Integrated development of fermentation and downstream processing or L-isoleucine production with Corynebacterium glutamicum

 Applying a genetic algorithm for the optimization of trace element composition in the medium for L-isoleucine production from glucose and DL-α-hydroxybutyric acid with Corynebacterium glutamicum resulted in a reduction of the byproduct L-valine. High L-isoleucine broth concentrations of 20 g/l within 72 h at an L-isoleucine/DL-α-hydroxy butyric acid yield of 70% (w/w) and an L-isoleucine/L-valine ratio of 100 were achieved, if closed-loop control of glucose and of DL-α-hydroxybutyric acid was applied. For the isolation of L-isoleucine from fermentation broth a specific downstream processing was developed and optimized up to semitechnical scale (ultrafiltration, reverse osmosis, first crystallization, active-carbon adsorption, electrodialysis, second crystallization). The economic model of this downstream processing, which was identified by coupling the mass balance and energy balance with the semi-empirical models of the unit operations, was used to quantify the isolation costs as a function of L-isoleucine concentration and L-isoleucine/L-valine ratio in the fermentation broth. A cost reduction for downstream processing from DM 55 to DM 25 (kg L-isoleucine)^-1 and an improvement of the L-isoleucine yield in downstream processing from 48% to 80% was achieved using this economic model as the objective function to be minimized by the fermentation process (scenario: production of 70 tonnes L-isoleucine/year). Received: 8 January 1996/Received revision: 22 April 1996/Accepted: 29 April 1996     

Modelling the influence of pH, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid production by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour

A kinetic model of the fermentative production of lactic acid from glucose by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour has been developed. The model consists of terms for substrate and product inhibition as well as for the influence of pH and temperature. Experimental data from fermentation experiments under different physical conditions were used to fit and verify the model. Temperatures above 30 °C and pH levels below 6 enhanced the formation of by-products and d-lactic acid. By-products were formed in the presence of maltose only, whereas d-lactic acid was formed independently of the presence of maltose although the amount formed was greater when maltose was present. The lactic acid productivity was highest between 33 °C and 35 °C and at pH 6. In the concentration interval studied (up to 180 g l⁻¹ glucose and 89  g l⁻¹ lactic acid) simulations showed that both substances were inhibiting. Glucose inhibition was small compared with the inhibition due to lactic acid. Received: 28 October 1997 / Received revision: 3 February 1998 / Accepted: 6 February 1998     

d-lactic acid production from cellooligosaccharides and β-glucan using l-LDH gene-deficient and endoglucanase-secreting Lactobacillus plantarum

In order to achieve direct fermentation of an optically pure d-lactic acid from cellulosic materials, an endoglucanase from a Clostridium thermocellum (CelA)-secreting plasmid was introduced into an l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (∆ldhL1) bacterial strain. CelA expression and its degradation of β-glucan was confirmed by western blot analysis and enzyme assay, respectively. Although the CelA-secreting ∆ldhL1 assimilated cellooligosaccharides up to cellohexaose (although not cellotetraose), the main end product was acetic acid, not lactic acid, due to the conversion of lactic acid to acetic acid. Cultivation under anaerobic conditions partially suppressed this conversion resulting in the production of 1.27 g/l of D-lactic acid with a high optical purity of 99.5% from a medium containing 2 g/l of cellohexaose. Subsequently, D-lactic acid fermentation from barley β-glucan was carried out with the addition of Aspergillus aculeatus β-glucosidase produced by recombinant Aspergillus oryzae and 1.47 g/l of D-lactic was produced with a high optical purity of 99.7%. This is the first report of direct lactic acid fermentation from β-glucan and a cellooligosaccharide that is a more highly polymerized sugar than cellotriose.     

