酶的工业保藏稳定性

保藏稳定性对于商品酶具有重要的实际意义。通过对碱性果胶酶的保护剂进行研究,最终筛选得到最优复合保护剂配方为：Tween201mL／L,六偏磷酸钠1．5g／L,明胶5g／L,甘油5％（口～）,CaCl22g／L;热稳定性提高18倍。此外在常温保藏时,在酶液中添加0．3‰山梨酸钾和0．3‰苯甲酸钠,能有效提高保藏效果,减少酶液染茵、混浊和发臭,最终常温保藏90d后酶活保留率为84．5％,达到工业化保藏的要求。     

Addition of protective agents for enhancing storage stabilization of alkaline polygalacturonate lyase

保藏稳定性对于商品酶具有重要的实际意义.通过对碱性果胶酶的保护剂进行研究,最终筛选得到最优复合保护剂配方为:Tween 20 1 mL/L,六偏磷酸钠1.5g/L,明胶5g/L,甘油5％(v/v),CaCl2 2 g/L;热稳定性提高18倍.此外在常温保藏时,在酶液中添加0.3‰山梨酸钾和0.3‰苯甲酸钠,能有效提高保藏效果,减少酶液染菌、混浊和发臭,最终常温保藏90d后酶活保留率为84.5％,达到工业化保藏的要求.     

Citramalate lyase of Clostridium tetanomorphum

Summary Assay methods and some properties of (+)-citramalate pyruvatelyase, an enzyme from Clostridium tetanomorphum that converts (+)-citramalate to pyruvate and acetate, are described. The enzyme is very active (0.8–1.2 units per mg protein) in freshly prepared extracts, but loses activity rapidly during storage. (+)-Citramalate is the only substrate found to be cleaved by the lyase; the equilibrium for the reaction permits almost complete cleavage at low substrate concentrations. A divalent cation is required as a cofactor. A sensitive and specific enzymic method for estimating (+)-citramalate is described.It is a pleasure to dedicate this paper to Prof. C. B. Van Niel who first awakened the interest of the author in the problems of bacterial metabolism and, more specifically, in the fermentation of glutamic acid.This work was supported in part by a research grant from the National Institutes of Health (AI-00563), U.S. Public Health Service, and by funds from the California Agricultural Experiment Station     

Similarities in the Action Patterns of Exopolygalacturonate Lyase and Pectinesterase from Clostridium multifermentans

Exopolygalacturonate lyase and pectinesterase from Clostridium multifermentans were assayed simultaneously in the same reaction mixture which contained a highly esterified pectin, polymethyl polygalacturonic acid methyl glycoside. Lyase is specific for unesterified galacturonide residues and cannot degrade this substrate in the absence of the esterase. The rate for esterase was twice the rate for lyase throughout the entire course of the combined reaction. Thus, the molar ratio of the two enzyme activities was the same since the product of the lyase is an unsaturated digalacturonic acid containing two free carboxyl groups. Since clostridial exopolygalacturonate lyase is known to degrade polygalacturonate in a linear manner beginning from the reducing ends of polygalacturonate chains, it was apparent that clostridial pectinesterase must hydrolyze methyl groups in highly esterified pectins with an action pattern similar to that of the lyase. Otherwise it would be impossible for the two enzyme rates to have corresponded on the basis of a 2:1 ratio.     

Purification and characterization of N-acetylneuraminate lyase from Clostridium perfringens.

Clostridium perfringens cells were cultivated on a large scale using an automatic system. 2) N-Acetylneuraminate lyase, which is a cytosolic enzyme, was liberated from the bacteria by cell lysis using lysozyme in hypotonic solution. The enzyme was purified 770-fold by precepitation with ammonium sulfate, filtration on Sephadex A-50 and final preparative electrophoresis in a 7.5% polyacrylamide gel. Yield: 12 mg from 1 kg wet cell paste; specific activity: 167 nkat/mg protein. 3) The enzyme preparation appeared homogeneous in analytical disc electrophoresis, in gel electrophroesis in 0.1% sodium dodecylsulfate or 8m urea and in immunoelectrophoresis. Contaminating enzyme activities were not detected. 4) The isoelectric point of pH 4.7 was found for the enzyme. At 278 nm a molar extinction coefficient of 6.4 x 10(4)M-1 Xcm-1 was determined. The enzyme exhibited a Km value for N-acetylneuraminic acid of 2.8mM at its pH optimum of pH 7.2. The pH dependence of the Km value gives evidence that an ionizing guoup in the active center of the enzyme with a pKe value of 6.4 may be involved in the catalytic reaction. Pyruvate inhibited the cleavage reaction of N-acetylneuraminic acid competitively; Ki = 2.9mM. 5) An average molecular weight of 99200 was determined for the native enzyme using different methods. After denaturation in sokium dodecylsulfate or urea, a mean molecular weight of only 50000 could be demonstrated, indicating the existence of two enzyme subunits. The lyase molecule was shown by electron microscopy, using a negative staining technique, to consist of two hemispherical parts. 6) Two active sites per native enzyme molecule, probably corresponding to one active site per subunit, were found by incubation of the enzyme with radioactive pyruvate followed by borohydride reduction. The results obtained from chemical modification of the lyase with 5-diazonium-1H-tetrazole and iodocaetamide under various conditionsare interpreted as evidence for the presence of two reactive histidine residues in the enzyme molecule. It is probable that one residue per subunit forms the nucleophilic group participating in enzyme catalysis. A model suggesting the mechanism of reversible cleavage of N-acylneuraminic acids by the lyase is presented.     

