MXPA96001606A - Process to prepare a leaf of bacillus cereusproductora de aminoacido l-aspart - Google Patents

Abstract

The invention relates to obtaining a new strain of Bacillus cereus bacteria, which synthesizes in different media the intracellular aspartase enzyme with high catalytic activity that responds to the demands of biocatalysts: it has a short crop cycle (6 hours) with a final product high index, obtained in the form of a pure optical isomer (L-form of aspartic acid [0] D20 = 25.5ø), ie a strain having racemase activity. The strain (biocatalyst) proposed in this application was selected from a number of pure strains of microorganisms, isolated from different natural substrates (water and soil) grown on nutrient agar. The strain is conserved in the collection of pure microorganisms, in the Department of Biotechnology of the Faculty of Chemical Sciences of the U.A.C. with No. SC 31

Description

PROCESS TO PREPARE A BACI LUS CEREUS RODUCTORA DE AMINOÁCIDO L-ASPARTICO.

BACKGROUND OF THE INVENTION The invention relates to a process for preparing a new strain of Bacillus cereus that produces L-aspartic amino acid useful in the microbiological industry.

Among the processes known to obtain the L-aspartic amino acid, there is an enzymatic process which uses a bacterial enzyme aspartase or intact cells that contain the same enzyme.

In the prior art the strain of Escherichia coli co or producer of amino acid L-aspartic acid is described, one of which being registered in ATCC No. 11303 and a Russian patent E. Coli 85 No. 2423976 / 28-13.

The strain of E. Coli ATCC-11303 is incubated by the deep method in a medium that contains in a liter: 30 gr .. of ammonium fumarate, 2.0 gr. K2HP04, 0.5 gr. MgS0".7H20, 0.5 gr. CaCO, and 40 gr. of corn extract, - pH of medium 7 over the course of 24 hours at 37 ° C.

The strain of E. coli of Russian origin is grown in a medium containing pepperonated meat broth pH-7 for 10-12 hrs. 30-35 ° C in flasks of 750 ml (volume of medium -50 ml); then, an inoculum of 2-3% is grown in a medium of the same composition, for 6 hrs. In the same conditions (the volume of the medium is 125 ml). The aspartase activity of E. coli whole cells, in different experiments, was 15.4-46 μM aspartic acid per milligrams of cellulai protein per hour.

The aspartase activity of bacterial intact cells, which were previously separated from their respective media, was assessed following the formation of L-aspartic acid from fumaia? of ammonium using a spectrophotometer to follow the consumption of ammonium fumarate. The enzymatic activity is represented in micromoles (μM) of L aspartic acid produced in one hour by intact bacterial cells; The aspartase-specific activity of E. coli? TC 11303 native cells cultured in the medium indicated above is 1.R1 μM of asylactic O-ic I produced in one hour per mg. Of protein The aspartase activity of E.coli-85 cells in a medium with corn extract is μM L-aspartic acid / 1 hr x mg. of cellular protein. While the aspartase activity of Bacillus cereus cells object of the invention is 60 μM L-aspartic acid produced in one hour per mg of cellular protein.

Disadvantages of the strain of E.coli-ATCC -11303. 1) Comparatively it has a long development cycle (24 hrs.) Which increases the cost of the final product. 2) The strain develops in a medium with corn extract, which complicates the separation of bacterial cells from the corn extract precipitate. This contributes to increase the costs of the final product. 3) - Comparatively it has an activity in the indicated medium of 1.81 M of L-aspartic acid produced / rng of cellular protein in a hoi.

Disadvantages of E.coli-85 strain No. 2423976 / 28-13. 1) Comparatively, it has a longer development cycle (total, LH hrs) 2) In comparison with the strain that is the reason for this patent, its activity corresponds to 73% (20 μM of L-aspartic acid produced / mg of cellular protein in one hour). The aim of the invention is to obtain a new strain of bacteria of Bacillus cereus, which synthesizes in different media the intracellular aspartase enzyme with high catalytic activity that meets the requirements of the biocatalyst is: it has a short crop cycle (6 hours) with a high index of final product, obtained in the form of a pure isomer (L-form of aspartic acid [ÓJβ20 = 25.5 °), that is to say a strain that does not possess racemase activity.

DETAILED DESCRIPTION OF THE INVENTION The strain of Bacillus cereus (biocatalyst) described in this patent application was selected from a number of pure strains of microorganisms, isolated from different natural substrates (water and soil) grown on nutrient agar.

The strain conserved in the collection of pure microorganisms, in t 1 Department of Biotechnology of the Faculty of Chemical Sciences of the U.A. of C. With No. SC 3115; Its cellular morphology is as follows: it occurs in isolated form or in short chains, with a foeinated ondospoi in the central or subterminal region with rounded ends, - length from 2.1 to 5.0μ, width from 0.5 to 2.0μ. The optimal growth is obseiva at a temperature of 21 to 30 ° C. The cells are not uniformly unifying, they are gram positive or gram variable. Lae colonies that foim are < a (is not irregular with a flat elevation, blunt and rough surface and eroded or lobed margins.

