Characterization of glucoamylase from Lactobacillus amylovorus ATCC 33621

Summary An intracellular glucoamylase, purified from Lactobacillus amylovorus, reacted selectively with polysaccharides. Kinetic studies indicated low affinity for maltose and maltotriose (K_m 58 g/ml and 178 g/ml) and higher affinity for starch and dextrin (K_m 0.01 g/ml and 0.02 g/ml). Glucoamylase was inhibited almost 50% by 10 mM glucose. Cu²⁺ and Pb²⁺ inhibited glucoamylase at 1.0 mM but EDTA and other metal chelators had no effect on the enzyme activity. Acarbose and Tris inhibited the enzyme by 84% and 98%, respectively at 1 mM, while iodoacetate and p-chloromecuribenzoic acid inhibited activity by 98% and 78%, respectively at 10 mM. The purified enzyme was thermolabile at temperatures greater than 55°C and thus has potential for application in the brewing industry.     

Purification of Glucoamylase from Lactobacillus amylovorus ATCC 33621

An intracellular glucoamylase (E.C. 3.2.1.3) was purified to homogeneity from Lactobacillus amylovorus on a Fast Protein Liquid Chromatography System (FPLC) with a Mono Q ion-exchanger and two Superose 12 gel filtration columns arranged in series. The enzyme activity was quantified with a specific, chromogenic substrate, p-nitrophenyl-β-maltoside. Preparative gel electrophoresis was then used to further purify active enzyme fractions. Native polyacrylamide gel electrophoresis (Native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of molecular weight 47 kDa. Glucoamylase activity of the purified protein was confirmed by its ability to degrade starch on a 0.025% starch-polyacrylamide gel stained with I₂/KI. Glucoamylase exhibited optimum catalytic activity at pH 6.0 and 45°C, and the enzyme had an isoelectric point near 4.39. The glucoamylase contained high levels of hydrophilic amino acids, comparable to fungal glucoamylases. Received: 12 July 1996 / Accepted: 10 September 1996     

Cloning and expression of a glucoamylase gene from Lactobacillus amylovorus ATCC 33621 in Escherichia coli

Summary The glucoamylase gene from Lactobacillus amylovorus was cloned and expressed in Escherichia coli. A genomic DNA library from Lactobacillus amylovorus was prepared by partially digesting genomic DNA with EcoRI and ligating random fragments to the EcoRI digested cloning vector, pZErO-1.1. Three E. coli transformants expressing glucoamylase were identified using a probe prepared from the STA2 glucoamylase gene from Saccharomyces cerevisiae var. diastaticus. The physical maps of the recombinant plasmids were constructed. These plasmids contained inserts of about 5.2 Kb, 5.9 Kb and 6.4 Kb respectively. Temperature and pH optima of 45°C and 6.0, respectively, were obtained for both recombinant and purified wild type glucoamylases. Also, the enzymes were found to be thermolabile at temperatures above 50°C.     

Genome sequence of Lactobacillus amylovorus GRL1112

Lactobacillus amylovorus is a common member of the normal gastrointestinal tract (GIT) microbiota in pigs. Here, we report the genome sequence of L. amylovorus GRL1112, a porcine feces isolate displaying strong adherence to the pig intestinal epithelial cells. The strain is of interest, as it is a potential probiotic bacterium.     

Comparison of amylolytic properties of Lactobacillus amylovorus and of Lactobacillus amylophilus

Partially purified amylases produced by Lactobacillus amylovorus and L. amylophilus were compared and they differed in several properties. The maximum amylase activity of L. amylovorus was higher than that of L. amylophilus. As estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the molecular mass of the enzymes was 140 kDa for L. amylovorus amylase and 100 kDa for L. amylophilus amylase. Maximum enzymatic activities were obtained when the strains were grown in the presence of CaCO₃, on maltose with L. amylovorus and on sucrose with L. amylophilus. Optimal activities were obtained at pH values between 5.0 and 6.0 for both amylases. The L. amylovorus amylase was stable at a higher temperature (50°C) than the L. amylophilus amylase (40°C). Of six substrates examined, greatest activity was obtained by both enzymes on soluble starch. Neither enzymes hydrolysed pullulan or - and \-cyclodextrins. With the exception of Hg²⁺, which partially inhibited both enzymes, various metal ions, such as 1 mm Ca²⁺ and Ba²⁺, stimulated L. amylophilus amylase activity whereas they inhibited L. amylovorus amylase activity. Correspondence to: J. Morlon-Guyot, ORSTOM     

Bacteriocin production with Lactobacillus amylovorus DCE 471 is improved and stabilized by fed-batch fermentation

