Purification and characterization of the NADH-dependent butanol dehydrogenase from Clostridium acetobutylicum (ATCC 824)

Two butanol dehydrogenases with different cofactor requirements and different pH ranges have been detected in Clostridium acetobutylicum ATCC 824. The NADH-dependent butanol dehydrogenase (NADH-BDH) was purified to near homogeneity and characterized. One striking feature of the enzyme is that Zn2+ was needed to obtain a significant recovery during purification. The enzyme was a dimer composed of two subunits with subunit molecular mass of 42 kDa and a native molecular mass of 82 +/- 2 kDa. The kinetics were studied in the direction of the reduction of butyraldehyde. Inhibition studies with S-NADH and butanol indicate that the NADH-BDH follows an ordered bibi mechanism with kinetic constants of 4.86 s-1, 0.18 mM, and 16 mM for Kcat, KNADH, and Kbutyraldehyde, respectively. Activity in the reverse direction was 50-fold lower than that in the forward direction. The NADH-BDH had higher activity with longer chained aldehydes and was inhibited by metabolites containing an adenine moiety.     

Autolysis of Clostridium acetobutylicum ATCC 824.

The optimum conditions for autolysis of Clostridium acetobutylicum ATCC 824 were determined. Autolysis was optimal at pH 6.3 and 55 degrees C in 0.1 M-sodium acetate/phosphate buffer. The ability of cells to autolyse decreased sharply at the end of the exponential phase of growth. Lysis was stimulated by monovalent cations and compounds that complex divalent cations, and inhibited by divalent cations. The autolysin of C. acetobutylicum, which was mainly cytoplasmic, was purified to homogeneity and characterized as a muramidase. The enzyme was identical to the extracellular muramidase in terms of M(r), isoelectric point and NH2-terminal amino acid sequence. The autolysin was inhibited by lipoteichoic acids and cardiolipin but not by phosphatidylethanolamine and phosphatidylglycerol. A mechanism of regulation and fixation involving lipoteichoic acid, cardiolipin and divalent cations is proposed.     

非离子表面活性剂对生物丁醇发酵的影响

传统的丙酮-丁醇发酵的产物浓度过低(丁醇终浓度约为1.3 wt%),导致后期分离成本过高,从而影响了该过程的经济性,限制了其工业化进程。本文研究了高添加量的小分子非离子表面活性剂对生物丁醇发酵的影响。以吐温80为例,实验表明,当表面活性剂添加量超过其临界胶束浓度后,丁醇发酵的终浓度会随着表面活性剂添加量的增加而增加。当添加量达到5 wt%时,丁醇终浓度可以达到1.6 wt%,远高于该菌种的抑制浓度(0.8 wt%)。为阐明表面活性剂的作用机理,实验考察了吐温80对丁醇的增溶效应以及对发酵菌体表面亲疏水性的影响。结果表明,吐温80对丁醇的增溶效果很小,而对菌体表面的亲疏水性有较明显的影响。     

Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum ATCC 824.

Butanol, at sub-growth-inhibitory levels, caused a ca. 20 to 30% increase in fluidity of lipid dispersions from Clostridium acetobutylicum. When grown in the presence of butanol or into stationary phase, C. acetobutylicum synthesized increased levels of saturated acyl chains at the expense of unsaturated chains.     

Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum ATCC 824

Butanol, at sub-growth-inhibitory levels, caused a ca. 20 to 30% increase in fluidity of lipid dispersions from Clostridium acetobutylicum. When grown in the presence of butanol or into stationary phase, C. acetobutylicum synthesized increased levels of saturated acyl chains at the expense of unsaturated chains.     

Biotransformation of furfural and 5-hydroxymethyl furfural (HMF) by Clostridium acetobutylicum ATCC 824 during butanol fermentation

The ability of fermenting microorganisms to tolerate furan aldehyde inhibitors (furfural and 5-hydroxymethyl furfural (HMF)) will enhance efficient bioconversion of lignocellulosic biomass hydrolysates to fuels and chemicals. The effect of furfural and HMF on butanol production by Clostridium acetobutylicum 824 was investigated. Whereas specific growth rates, μ, of C. acetobutylicum in the presence of furfural and HMF were in the range of 15-85% and 23-78%, respectively, of the uninhibited Control, μ increased by 8-15% and 23-38% following exhaustion of furfural and HMF in the bioreactor. Using high performance liquid chromatography and spectrophotometric assays, batch fermentations revealed that furfural and HMF were converted to furfuryl alcohol and 2,5-bis-hydroxymethylfuran, respectively, with specific conversion rates of 2.13g furfural and 0.50g HMF per g (biomass) per hour, by exponentially growing C. acetobutylicum. Biotransformation of these furans to lesser inhibitory compounds by C. acetobutylicum will probably enhance overall fermentation of lignocellulosic hydrolysates to butanol.     