Biochemical characteristics In relation to carbohydrates: dissociates glucose and sucrose with acid formation, but not lactose, xylose, arabinose and mannitol; no gas formation with aiabmosa, xylose, glucose, lactose, sucrose and mannitol, reduces methylene blue. In relation to other organic substances, it uses propionate, citrate, hydrolyzes starch, liquefies gelatin, hydrolyzes casein, does not hydrolyze hippurate, does not reoxidize methylene blue, reduces nitrates to nitrites, VP tests (5198 fil and 5331) are positive, decomposes hydrogen peroxide, does not produce indole, decomposes tyrosine, the reaction in acidic litmus medium is negative, in coagulated, peptonized, alkaline and reduced litmus, are positive, has strong lecithinase activity.

In comparison with the already known strains of E.coli ATCC 11303 and V. cuJ i 85, the aspartase activity determined by a general method, of an-. suspension of bacteria cells incubated for one hour in ammonium fumarate, is higher in the proposed strain since it forms more amount of L-aspartic acid. The intensity of development and activity aspartase in the proposed strain is greater than in the strains already known.

The amount of aspartic acid formed from ammonium fumarate, from a cell suspension of the strain in se pi opone and from those already known, is presented in Table 1. Table 1 COMPARISON OF PRODUCTION OF THE L-ASPARTIC AMINO ACID LA CEPA Bac ± llus cereus REASON FOR THIS PATENT APPLICATION, WITH OTHER REGISTERED SCOPE.

STRAINS OF DIFFERENT ORIGIN SPECIFIC ACTIVITY ASPARTASE (μM aspartic acid / hr / mg protein) E. Coli ATCC No. 11303 1.81 E. Coli 85 No. 2423976 / 28-3 20 Bacillus cereus (reason for this patent) 60 According to the data in Table 1, Bacillus cereus SC3115 s differs from E.coli ATCC 11303 and from E.coli-85 due to its high aspartase activity.

Characteristics of the soils of the Saltillo region where the samples were raised for the development of cultures of bacteria with aspartate ammonia-coliase activity.

In the intermontane valleys (Los Lirios, Carbonera, San Antonio, Huachichil) the soils are dark, which in the FAO system are classified in Chernozem, Feoze and Castañozem (from higher to lower content of organic matter); in the American system they are grouped in the order Molisoles and Molo epipedon. Soil samples known as Fluvisols were also taken according to FAO definitions. The bacteria selected for the study were the following: - Serratia marscens - Proteus vulgaris - Micro coccus sp - Citrobacter freundii - Bacillus cereus - Enterobacter cloacae - Penicillum sp. - Aspergillus niger The following example describes the process to prepare the bacillus cereus strain SC3115. Example: Several water and soil samples from the northern region of the cd were used to obtain the bacillus cereus strain. From Saltillo, Coatí. Of cells developed 24 hrs. On nutritive agar, one foot or a suspension with 1-2 ml of sterile liquid medium and with a sterile pipette, a liquid culture medium was inoculated with the following composition (%): nutritive broth - 0.8, peptone -0.3, extract of yeast -025, L-aspartic acid -0.1. They were incubated for hours at a temperature of 30-32 ° C in one liter Erlenmeyer flasks under constant agitation (250 r.p.m.). At this time, the cells possess an aspartase activity of 60-100 ^ M aspatic acid per 100 g protein per hour. The cells are separated from the culture medium with the aid of a Beckman J2-HS refrigerated centrifuge, washed three times with sterilized water, suspended in a tosiol buriei. iμM i pli / the aspartase activity is determined by the incubation method of a suspension of whole bacteria cells, with ammonium fumarate. The specific cell-like aspartase activity of baciJlus f ^? Eu < is from 60.0 to 100 μM of aspartic acid formed by miLrqiaiiio d < . protein in one hour.

CHARACTERISTICS OF Bacillus cereus.

Cell morphology: The mobile cells are alone or in short chains, ton uni endospore formed in the central or subterminal region. The cells do not have a uniform Gram stain, they are Grampositive or Gramvariable.

Morphology of the colony: The colonies are opaque and irregular with a flat to blunt elevation, rough surface and eroded or lobed margins.

Characterization data Swabs + parasporal crystals Straight sticks + Gram stain Curved sticks + Gram-positive 2.1.-3.0 + Gramnegat iva Length 3.1.-4.0 + Long Gram-variable 4.1-5.0 + Translucent cologne Length 5.0-10.0 Colony transparent Width below 5.0 Opaque colony Width 0-.5-1.0 + Entire colony Width 1.1.-2.0 + Colony eiosionada Isolated cells + rare Rhizoid colony Chain cells + irregular colony Elongated colonies Lobed colony Rounded finishes + low convex colony Square terminations High convex colony Endospore formation + Convoked colony Endoesporangio Flat colony A spore per cell High colony Round spore Mobile colony as a unit Cylindrical spore Dissociated colonies Oval spore Twinkling colony Central spore Roma colony Terminal spore Dry colony Subterminal spore Soft colony * No parasporal crystals were observed at 24, 48 and 72 hours when cultured on plates with KTB Thuringensis agar (ATCC No, .1348) and incubated aerobically at 30 ° C. ** Non-uniform coloring.