Amylovorin L471 is a small, heat-stable, and hydrophobic bacteriocin produced by Lactobacillus amylovorus DCE 471. The nutritional requirements for amylovorin L471 production were studied with fed-batch fermentations. A twofold increase in bacteriocin titer was obtained when substrate addition was controlled by the acidification rate of the culture, compared with the titers reached with constant substrate addition or pH-controlled batch cultures carried out under the same conditions. An interesting feature of fed-batch cultures observed under certain culture conditions (constant feed rate) is the apparent stabilization of bacteriocin activity after obtaining maximum production. Finally, a mathematical model was set up to simulate cell growth, glucose and complex nitrogen source consumption, and lactic acid and bacteriocin production kinetics. The model showed that bacterial growth was dependent on both the energy and the complex nitrogen source. Bacteriocin production was growth associated, with a simultaneous bacteriocin adsorption on the producer cells dependent on the lactic acid accumulated and hence the viability of the cells. Both bacteriocin production and adsorption were inhibited by high concentrations of the complex nitrogen source.     

Modelling the growth and bacteriocin production by Lactobacillus amylovorus DCE 471 in batch cultivation

A model was set up to describe the production of amylovorin L471 by Lactobacillus amylovorus DCE 471, on a laboratory scale, in which the cells are grown in MRS (deMau-Rogosa-Sharpe) broth. The main features of the dynamic model are : (i) increase of the biomass according to a logistic equation ; (ii) non-growth-associated consumption of substrate (maintenance metabolism) ; and (iii) primary metabolite kinetics for the bacteriocin production. The main purpose was to set up a simple empirical model to examine growth and bacteriocin production in different conditions. Parameters estimated from a fermentation with 20 g l⁻¹ glucose (w/v) could be used to predict the evolution of cell dry mass, glucose and lactic acid concentration of fermentations, performed with 5, 30, 40 and 60 g l⁻¹ initial glucose. The influence of the operating temperature (30, 37 and 45 °C) on the model parameters was also investigated. The proposed model was able to describe growth and bacteriocin production in all cases. The specific bacteriocin production rate was found to vary strongly with temperature, with 30 °C as the best value. Variation of the operating temperature from 37 to 30 °C appeared to significantly enhance the specific bacteriocin production.     

Influence of temperature on flavour compound production from citrate by Lactobacillus rhamnosus ATCC 7469

The citrate utilization by Lactobacillus rhamnosus ATCC 7469 was found to be temperature-dependent. The maximum citrate utilization and incorporation of [1,5-14C]citrate rate were observed at 37 degreesC. At this temperature, maximum citrate lyase activity and specific diacetyl and acetoin production (Y(DA%)) were observed. The high levels of alpha-acetolactate synthase and low levels of diacetyl reductase, acetoin reductase and L-lactate dehydrogenase found at 37 degreesC led to an accumulation of diacetyl and acetoin. Optimum lactic acid production was observed at 45 degreesC, according to the high lactate dehydrogenase activity. The NADH oxidase activity increased with increasing culture temperature from 22 degreesC to 37 degreesC. Thus there are greater quantities of pyruvate available for the production of alpha-acetolactate, diacetyl and aceotin, and less diacetyl and acetoin are reduced.     

Culture conditions for the production of esterase from Lactobacillus casei CL96

Culture conditions in growth and esterase production by a newly isolated Lactobacillus casei CL96 were investigated using a dextrose-free MRS medium supplemented with different sugars in a 2 l fermentor at different pHs (4.0-9.0) and temperatures (20-50°C). The optimal growth was obtained in basal MRS medium containing 1% (w/v) lactose at pH 7.0 and 30°C. The maximal esterase production was obtained intracellularly during the late logarithmic phase, but during the stationary phase, the esterase activity was released in the culture medium. The enzyme activity was maximal at pH 7.0 and 37°C. Among various substrates (C₂-C₁₆) tested, the highest activity was towards C₆ and C₈. Though the enzyme was produced constitutively, the tributylin induced the enzyme production by 2.5 fold. L. casei CL96 esterase was very active at neutral pH and ambient temperature and might be suitable for biotechnological applications in the dairy industry.     

Genome sequence of Lactobacillus amylovorus GRL1118, isolated from pig ileum

Lactobacillus amylovorus is a common member of the beneficial microbiota present in the pig gastrointestinal tract. Here, we report the genome sequence of the surface layer (S-layer) protein-carrying and potentially probiotic strain L. amylovorus GRL1118, which was isolated from porcine ileum and which shows strong adherence to pig intestinal epithelial cells.     