Modeling the specific growth rate of Lactobacillus plantarum in cucumber extract

Extensive empirical research has been published on the fermentation of vegetables, but little predictive modeling of the process is available. The objectives of this study were to assess the effects of key variables involved in cucumber fermentation and to develop models for predicting the growth of Lactobacillus plantarum in pure and mixed culture fermentations. The growth medium for the studies was cucumber juice. The effects of various concentrations of lactic, acetic, and hydochloric acids and sodium chloride on growth at 30° C were determined in batch culture. Limiting conditions for growth were pH 3.37 (lower limit), 69 mm undissociated lactic acid, 150 mm undissociated acetic acid, or 11.8% NaCl. Acetic acid was stimulatory to growth at low concentrations (up to 40 mm) but inhibitory at higher concentrations. Lactic acid was more inhibitory than acetic acid, whether total or undissociated concentrations were used as the basis of comparison. A predictive equation for specific growth rate was developed, tested, and shown to predict growth of L. plantarum in batch processes reasonably well.Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U. S. Department of Agriculture or North Carolina Agricultural Research Service, nor does it imply approval to the exclusion of other products that may be suitable Correspondence to: H. P. Fleming     

Intracellular Proteases of Bacillus thuringiensis subsp. kurstaki and a Protease-Deficient Mutant Btk-q

The commencement of intracellular protease synthesis was studied by gelatin zymography in Bacillus thuringiensis (Btk) HD1, Btk HD73, and a protease-deficient mutant Btk-q derived from the former strain. By gelatin zymography, a 92-kDa protease was detected first at 3 h of sporulation, which continued until 48 h, whereas two other proteases of mol wt 78 and 69 kDa were detectable from 6 h onwards and continued until 48 h of growth in Btk HD1. Similar studies revealed the presence of two major intracellular proteases in Btk HD73 by gelatin zymography, which first appeared at 6 h of sporulation and continued until 48 h of growth. The quantitative azocasein assay confirmed that the total protease activity increases from 3 to 21 h, thereafter reaching a plateau up to 48 h of growth examined, in HD1 and HD73 strains. Btk-q, a protease-deficient mutant, showed traces of protease activity by azocasein analysis that could not be detected by gelatin zymography. The free amino acid pool content was also increased parallel to the way that the protease activity increased in all three strains. However, this increase was found to be low (16-fold) in Btk-q when compared with Btk HD1 and HD73 strains. The following amino acids were detected by paper chromatography in Btk HD1: DL-alanine, L-glutamic acid, L-aspartic acid, tyrosine, tryptophan/methionine/valine, arginine, leucine/norleucine/isoleucine, and glycine, whereas only DL-alanine, L-glutamic acid, and L-aspartic acid were in Btk-q at 24 and 48 h, when the protease activity was maximum. Received: 4 January 2002 / Accepted: 6 March 2002     

The aerobic growth and product formation of Lactobacillus,Leuconostoc, Brochothrix,and Carnobacterium in batch cultures

Summary The aerobic growth and metabolism of eleven homofermentative and three heterofermentative Lactobacillus strains, three Leuconostoc strains, two Brochothrix thermosphacta strains and two Carnobacterium strains were studied in batch cultures at pH 6.0 and 25°C on a complex substrate containing 10.0 g glucose per litre. All strains, except Carnobacterium divergens 69, grew well aerobically. An oxygen consumption was registered for 18 of the strains—the exceptions being Lactobacillus alimentarius DSM 20249^T, Lactobacillus farciminis DSM 20284^T and Lactobacillus sharpeae DSM 20505^T. The homofermentative lactobacilli showed a maximal oxygen consumption during the stationary growth phase and this was coupled with a low final viable count. Leuconostoc strains, heterofermentative lactobacilli, Brochothrix thermosphacta and Carnobacterium strains showed a maximal oxygen consumption during the exponential growth phase together with a high final viable count. The maximum specific growth rate varied from 0.19 to 0.54 h^-1 while the growth yield varied from 19 to 86 g dry weight per mol glucose consumed. In general, homofermentative lactobacilli produced dl-lactic acid, acetic acid and acetoin. The three heterofermentative lactobacilli produced dl-lactic acid and acetic acid, two strains also produced ethanol Leuconostoc spp. formed d-lactic acid, acetic acid, and ethanol. B. thermosphacta produced acetoin, acetic acid, formic acid, isobutyric acid and isovaleric acid but no lactic acid. Carnobacterium produced l-lactic acid, acetic acid and acetoin. All strains accumulated hydrogen peroxide except L. alimentarius DSM 20249^T, Carnobacterium piscicola 3 and B. thermosphacta.née Blickstad     

