Purification and characterization of a thermostable esterase from the moderate thermophile Bacillus circulans.

The thermostable esterase from the moderate thermophile Bacillus circulans was purified to homogeneity using a four-step procedure. Esterase activity was associated with a protein of molecular mass 95 kDa, composed of three identical subunits of 30 kDa. The esterase activity was thermostable with a maximum activity at 55 degrees C using initial rate assay. The half-inactivation temperature was 71 degrees C after a 1-h treatment, which compared favorably to that of other enzymes. Activity at temperatures of 30-37 degrees C was high (about half of maximum), making this new enzyme very attractive for applications in this moderate temperature range. The esterase also showed high activity at a rather alkaline pH (higher than 10). The specificity pattern showed a marked specificity for mid-chain-length fatty acids (3-8 carbon atoms), which classified the enzyme as a carboxylesterase.

D-Cycloserine biases enrichment for auxotrophic mutants of Zymomonas mobilis.

Contrary to its effect on rich medium, D-cycloserine showed no bactericidal effect on Zymomonas mobilis cells cultured on mineral medium. Addition of a mixture of glycine and glutamic acid to the mineral medium restored its bactericidal action. However, mutant enrichments run in these conditions were biased, with mostly methionine mutants isolated. A decrease of the D-cycloserine concentration only reduced the bias.

Isolation and Properties of Mutants of Zymomonas mobilis Deficient in Sugar Assimilation.

An enrichment method using d-cycloserine was designed for the isolation of spontaneous mutants of Zymomonas mobilis deficient in glucose or fructose utilization. The mutants could easily be isolated since they represented 80 to 90% of the population after two and three enrichment cycles. Glucokinase and fructokinase activities in the mutants were affected.

Overexpression of extracellular sucrase (SacC) of Zymomonas mobilis in Escherichia coli.

The extracellular sucrase (SacC) gene of Zymomonas mobilis was overexpressed in Escherichia coli BL21 using the T7 polymerase expression system. A low cell density induction method was designed to have maximum expression, and the conditions (IPTG concentration, ampicillin addition) were optimised to overexpress to the level of more than 60% of the total cellular protein representing SacC protein.

Molecular cloning and characterization of the extracellular sucrase gene (sacC) of Zymomonas mobilis.

The Zymomonas mobilis gene sacC that encodes the extracellular sucrase (protein B46) was cloned and expressed in Escherichia coli. The gene was found to be present downstream to the already described levansucrase gene sacB in the cloned chromosomal fragment of Z. mobilis. The expression product was different from SacB and exhibited sucrase but not levansucrase activity; therefore, SacC behaves like a true sucrase. Expression of sacC in E. coli JM109 and XL1 was very low; overexpression was observed in E. coli BL21 after induction of the T7 polymerase expression system with IPTG. Subcellular fractionation of the E. coli clone carrying plasmid pLSS2811 showed that more than 70% of the sucrase activity could be detected in the cytoplasmic fraction, suggesting that the enzyme was soluble and not secreted in E. coli. The nucleotide sequence analysis of sacC revealed an open reading frame 1239bp long coding for a 413 amino acid protein with a molecular mass of 46 kDa. The first 30 deduced amino acids from this ORF were identical with those from the N-terminal sequence of the extracellular sucrase (protein B46) purified from Z. mobilis ZM4. No leader peptide sequence could be identified in the sacC gene. The amino acid sequence of SacC showed very little similarity to those of other known sucrases, but was very similar to the levansucrases of Z. mobilis (61.5%), Erwinia amylovora (40.2%) and Bacillus subtilis (25.6%).

Amylase production by three Lactobacillus strains isolated from chicken crop.

Three amylolytic Lactobacillus strains designated LEM 220, LEM 207 and LEM 202 were isolated from the chicken crop. They belonged to the subgenus Thermobacterium. Strain LEM 220 resembled Lact. acidophilus. Amylase production was more abundant in cells grown in media containing amylopectin or starch than in media containing glucose or maltose. Optimum pH and temperature of the amylase were 5.5 and 55 degrees C respectively. Hydrolysis of amylopectin gave maltose, maltotriose and small amounts of glucose. Stain LEM 207 also resembled Lact. acidophilus, but differed from strain 220. It had a lower amylase activity. Optimum pH and temperature of the amylase were 6.4 and 40 degrees C, respectively, and hydrolysis of amylopectin gave maltose, maltotriose and carbohydrates higher than maltopentaose. Strain LEM 202 was similar to Lact. vitelinus. It had the lowest amylase activity which was increased only in presence of maltose. Amylase properties were similar to those of LEM 220.

[Role of five Lactobacillus strains on carbohydrate degradation in monoxenic chickens]. / Rôle de cinq souches de lactobacilles sur la dégradation des glucides chez le poulet monoxénique.

Five strains of Lactobacillus were isolated in holoxenic roosters; two of these which had an alpha-amylase were inoculated separately into 5 groups of axenic chickens fed the same diet. Some differences among these 5 groups were noted. Lactobacillus proliferation varied between ten and a thousand-fold, depending on the strain, and for the same strain depending on whether the crop, caecum or faeces was examined. Amylolytic lactobacilli in vivo played a role in starch degradation in various ways related to the specific properties of their amylase. Lactic acid production in the crop was higher with the three strains producing the two lactic acid isomers than with the two strains producing only one of the isomers. Finally, monoxenic caecal digestion was different from that of both the axenic and the holoxenic.