BACTERIAL CHARACTERIZATION Sol. Of brown pigment Sucrose acid Sol. of black pigment Sucrose gas Sol. of pigment Yellow D-mannitol acid Insol. of pigment Acid passing > 14 days coffee Insol. of pigment D-mannitol gas Black Insol. of pigment Ut lization of + yellow propionate Insol. of pigment Use of citrate + orange Insol. of pigment Hydrolysis of hippurate - red Cells mobile Weak hydrolysis of hippurate Mobile by flagellum Hydrolyzed starch + temp. Opt. 0-10 ° C Jellied liquefaction Temp. Opt. 11-20 ° C Hydrolyzed Casein + Temp. Opt. 21-30 ° C Methylene blue + reduced Temp. Opt. 31-40 ° C Methylene blue + reoxidized Temp. Opt. 41-50 ° C NO, reduced to NO, i Growth at 15 ° C VP (5198) positive + Growth at 20 ° C VP (5198 fil) positive + Growth at 25 ° C VP (5331) positive + Growth at 30 ° C Decomposition of H ^ 0, + II Growth at 37 ° C + Indole Growth at 45 ° C + Degradation of + tyrosine Growth at 50 ° C Acidic Litmus Milk Growth at 55 ° C Coagulated Litmus Milk Growth at 60 ° C Litmus + alkaline milk Growth at 65 ° C C Litmus + peptonized milk Growth at 70 ° C Litmus milk reduced i Growth at 5% + Growth at pH 6.0 t NaCl Growth at 7% -t- pH VP 5198 6.0 or less ^ of NaCl Growth at 10% pH VP 5198 7.0 +/- 0.5 NaCl Acid of L-arabinose pH VP 5198 8.0 or more Acid passing > 14 days Aerobio Gas from L-arabinose Optional i Acid from D-xyls Microaerophilic + Acid passing > 14 days Anaerobic D-xylose gas Growth 0.02% + azide D-glucose + gas acid from nitrate + sealed D-glucose gas - sealed glucose growth Lactose acid. _ - Lecithinase + S Acid passing > 14 days Lactose gas * Microaerophilic band formed 1/2"below A.C. stab S = strong

Claims (5)

Having described the invention is considered a novelty and therefore the content is claimed in the following: CLAIMS

1. - A strain SC 3115 of Bacillus cereus producing L-aspartic acid.

2. - A process for preparing a strain of Bacillus cereus, which comprises growing cells grown for 24 hours on nutritious agar from water and soil samples from the northern region of Coahuila in the following steps: A suspension was prepared with 1-2 ml of liquid medium sterilized and with a sterile pipette, a liquid culture medium was inoculated with the following composition (%): nutrient broth 0.8, peptone 0. 3, yeast extract 0.25, L-aspartic acid 0.1; * * -incubaron for 6 hrs. At a temperature of 30 - 32 ° C in mat aces

One liter Erlenmeyes under constant agitation (250 r.p.), at f-ar < -time, the cells possess an aspartase activity of 60 μl OμM aspartic acid per milligram of protein per hour; the cells are separated from the culture medium with the aid of a refrigerated Beckman J2-HS centrifuge; they are washed three times with sterile water, suspended in phosphate buffer O.OlμM of pH 7 and the aspartase activity is determined by the incubation method of a suspension of whole bacteria cells, with ammonium fumarate, aspartase activity. Bacillus cereus complete cell specific is from 60.0 to 100 μM aspartic acid formed per milligram of protein in one hour. • 3.- A process to prepare a Bacillus cereus strain, according to clause 2, characterized in that the soil samples from the Saltillo region for the development of cultures of bacteria with aspartate ammonia-coliase activity are osseous soils of the Chernozem type , Feozem and Castañozem and are grouped in the American system as Molisoles and Molo epipedon and compared in samples of 10 soils known as Fluvisols.

4. - A process for preparing a Bacillus cereus strain, in accordance with clause 3, characterized in that the bacteria selected for the study of used soils are Serratia maiscens, 15 Proteus vulgaris, Micro coecus sp, Citrobacter fieunriii, Racillus cereus, Enterobacter cloacae, Penicillum sp., Aspergillus nigei twenty 25 EXTRACT The invention relates to obtaining a new strain of Bacillus cereus bacteria, which synthesizes in different media the intracellular aspartase enzyme with high catalytic activity that responds to the requirements of biocatalysts: it has a short crop cycle (6 hours) with a high Final product index, obtained in the form of a pure optical isomer (L-form of aspartic acid [0] = 25.5 °), that is to say a ce D pa that does not possess racemase activity. The strain (biocatalyst) proposed in this application was selected from a number of pure strains of microorganisms, isolated from different natural substrates (water and soil) grown on nutrient agar. The strain is conserved in the collection of pure microorganisms, in the Department of Biotechnology of the Faculty of Chemical Sciences of the U.A.C. with NQ SC 3115.