A higher optical purity of l-lastic acid produced in Streptococcus faecalis

In fermentation of lactic acid with Streptococcus faecalis, which produces mainly l-lactic acid, the optical purity of the l-lactic acid produced was improved from 97.1% to 99.8% by the addition of 0.5 g/l of diammonium hydrogen phosphate. The fermentation time was reduced from 130 h to 47 h b9y the improved method. Correspondence to: H. Ohara     

The effect of water activity on growth and end-product formation of two Lactobacillus spp. and Brochothrix thermosphacta ATCC 11509T

Summary The effect of water activity (a_w) on the growth and end-product formation of Lactobacillus viridescens SMRICC 174, Lactobacillus SMRICC 173 (homofermentative) and Brochothrix thermosphacta ATCC 11509^T was studied. All strains orginated from meat or meat products. The a_w was adjusted in the range 0.94–0.99 with NaCl or glycerol. A greater reduction in growth rates was found for L. viridescens and B. thermosphacta when a_w was regulated with NaCl rather than with glycerol, the opposite was true for Lactobacillus 173. L. viridescens grew at a_w >-0.94. At 0.94 a_w B. thermosphacta was totally inhibited when NaCl was the solute and Lactobacillus 173 when glycerol was the solute. Only minor variations in the end-product formation of the Lactobacillus spp. were found at different a_w values. In aerobic culture B. thermosphacta produced less l-lactic acid and more acetic acid as the a_w was decreased with NaCl, while the yields were unaffected when glycerol was used.     

Evaluation ofl-lactic acid production in batch, fed-batch, and continuous cultures ofRhizopus sp. MK-96-1196 using an airlift bioreactor

Various processes which producel-lactic acid using ammonia-tolerant mutant strain,Rhizopus sp. MK-96-1196, in a 3 L airlift bioreactor were evaluated. When the fed-batch culture was carried out by keeping the glucose concentration at 30 g/l, more than 140 g/l ofl-lactic acid was produced with a product yield of 83%. In the case of the batch culture with 200 g/l of initial glucose concentration, 121 g/L ofl-lactic acid was obtained but the low product yield based on the amount of glucose consumed. In the case of a continuous culture, 1.5 g/l/h of the volumetric productivity with a product yield of 71% was achieved at dilution rate of 0.024 h⁻¹. Basis on these results three processes were evaluated by simple variable cost estimation including carbon source, steam, and waste treatment costs. The total variable costs of the fed-batch and continuous cultures were 88% and 140%, respectively, compared to that of batch culture. The fed-batch culture with highl-lactic acid concentration and high product yield decreased variable costs, and was the best-suited for the industrial production ofl-lactic acid.     

Production of optically pure d-lactic acid from brown rice using metabolically engineered Lactobacillus plantarum

Simultaneous saccharification and fermentation (SSF) of d-lactic acid was performed using brown rice as both a substrate and a nutrient source. An engineered Lactobacillus plantarum NCIMB 8826 strain, in which the ʟ-lactate dehydrogenase gene was disrupted, produced 97.7 g/L d-lactic acid from 20% (w/v) brown rice without any nutrient supplementation. However, a significant amount of glucose remained unconsumed and the yield of lactic acid was as low as 0.75 (g/g-glucose contained in brown rice). Interestingly, the glucose consumption was significantly improved by adapting L. plantarum cells to the low-pH condition during the early stage of SSF (8–17 h). As a result, 117.1 g/L d-lactic acid was produced with a high yield of 0.93 and an optical purity of 99.6% after 144 h of fermentation. SSF experiments were repeatedly performed for ten times and d-lactic acid was stably produced using recycled cells (118.4–129.8 g/L). On average, d-lactic acid was produced with a volumetric productivity of 2.18 g/L/h over 48 h.