Lactic acid production from enzyme-thinned corn starch usingLactobacillus amylovorus

Summary An alternative process for industrial lactic acid production was deveooped using a starch degrading lactic acid producing organism,Lactobacillus amylovorus B-4542. In this process, saccharification takes place during the fermentation, eliminating the need for complete hydrolysis of the starch to glucose prior to fermentation. The cost savings of this alternative are substantial since it eliminates the energy input, separate reactor tank, time, and enzyme associated with the typical pre-fermentation saccharification step. The only pre-treatment was gelatinization and enzyme-thinning of the starch to overcome viscosity problems associated with high starch concentrations and to make the starch more rapidly degradable. This fermentation process was optimized for temperature, substrate level, nitrogen source and level, mineral level, B-vitamins, volatile fatty acids, pH, and buffer source. The rate of the reaction and the final level of lactic acid obtained in the optimized liquefied starch process was similar to that obtained withL. delbrueckii B-445 using glucose as the substrate.     

Effects of Lactobacillus amylovorus and Bifidobacterium breve on cholesterol

To determine the validity of the hypothesis of assimilation and/or precipitation of cholesterol by Lactobacillus and Bifidobacterium species, culture were undertaken in TPY medium containing oxgall or taurocholic acid. In the case of growing cells, both strains were able to remove cholesterol in the presence of bile salts. Nevertheless, the behaviour was different according to the kind of bile salt. In the presence of taurocholic acid, the removal of cholesterol was due to both bacterial uptake and precipitation. In the presence of Oxgall, bacterial uptake and precipitation were observed for Lactobacillus but only precipitation occurred for Bifidobacterium.     

Optimization of lactic acid production in SSF by Lactobacillus amylovorus NRRL B-4542 using Taguchi methodology

Lactic acid production parameter optimization using Lactobacillus amylovorus NRRL B-4542 was performed using the design of experiments (DOE) available in the form of an orthogonal array and a software for automatic design and analysis of the experiments, both based on Taguchi protocol. Optimal levels of physical parameters and key media components namely temperature, pH, inoculum size, moisture, yeast extract, MgSO4 . 7H20, Tween 80, and corn steep liquor (CSL) were determined. Among the physical parameters, temperature contributed higher influence, and among media components, yeast extract, MgSO4 . 7H20, and Tween 80 played important roles in the conversion of starch to lactic acid. The expected yield of lactic acid under these optimal conditions was 95.80% and the actual yield at optimum conditions was 93.50%.     

Mapping of three plasmids from Lactobacillus helveticus ATCC 15009

A 1.4 kb DNA fragment from the chromosomal DNA of Penicillium nalgiovense was isolated which confers proteolytic activity to E. coli DH5α cells when cloned under the control of the E. coli lacZ promoter. The protein was excreted by the cells as was shown by the formation of a clearing zone in skim milk medium. A retransformation of the plasmid carrying the protease gene into P. nalgiovense leads to transformants with both increased and with nearly no proteolytic activity under neutral conditions. Southern blotting experiments revealed that the transforming plasmid had apparently integrated into the homologous locus and thereby inactivated the residual gene.     

Adhesion of Lactobacillus amylovorus to Insoluble and Derivatized Cornstarch Granules

Approximately 70% of the cells in a suspension of the amylolytic bacterium Lactobacillus amylovorus bind to cornstarch granules within 30 min at 25°C. More than 60% of the bound bacteria were removed by formaldehyde (2%) or glycine (1 M) at pH 2.0. More than 90% of the bound bacteria were removed by MgCl₂ (2 M; pH 7.0). Binding of L. amylovorus to cornstarch was inhibited in heat-killed cells and in cells that had been pretreated with glutaraldehyde, formaldehyde, sodium azide, trypsin, or 1% soluble potato starch. Bacterial binding to cornstarch appeared to correlate with both the concentration of cornstarch in the suspension and the amylose content in the granules. The ability of L. amylovorus to adhere to cornstarch granules was reduced for granules that had been extracted with HCl-ethanol, HCl-methanol, HCl-propanol, or HCl-butanol. Chemical derivatization of cornstarch resulted in a wide variety of adhesion responses by these bacteria. For example, 2-O-butyl starch (degree of substitution, 0.09) enhanced adhesion, whereas two palmitate starches (degree of substitution, 0.48 and 0.09) exhibited reduced adhesion activities. 2-O-(2-hydroxybutyl) starch and starch-poly(ethylene-co-acrylic acid) ester showed adhesion activities similar to those of the nonderivatized starch controls.     

