Jacqueline Millet

Purification and properties of an endo-β-1,4-glucanase from Clostridium thermocellum

The extracellular cellulolytic enzymes of the thermophilic anaerobe Clostridium thermocellum occur as a protein complex or aggregate which, until now, has not been resolved into individual enzyme components. By using QAE-Sephadex A50 chromatography in the presence of 6 M urea, it was possible to split the complex into distinct protein fractions. One of these fractions contained an endo-beta-1,4-glucanase which was isolated at a high degree of purity and was identified by its ability to hydrolyze trinitrophenylated carboxymethylcellulose. The enzyme is of monomeric nature, with a molecular weight of 56,000. It has an isoelectric pH of 6.2 and an optimum pH of 6.0. It hydrolyzed carboxymethylcellulose and, at a slower rate, cellulose powder. The major end products of cellulose degradation are glucose, cellobiose and cellotriose; cellotetrose is formed as an intermediate product. No specific small molecular weight activator or inhibitor was found except cellobiose and, to a lesser extent, glucose, which at high concentrations partially inhibit the activity of the enzyme. The temperature dependence of the enzyme is related to the thermophilic character of the producing microorganism.

High activity of inclusion bodies formed in Escherichia coli overproducing Clostridium thermocellum endoglucanase D

The formation of cytoplasmic inclusion bodies by Escherichia coli overproducing Clostridium thermocellum endoglucanase D (EGD) was investigated. EGD was found in inclusion bodies as a 68 kDa form, whereas the size of the cytoplasmic form was 65 kDa. Upon solubilization with urea followed by dialysis, the 68 kDa form was converted to the 65 kDa species. Proteolysis occurred within the COOH‐terminal, reiterated region of the 68 kDa form, which is conserved among most C. thermocellum endoglucanase, but is not required for catalytic activity. The specific activity of the enzyme embedded in inclusion bodies was close to that of the purified protein. Thus, inclusion body formation does not involve denaturation of the catalytic domain of EGD, but more likely, the participation of the reiterated, conserved region in intermolecular interactions.

Purification and properties of the endoglucanase C of Clostridium thermocellum produced in Escherichia coli

The celC gene, which codes for a new endoglucanase of Clostridium thermocellum, termed endoglucanase C, was found to be expressed when cloned in Escherichia coli. The enzyme was purified to electrophoretic homogeneneity from E. coli and its biochemical properties were studied. It differs from the previously studied endoglucanases A and B. In particular, endoglucanase C displays features common to endo- and exoglucanases, since it had a high activity on carboxymethylcellulose and on p-nitrophenyl-beta-D-cellobioside where only the agluconic bond was split. In addition, the enzyme was able to release cellobiose units from G3, G4 and G5 cellodextrins. Endoglucanase C was characterized by Western blot in a culture supernatant from C. thermocellum grown on cellulose, using an antiserum raised against the enzyme produced by E. coli.

Identification of the endoglucanase encoded by the celB gene of Clostridium thermocellum

The endoglucanase encoded by the celB gene of Clostridium thermocellum was purified from an E. coli strain carrying and expressing the C. thermocellum gene cloned in the plasmid pBR322. The preparation showed two active bands, with Mr 55,000 and 53,000, presumably derived from the primary translation product by proteolysis. Specific antiserum raised against these bands was used to identify the corresponding antigen in the culture supernatant of C. thermocellum: in a double immunodiffusion test (Ouchterlony), a precipitin line was observed which fused completely with that formed by an E. coli extract containing endoglucanase B expressed from the cloned gene. Proteins from C. thermocellum supernatant were further analyzed by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose sheet. After incubating the nitrocellulose blot with antiserum and subsequently with 125I-labeled protein A, a band with Mr 66,000, corresponding to the celB gene product expressed by C. thermocellum, was detected by autoradiography.

Characterization of an intracellular protease in B.,subtilis during sporulation

Abstract An intracellular serylprotease has been characterized in B. , subtilis , Marburg strain. Like the extracellular serylprotease of that strain, it occurs only during sporulation, but otherwise differs in many characteristics, including its migration on electrophoresis, its absolute requirement for calcium and its narrower specificity toward esters. On the other hand, it is quite similar to the cytoplasmic serylprotease of B. , meqaterium also synthesized during sporulation. Since the latter enzyme can specifically modify the B. , subtilis vegetative RNA polymerase in vitro , it is conceivable that the intracellular B. subtilis serylprotease studied here carries out this modification in , vivo .