     

Enzymatic preparation of optically active 3-trimethylsilylalanine

 We constructed an efficient system for preparing optically active 3-trimethylsilylalanine (TMS-Ala) by kinetic resolution with acylase I (aminoacylase; N-acylamino-acid amidohydrolase, EC 3.5.1.14). Racemic TMS-Ala was chemically synthesized and acetylated. Enantioselective deacetylation of N-acetyl-DL-TMS-Ala with acylase I from porcine kidney or from Aspergillus melleus was then attempted. Both enzymes could catalyze the deacetylation of N-acetyl-DL-TMS-Ala, and the porcine enzyme was found to have much higher activity than the enzyme from A. melleus. The optimum pH of the porcine-acylase-catalyzed reaction was 7.5, and the addition of 0.5 mM Co²⁺ accelerated the reaction. Optically pure L-TMS-Ala (>99% enantiomeric excess, ee) was obtained in 72% yield under the optimized conditions. Furthermore, highly optically pure D-TMS-Ala (96% ee) could also be obtained in 76% yield by chemically hydrolyzing the residual substrate. Received: 6 June 1995/Received revision: 3 July 1995/Accepted: 19 July 1995     

Diacetyl and acetoin production from the co-metabolism of citrate and xylose by Leuconostoc mesenteroides subsp. mesenteroides

The co-metabolism of citrate plus xylose by Leuconostoc mesenteroides subsp. mesenteroides results in a growth stimulation, an increase in d-lactate and acetate production and repression of ethanol production. This correlated well with the levels of key enzymes involved. A partial repression of alcohol dehydrogenase and a marked stimulation of acetate kinase were observed. High citrate bioconversion yields in diacetyl plus acetoin were obtained at pH 5.2 in batch (11.5%) or in chemostat (up to 17.4%) culture. In contrast, no diacetyl or acetoin was detected in citrate plus glucose fermentation. Received: 6 December 1996 / Received revision: 14 February 1997 / Accepted: 14 February 1997     

Die Aminosäuresequenz des DAP-haltigen Mureins von Lactobacillus plantarum und Lactobacillus inulinus

Zusammenfassung Von L. plantarum und L. inulinus wurden die Zellwände isoliert und durch Inkubation mit Trypsin gereinigt. Durch Extraktion mit TES und Formamid konnte das Murein (Peptidoglycan) bis zu rund 85% der Trockenmasse angereichert werden. Die Zellwände von L. plantarum enthielten rund 30% Teichonsäure des Ribit-Typs, die von L. inulinus waren frei von Teichonsäure.Im Hydrolysat der teichonsäurefreien Zellwände ergaben sich folgende aufbzw. abgerundete Molverhältnisse Mur: GlNH₂:Glu:DAPl-Alad-Ala=1:1:1:1:1:0,5. Außerdem waren 2 Mole Ammoniak enthalten, was das Vorliegen von Glu und DAP als Amide anzeigt. Die durch Hemmung mit d-Cycloserin angereicherte unvollständige Mureinvorstufe hatte ein Molverhältnis von UDP:Murl-Ala:Glu:DAP=1:1:1:1:1.Nach Dinitrophenylierung der Zellwand ließen sich rund 50% der gesamten DAP als mono-DNP-DAP nachweisen. Die Hydrazinolyse der Zellwand zum Nachweis C-terminaler Aminosäuren ergab 4% freies DAP und 0,8% freies Alanin.Durch die Analyse der in Partialhydrolysaten der Zellwand auftretenden Peptide konnte die folgende Aminosäuresequenz des an die Muraminsäure gebundenen Tetrapeptides bestimmt werden: l-Ala-d-Glu-l-Lys-d-Ala. Im Murein ist vermutlich nur etwa die Hälfte der Muraminsäure mit einem Tetrapeptid, die andere Hälfte mit einem Tripeptid, dessen d-Alanin fehlt, substituiert.Die Quervernetzung erfolgt zwischen der 2. Aminogruppe der DAP und der Carboxylgruppe des d-Alanins eines benachbarten Tetrapeptids.