Improved production of alkaline polygalacturonate lyase by homologous overexpression pelA in Bacillus subtilis

A polygalacturonate lyase (PGL), PelA, was purified from the culture broth of Bacillus subtilis 7-3-3, with a molecular weight, optimal temperature, and pH of approximately 45 kDa, 55 °C, and 9.4, respectively. The PGL gene (pelA) was homologously overexpressed in B. subtilis 7-3-3 to increase the gene copies and enhance the PGL production. The resulting PGL activity was 2138 U mL^?1 at 44 h, and the productivity reached 48.58 U (mL h)^?1 through the homologous overexpression of strain B-pN-pelA in a 7.5 L fermentor, the highest PGL production compared to those reported in literature to the best of our knowledge. Crude enzyme has high PGL and PGase activity, which can remove 50.58% of pectin in unpretreatment ramie fibers at 50 °C for 4 h. Meanwhile, the enzyme system with a low level hemicellulase and almost no cellulase will further help in enhancing the efficiency of degumming besides maintaining tenacity of plant fiber. The B. subtilis B-pN-pelA shows high genetic stability and has great potential in the textile industry.     

Characterization of citrate lyase from Clostridium sporosphaeroides

Cells of Clostridium sporosphaeroides which were grown on citrate contained citrate lyase and citrate lyase acetylating enzyme, but no detectable citrate synthase and citrate lyase deacetylase activities. Citrate lyase from C. sporosphaeroides was purified to homogeneity as judged by polyacrylamide gel electrophoresis and high performance liquid chromatography. In contrast to the enzyme from Clostridium sphenoides, the addition of l-glutamate was not necessary for activity and stabilization of the enzyme. The purified enzyme had a specific activity of 34 U/mg protein and was comparable to other citrate lyases with respect to its molecular weight and subunit composition. Electron microscopic investigations showed that similar to the lyase from C. sphenoides and in contrast to all other citrate lyases examined so far, the majority of the enzyme molecules was present in star form.     

混合碳源流加对重组毕赤酵母生产碱性果胶酶的影响

为提高重组毕赤酵母生产碱性果胶酶(PGL)的产量和生产强度,在诱导期采用多种碳源与甲醇混合添加的模式。实验结果发现:甘油、山梨醇、乳酸与甲醇的混合添加均可以提高PGL的产量,其中山梨醇与甲醇的混合流加效果最为显著。研究表明,通过双碳源混合流加可以提高细胞活力,增强醇氧化酶活力,提高毕赤酵母表达外源蛋白效率。当山梨醇的流速为3.6g/(h·L)时,PGL酶活可达1593U/mL,生产强度为16.7U/(mL·h),比对照分别提高了84.6%和45.2%,实现了碱性果胶酶的高效生产。     

Overproduction of alkaline polygalacturonate lyase in recombinant Escherichia coli by a two-stage glycerol feeding approach

This work aims to achieve the overproduction of alkaline polygalacturonate lyase (PGL) with recombinant Escherichia coli by a two-stage glycerol feeding approach. First, the PGL coding gene from Bacillus subtilis WSHB04-02 was expressed in E. coli BL21 (DE3) under the strong inducible T7 promoter of the pET20b (+) vector. And then the influence of media composition, induction temperature, and inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration on cell growth and PGL production was investigated. Finally, a two-stage glycerol feeding strategy was proposed and applied in a 3-L fermenter, where cultivation was conducted at a controlled specific growth rate (μset=0.2) during pre-induction phase, followed by a constant glycerol feeding rate of 12 ml h(-1) at post-induction phase. The total PGL yield reached 371.86 U mL(-1), which is the highest PGL production by recombinant E. coli expression system.     

On the mechanism of action of isocitrate lyase.

1. The enzymes citrate lyase and isocitrate lyase catalyse similar reactions in the cleavage of citrate to acetate plus oxaloacetate and of isocitrate to succinate plus glyoxylate, respectively. 2. Nevertheless, the mechanism of action of each enzyme appears to be different from each other. Citrate lyase is an acyl carrier protein-containing enzyme complex whereas isocitrate lyase is not. The active form of citrate lyase is an acetyl-S-enzyme but that of isocitrate lyase is not a corresponding succinyl-S-enzyme. 3. In contrast to citrate lyase, the isocitrate enzyme is not inhibited by hydroxylamine nor does it acquire label if treated with appropriately labelled radioactive substrate. 4. Isotopic exchange experiments performed in H18-2O with isocitrate as a substrate produced no labelling in the product succinate. This was shown by mass-spectrometric analysis. 5. The conclusion drawn from these results is that no activation of succinate takes place on the enzyme through transient formation of succinic anhydride or a covalently-linked succinyl-enzyme, derived from this anhydride.     

Immobilization of alkaline polygalacturonate lyase from Bacillus subtilis on the surface of bacterial polyhydroxyalkanoate nano-granules

Applied Microbiology and Biotechnology - Alkaline polygalacturonate lyase (PGL), one of the pectinolytic enzymes, has been widely used for the bioscouring of cotton fibers, biodegumming, and...