Enhanced butanol production and reduced autolysin activity after chloramphenicol treatment of Clostridium acetobutylicum ATCC 824

Summary Release of autolysin during the late exponential growth phase of Clostridium acetobutylicum resulted in early lysis of the culture and reduction of solvent formation. A simple and effective way of reducing autolysin activity and increasing solvent production is partial inhibition of protein synthesis with chloramphenicol (CAP). The extracellular autolytic activity in the culture, determined by following loss of turbidity of washed clostridial cells in 0.04m sodium phosphate buffer at 37° C, was decreased by 40% after CAP treatment. This caused an extension of cell viability by 12 h and an increase in butanol production by 30%. The optimal time of CAP addition was 12 h of incubation, and the optimal antibiotic concentration was 120 g/ml. The effects of CAP on the fermentation are due to the inhibition of protein synthesis leading to a decrease in autolysin level in the culture. The results obtained provide economic advantages for industrial production of solvents by minimizing autolysin activity and maximizing solvent yield during the critical solvent-producing phase. Correspondence to: R. W. Traxler     

Purification of acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824 and cloning of the acetoacetate decarboxylase gene in Escherichia coli.

In Clostridium acetobutylicum ATCC 824, acetoacetate decarboxylase (EC 4.1.1.4) is essential for solvent production, catalyzing the decarboxylation of acetoacetate to acetone. We report here the purification of the enzyme from C. acetobutylicum ATCC 824 and the cloning and expression of the gene encoding the acetoacetate decarboxylase enzyme in Escherichia coli. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was screened by plaque hybridization, using oligodeoxynucleotide probes derived from the N-terminal amino acid sequence obtained from the purified protein. Phage DNA from positive plaques was analyzed by Southern hybridization. Restriction mapping and subsequent subcloning of DNA fragments hybridizing to the probes localized the gene within an approximately 2.1 kb EcoRI/Bg/II fragment. A polypeptide with a molecular weight of approximately 28,000 corresponding to that of the purified acetoacetate decarboxylase was observed in both Western blots (immunoblots) and maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. Although the expression of the gene is tightly regulated in C. acetobutylicum, it was well expressed in E. coli, although from a promoter sequence of clostridial origin.     

Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis.

Phosphotransbutyrylase (phosphate butyryltransferase [EC 2.3.1.19]) from Clostridium acetobutylicum ATCC 824 was purified approximately 200-fold to homogeneity with a yield of 13%. Steps used in the purification procedure were fractional precipitation with (NH4)2SO4, Phenyl Sepharose CL-4B chromatography, DEAE-Sephacel chromatography, high-pressure liquid chromatography with an anion-exchange column, and high-pressure liquid chromatography with a hydrophobic-interaction column. Gel filtration and denaturing gel electrophoresis data were consistent with a native enzyme having eight 31,000-molecular-weight subunits. Within the physiological range of pH 5.5 to 7, the enzyme was very sensitive to pH change in the butyryl phosphate-forming direction and showed virtually no activity below pH 6. This finding indicates that a change in internal pH may be one important factor in the regulation of the enzyme. The enzyme was less sensitive to pH change in the reverse direction. The enzyme could use a number of substrates in addition to butyryl coenzyme A (butyryl-CoA) but had the highest relative activity with butyryl-CoA, isovaleryl-CoA, and valeryl-CoA. The Km values at 30 degrees C and pH 8.0 for butyryl-CoA, phosphate, butyryl phosphate, and CoASH (reduced form of CoA) were 0.11, 14, 0.26, and 0.077 mM, respectively. Results of product inhibition studies were consistent with a random Bi Bi binding mechanism in which phosphate binds at more than one site.     