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The amino acid sequence of the DAP-containing murein of Lactobacillus plantarum and Lactobacillus inulinus

Summary Cell walls of L. plantarum and L. inulinus were isolated and purified by incubation with trypsin. After extraction with TCA and formamide, 85% of the dry weight consists of murein (peptidoglycan).The cell walls of L. plantarum contained about 30% teichoic acid (ribit-type), whereas no teichoic acid was present in the cell walls of L. inulinus.The quantitative determination of amino sugars and amino acids in the hydrolysate of the cell walls showed the following molar ratios: Mur: Gl-NH₂:Glu:DAP l-Alad-Ala=1:1:1:1:1:0.5. In addition, 2 mols of NH₃ were found per mol of glutamic acid, indicating, that DAP as well as glutamic acid are present as amides.The UDP-activated cell wall precursor which was accumulated by inhibiting the cells by d-cycloserine showed the following molar ratios: UDP:Murl-Ala: Glu:DAP=1:1:1:1:1.After dinitrophenylation and hydrolysation of the cell wall 50% of the DAP were present as mono-DNP-DAP. Hydrozinolysis of the cell wall yielded 4% free DAP and 0.8% free alanine. This shows that only a very small amount of these amino acids are C-terminal in the whole murein.The analysis of various peptides from acid partial hydrolysates of the cell wall indicates the following amino acid sequence of the tetrapeptides attached to muramic acid: l-Ala-d-Glu-meso-DAP-d-Ala. Only half of the muramic acid molecules are substituted by tetrapeptides, while the other half carries a tripeptide in which the terminal d-alanine is missing.The cross-linking of the muropeptides is achieved by a peptide-bond between the second amino group of DAP and the carboxylgroup of the d-alanine of an adjacent muropeptide.

     

Production of optically pure d-lactate by Lactobacillus bulgaricus and purification by crystallisation and liquid/liquid extraction

Optically pure d-lactic acid was produced by fermentation of lactose with Lactobacillus bulgaricus Lb-12, and purified by crystallisation as magnesium d-lactate followed by extraction with butanol. The yield of d-lactate and contaminations with nitrogen and phosphorus were mapped during the purification procedure. The overall yield of d-lactic acid was 72% and the purity was more than 99%. Contaminations in the final d-lactic acid with nitrogen, phosphorus and l-lactic acid were only 0.032% w/w, 0.018% w/w and 0.04% w/w respectively.     

Efficient synthesis of enantiomeric ethyl lactate by <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B (CALB)-displaying yeasts

The whole-cell biocatalyst displaying Candida antarctica lipase B (CALB) on the yeast cell surface with α-agglutinin as the anchor protein was easy to handle and possessed high stability. The lyophilized CALB-displaying yeasts showed their original hydrolytic activity and were applied to an ester synthesis using ethanol and l-lactic acid as substrates. In water-saturated heptane, CALB-displaying yeasts catalyzed ethyl lactate synthesis. The synthesis efficiency increased depending on temperature and reached approximately 74% at 50°C. The amount of l-ethyl lactate increased gradually. l-Ethyl lactate synthesis stopped at 200 h and restarted after adding of l-lactic acid at 253 h. It indicated that CALB-displaying yeasts retained their synthetic activity under such reaction conditions. In addition, CALB-displaying yeasts were able to recognize l-lactic acid and d-lactic acid as substrates. l-Ethyl lactate was prepared from l-lactic acid and d-ethyl lactate was prepared from d-lactic acid using the same CALB-displaying whole-cell biocatalyst. These findings suggest that CALB-displaying yeasts can supply the enantiomeric lactic esters for preparation of useful and improved biopolymers of lactic acid.     

d-p-Hydroxyphenylglycine production from dl-5-p-hydroxyphenylhydantoin by Agrobacterium sp.