Characterization of a Mutant from Lactobacillus amylovorus JCM 1126T with Improved Utilization of Sucrose

A strain YF43, which can grow on sucrose as rapidly as glucose, was isolated by mutation from Lactobacillus amylovorus JCM 1126, the type strain defective in sucrose utilization. Exogenous sucrose stimulated the production of invertase by strains YF43 and JCM 1126 simultaneously. In a medium containing fructooligosaccharide as the sole carbon source, the cells of strain YF43 showed high invertase activity in spite of poor growth. The two invertases produced in the cells grown on sucrose and fructooligosaccharide were an identical β-fructofuranosidase, as judged from properties of partially purified enzymes. These observations indicated that strain YF43 is a mutant improved for permeation of sucrose and not derepressed for the synthesis of invertase. Received: 23 May 2000 / Accepted: 26 June 2000     

Development of an amylolytic Lactobacillus plantarum silage strain expressing the Lactobacillus amylovorus alpha-amylase gene.

An amylolytic Lactobacillus plantarum silage strain with the starch-degrading ability displayed by Lactobacillus amylovorus was developed. An active fragment of the gene coding for alpha-amylase production in L. amylovorus was cloned and integrated into the chromosome of the competitive inoculant strain L. plantarum Lp80 at the cbh locus. The alpha-amylase gene fragment was also introduced into L. plantarum Lp80 on an autoreplicative plasmid. Both constructions were also performed in the laboratory strain L. plantarum NCIB8826. All four recombinant strains secreted levels of amylase ranging from 23 to 69 U/liter, compared with 47 U/liter for L. amylovorus. Secretion levels were higher in L. plantarum NCIB8826 than in L. plantarum Lp80 derivatives and were higher in recombinant strains containing autoreplicative plasmids than in the corresponding integrants. The L. plantarum Lp80 derivative containing the L. amylovorus alpha-amylase gene fragment integrated into the host chromosome secreted alpha-amylase to a level comparable to that of L. amylovorus and was stable over 50 generations of growth under nonselective conditions. It grew to a higher cell density than either the parent strain or L. amylovorus in MRS medium containing a mixture of starch and glucose as the fermentable carbohydrate source. This recombinant alpha-amylolytic L. plantarum strain would therefore seem to have considerable potential as a silage inoculant for crops such as alfalfa, in which water-soluble carbohydrate levels are frequently low but starch is present as an alternative carbohydrate source.     

Culture condition for production of glucoamylase from rice bran byAspergillus terreus

Summary Optimal conditions for the production of glucoamylase from rice bran usingAspergillus terreus in stationary culture were a medium containing 20 g rice bran/l, 0.3% (w/v) (NH₄)₂SO₄ and 0.2% (w/v) peptone at 30°C with an initial pH of 3.0. Enzymatic activity was maximal after 4 d. Glucose was the major reducing sugar produced by hydrolysis of starch. Carbohydrates favouring induction of glucoamylase were, in order: maltose, starch, cellobiose, lactose, glucose, fructose and galactose. Amino acids, in particular glycine, lysine, isoleucine and histidine, were vital for glucoamylase synthesis. Tween 80 and Triton X-100 enhanced the growth but suppressed glucoamylase synthesis.

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Conditions de culture pour la production de glucoamylase à partir de son de riz parAspergillus terreus

Résumé Les conditions optimales pour la production de glucoamylase à partir de son de riz en utilisantAspergillus terreus en culture en état stationnaire, consistent en un milieu contenant 20 g de son de riz par litre, 0.3 % (poids/vol.) de (NH₄)₂ SO₄ et 0.2 % (poids/vol.) de peptone, à 30 °C avec un pH initial de 3.0. L'activité enzymatique est maximum après 4 jours. Le glucose est le principal sucre réducteur produit par hydrolyse de l'amidon. Les hydrates de carbone qui favorisent l'induction de la glucoamylase, sont, dans l'ordre: le maltose, l'amidon, la cellobiose, le lactose, le glucose, le fructose et le galactose. Les acides aminés, en particulier la glycine, la lysine, l'isoleucine et l'histidine sont vitales pour la synthèse de glucoamylase. Le tween 80 et le triton X-100 augmentent la croissance mais suppriment la synthèse de glucoamylase.

     

Culture conditions of tannase production by Lactobacillus plantarum

Lactobacillus plantarum produced an extracellular tannase after 24 h growth on minimal medium of amino acids containing 2 g tannic acid l^–1. Enzyme production (6 U ml^–1) was optimal at 37 °C and pH 6 with 2 g glucose l^–1 and 7 g tannic acid l^–1 in absence of O₂.