Molecular cloning of a gene for a thermostable β-glucosidase from Clostridium thermocellum into Escherichia coli

Abstract Three Escherichia coli clones containing Clostridium thermocellum DNA were isolated by screening a partial Sau IIIA library in pBR325 for β-glucosidase using a methylumbelliferyl-β- d -glucoside ( MUG ) overlay. All of the clones also hydrolysed cellobiose to glucose. Restriction analysis of the C. thermocellum DNA from these clones showed that in each case an Eco R1 fragment ( 4.0kb ) previously cloned in pCT401 1 was present as part of the insert. Cell extracts of E. coli carrying pCT401 or an otherwise identical plasmid ( pCT402 ) with the same Eco R1 insert in the opposite orientation hydrolysed MUG but not cellobiose. The present finding demonstrates that the genetic functions encoding hydrolysis of MUG and cellobiose are associated closely on the C. thermocellum chromosome. However, whether one or more protein is involved in the hydrolysis of the two substrates remains to be determined .

Proteolytic conversion in vitro of B. subtilis vegetative RNA polymerase into the homologous spore enzyme

It was previously shown that the structural difference between the two DNA-dependent RNA polymerases isolated from B. subtilis vegetative cells and from dormant spores resides in the replacement of one of the 0 subunits in the vegetative enzyme (146,000 daltons each) by a polypeptide with the molecular weight of 129,000 daltons present in the spore RNA polymerase [l] . A similar difference in the RNA polymerase was observed by Losick and Sonensheim in sporulating cells of B. subtilis strain 36 10 [2] . One of the questions raised by these findings was how the 0 modification could take place in vivo. By analogy to the studies of Sadoff et al. [3] on a protease catalyzed interconversion of the B. cereus vegetative aldolase into the spore form of the same enzyme, it was suggested that a similar mechanism may operate during sporulation leading to the observed RNA polymerase modification. This hypothesis was further strengthened by the experiments reported by Leighton et al. in which they observed the /3 modification in vitro by incubating purified B. subtilis vegetative RNA polymerase in the presence of the “serine protease” isolated from the same microorganism [4]. However, the question which was still left to be clarified was to know how the serine protease which, as shown by Millet [5] is an extracellular enzyme, could act in vivo on the DNA-dependent RNA polymerase located in the nuclear fraction of the cell [6] . Therefore, we thought that, in vivo, the

Crystallization and preliminary X-ray diffraction study of an endoglucanase from Clostridium thermocellum

Endoglucanase D, a cellulose degradation enzyme from Clostridium thermocellum has been cloned in Escherichia coli, purified and crystallized. The crystals are trigonal, space group P3(1)12 (or P3(2)12) with a = 57.7 (+/- 0.1) A, c = 192.1 (+/- 0.2) A, and diffract X-rays to a resolution of 2.8 A. They are suitable for a high-resolution X-ray diffraction analysis.

Mutant thermosensible de B. subtilis affecté dans la sporulation et la sérylprotéase extracellulaire

Summary Isolation and properties of B. subtilis ts 19 mutant, isolated as thermosensitive for sporulation, are described. At the non permissive temperature (42°C), the mutant cells are blocked at stage zero of sporulation and do not excrete extracellular enzymes such as serylprotease and esterase. At the permissive temperature (30°C), sporulation and excretion of extracellular enzymes are normal but the serylprotease is modified in its structure. Two molecular forms of this enzyme can be separated by polyacrylamide electrophoresis, both more thermolabile than the corresponding enzyme of the mother strain. Experiments of reversion and of transformation for the sporulation character have suggested that ts 19 contained two independent thermosensitive mutations. One of them is responsible for the pleiotropic Spo O A phenotype at the non permissive temperature. The other mutation is likely to reside in the structural gene coding for the extracellular serylprotease and leads to the formation of a modified enzyme which hydrolyzes itself into at least two types of more stable molecules. No conclusion can be drawn with certainty concerning the physiological role of the extracellular serylprotease in sporulation. It may be pointed out however that transformants for the Sp + character at 42°C keep the same impaired serylprotease as the ts 19 mutant and sporulate, at any temperature, as well as the wild strain.

Characterization of an inhibitor of the intracellular protease from Bacillus subtilis

A specific inhibitor of intracellular serylprotease from Bacillus subtilis has been isolated from both growing and sporulating cells. Like other protease inhibitors isolated from eukaryotic cells, the inhibitor from B. subtilis is a thermostable protein. A purification method is described. The molecular weight estimated by Biogel filtration and SDS gel electrophoresis is about 15,500. Both proteolytic and esterolytic activities of intracellular protease are equally sensitive to inhibition. With azocoll or Z-tyrosine p-nitrophenylester as substrates, noncompetitive inhibition patterns are observed. The inhibitor has no effect on the proteolytic or esterolytic activities of the extracellular serylprotease. A similar thermostable inhibitor is also present in Bacillus megaterium.