Purification of acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824 and cloning of the acetoacetate decarboxylase gene in Escherichia coli

In Clostridium acetobutylicum ATCC 824, acetoacetate decarboxylase (EC 4.1.1.4) is essential for solvent production, catalyzing the decarboxylation of acetoacetate to acetone. We report here the purification of the enzyme from C. acetobutylicum ATCC 824 and the cloning and expression of the gene encoding the acetoacetate decarboxylase enzyme in Escherichia coli. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was screened by plaque hybridization, using oligodeoxynucleotide probes derived from the N-terminal amino acid sequence obtained from the purified protein. Phage DNA from positive plaques was analyzed by Southern hybridization. Restriction mapping and subsequent subcloning of DNA fragments hybridizing to the probes localized the gene within an approximately 2.1 kb EcoRI/Bg/II fragment. A polypeptide with a molecular weight of approximately 28,000 corresponding to that of the purified acetoacetate decarboxylase was observed in both Western blots (immunoblots) and maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. Although the expression of the gene is tightly regulated in C. acetobutylicum, it was well expressed in E. coli, although from a promoter sequence of clostridial origin.     

Purification and characterization of the extracellular alpha-amylase from Clostridium acetobutylicum ATCC 824.

The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography (mono Q) and gel filtration (Superose 12). The enzyme had an isoelectric point of 4.7 and a molecular weight of 84,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a monomeric protein, the 19-amino-acid N terminus of which displayed 42% homology with the Bacillus subtilis saccharifying alpha-amylase. The amino acid composition of the enzyme showed a high number of acidic and hydrophobic residues and only one cysteine residue per mole. The activity of the alpha-amylase was not stimulated by calcium ions (or other metal ions) or inhibited by EDTA, although the enzyme contained seven calcium atoms per molecule. alpha-Amylase activity on soluble starch was optimal at pH 5.6 and 45 degrees C. The alpha-amylase was stable at an acidic pH but very sensitive to thermal inactivation. It hydrolyzed soluble starch, with a Km of 3.6 g . liter-1 and a Kcat of 122 mol of reducing sugars . s-1 . mol-1. The alpha-amylase showed greater activity with high-molecular-weight substrates than with low-molecular-weight maltooligosaccharides, hydrolyzed glycogen and pullulan slowly, but did not hydrolyze dextran or cyclodextrins. The major end products of maltohexaose degradation were glucose, maltose, and maltotriose; maltotetraose and maltopentaose were formed as intermediate products. Twenty seven percent of the glucoamylase activity generally detected in the culture supernatant of C. acetobutylicum can be attributed to the alpha-amylase.     

Purification and Characterization of Two Endoxylanases from Clostridium acetobutylicum ATCC 824

Two endoxylanases produced by C. acetobutylicum ATCC 824 were purified to homogeneity by column chromatography. Xylanase A, which has a molecular weight of 65,000, hydrolyzed larchwood xylan randomly, yielding xylohexaose, xylopentaose, xylotetraose, xylotriose, and xylobiose as end products. Xylanase B, which has a molecular weight of 29,000, also hydrolyzed xylan randomly, giving xylotriose and xylobiose as end products. Xylanase A hydrolyzed carboxymethyl cellulose with a higher specific activity than xylan. It also exhibited high activity on acid-swollen cellulose. Xylanase B showed practically no activity against either cellulose or carboxymethyl cellulose but was able to hydrolyze lichenan with a specific activity similar to that for xylan. Both xylanases had no aryl-β-xylosidase activity. The smallest oligosaccharides degraded by xylanases A and B were xylohexaose and xylotetraose, respectively. The two xylanases demonstrated similar K_m and V_max values but had different pH optima and isoelectric points. Ouchterlony immunodiffusion tests showed that xylanases A and B lacked antigenic similarity.     

Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis

Phosphotransbutyrylase (phosphate butyryltransferase [EC 2.3.1.19]) from Clostridium acetobutylicum ATCC 824 was purified approximately 200-fold to homogeneity with a yield of 13%. Steps used in the purification procedure were fractional precipitation with (NH4)2SO4, Phenyl Sepharose CL-4B chromatography, DEAE-Sephacel chromatography, high-pressure liquid chromatography with an anion-exchange column, and high-pressure liquid chromatography with a hydrophobic-interaction column. Gel filtration and denaturing gel electrophoresis data were consistent with a native enzyme having eight 31,000-molecular-weight subunits. Within the physiological range of pH 5.5 to 7, the enzyme was very sensitive to pH change in the butyryl phosphate-forming direction and showed virtually no activity below pH 6. This finding indicates that a change in internal pH may be one important factor in the regulation of the enzyme. The enzyme was less sensitive to pH change in the reverse direction. The enzyme could use a number of substrates in addition to butyryl coenzyme A (butyryl-CoA) but had the highest relative activity with butyryl-CoA, isovaleryl-CoA, and valeryl-CoA. The Km values at 30 degrees C and pH 8.0 for butyryl-CoA, phosphate, butyryl phosphate, and CoASH (reduced form of CoA) were 0.11, 14, 0.26, and 0.077 mM, respectively. Results of product inhibition studies were consistent with a random Bi Bi binding mechanism in which phosphate binds at more than one site.     

Resonance assignments of cohesin and dockerin domains from Clostridium acetobutylicum ATCC824

Cohesin and dockerin domains are critical assembling components of cellulosome, a large extracellular multienzyme complex which is used by anaerobic cellulolytic bacteria to efficiently degrade lignocellulose. According to sequence homology, cohesins can be divided into three major groups, whereas cohesins from Clostridium acetobutylicum are beyond these groups and emanate from a branching point between the type I and type III cohesins. Cohesins and dockerins from C. acetobutylicum show low sequence homology to those from other cellulolytic bacteria, and their interactions are specific in corresponding species. Therefore the interactions between cohesins and dockerins from C. acetobutylicum are meaningful to the studies of both cellulosome assembling mechanism and the construction of designer cellulosome. Here we report the NMR resonance assignments of one cohesin from cellulosome scaffoldin cipA and one dockerin from a cellulosomal glycoside hydrolase (family 9) of C. acetobutylicum for further structural determination and functional studies.     

Reconstruction and expression of the autolytic gene from Clostridium acetobutylicum ATCC 824 in Escherichia coli

The complete lyc gene encoding the autolytic lysozyme of Clostridium acetobutylicum ATCC 824 was reconstructed from two overlapping DNA fragments and cloned into a suitable plasmid enabling Escherichia coli to produce this lytic enzyme under the control of the lac promoter. A polypeptide with an apparent M(r) of 35,000, corresponding to that predicted from the nucleotide sequence, was observed by maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. The enzyme yield was shown to depend on the pH of the culture medium, since the protein was unstable at alkaline pH. The expression of the lyc gene was not increased by using the E. coli strong promoter, lpp-lac, probably due to the limit imposed by the extreme differences in codon usage. Although the LYC lysozyme does not contain a cleavable signal peptide, most of the protein was found in the periplasmic fraction of E. coli suggesting that this enzyme was secreted through a specific mechanism, as already observed for other autolysins.     

Cloning of the Clostridium acetobutylicum ATCC 824 acetyl coenzyme A acetyltransferase (thiolase; EC 2.3.1.9) gene.

Thiolase (acetyl coenzyme A acetyltransferase; EC 2.3.1.9) from Clostridium acetobutylicum is a key enzyme in the production of acids and solvents in this organism. The purification and properties of the enzyme have already been described (D. P. Wiesenborn, F. B. Rudolph, and E.T. Papoutsakis, Appl. Environ. Microbiol. 54:2717-2722, 1988). The thl gene encoding the thiolase has been cloned by using primary antibodies raised to the purified enzyme. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was prepared and screened by immunoblots with the antithiolase antibodies. Phage DNA was purified from positive plaques, and restriction enzyme digests identified an approximately 4.8-kb AccI fragment common to all positive plaques. A corresponding fragment was also found in AccI digests of C. acetobutylicum chromosomal DNA. The fragment was purified and EcoRI linkers were attached before being subcloned into pUC19. Maxicell analysis showed the production of an approximately 42-kDa protein, whose size corresponded to the molecular size of the purified thiolase, from the clostridial insert. Enzyme activity assays and Western blot (immunoblot) analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell extracts of Escherichia coli harboring the cloned thl confirmed the presence of the thiolase encoded within the cloned DNA.