Summary A bacterium that stereospecifically produces D-p-hydroxyphenylglycine (D-PHPG) from DL-5-p-hydroxyphenylhydantoin (DL-5-PHPH) was isolated from soil and identified as Agrobacterium sp. IP-I 671. The hydantoinase and the N-carbamyl-amino acid amido-hydrolase involved in this biotransformation process were both strictly D-stereospecific. Their biosynthesis was found to be inducible by addition of 2-thiouracil to the cultivation media, or to a lesser extent by uracil. The amidohydrolase activity of Agrobacterium sp. was strongly inhibited by ammonium ions co-produced with D-PHPG, whereas the hydantoinase activity under the same conditions was unaffected. Optimum temperature and pH were respectively 55° C and 10 for the partially purified hydantoinase, 45° and 6.75 when resting cells were used. Biotransformation under these slightly acidic conditions allowed to complete conversion of 30 g/1 DL-5-PHPH into 25 g/l of D-PHPG (molar yield 96%) and involved enzymatic racemization of DL-5-PHPH. Offprint requests to: S. Runser     

Preferential production of rapamycin vs prolylrapamycin by Streptomyces hygroscopicus

A trace of prolylrapamycin is often produced in rapamycin fermentations carried out by strains of Streptomyces hygroscopicus. Prolylrapamycin was produced as the major rapamycin when L-proline was added to the fermentation medium. Addition of proline plus thiazolidine-2-carboxylic acid (T2CA), a sulfur-containing proline analog, prevented rapamycin production and stimulated prolylrapamycin production, thereby resulting in an even greater selective production of prolylrapamycin. T2CA addition inhibited rapamycin production even in the presence of L-lysine which is converted into pipecolic acid intracellularly and normally stimulates rapamycin formation. Addition of the rapamycin precursor, DL-pipecolic acid, surprisingly failed to stimulate rapamycin production. However, when DL-pipecolic acid was added with L-proline, it reduced the formation of prolylrapamycin and stimulated rapamycin production; this was evident especially in the presence of T2CA. The evidence suggests that T2CA suppresses rapamycin production by inhibiting intracellular conversion of L-lysine into pipecolate. Furthermore, the data suggest that uptake of pipecolate into the cell is stimulated or induced by growth in the presence of L-proline and/or T2CA. Received 24 December 1997/ Accepted in revised form 12 May 1998     

Influence of amino acids on the biosynthesis of cyclosporin A by Tolypocladium inflatum

 An indigenously isolated strain of Tolypocladium inflatum, when grown as a suspension culture in semi-synthetic and synthetic media, produced cyclosporin A. Biosynthesis of this well-known immunosuppressive agent was found to be influenced heavily by the external addition of the amino acid constituents of the molecule. In synthetic media, L-leucine and L-valine were found to act as strong inducers of drug production. L-Valine increased the specific production of cyclosporin A by 75% in semi-synthetic medium and by ten times in synthetic medium compared to an unsupplemented control culture. D-Valine had no stimulating effect on the production. The presence of amino acids in the exponential growth phase ensured optimal production, as was indicated in the experiment in which L-valine was added at different times; 4 g/l was the optimum concentration of exogenous L-valine. On the other hand, exogenous sarcosine and L-methionine tended to diminish drug production. Received: 23 October 1995/Received revision: 23 January 1996/Accepted: 29 January 1996     

Mutation-Screening in <Emphasis Type="SmallCaps">l</Emphasis>-(+)-Lactic Acid Producing Strains by Ion Implantation

In this paper, in order to obtain some industrial strains with high yield of l-(+)-lactic acid, the wild type strain Lactobacillus casei CICC6028 was mutated by nitrogen ions implantation. By study, it was found that the high positive mutation rate was obtained when the output power was 10 keV and the dose of N⁺ implantation was 50 × 2.6 × 10¹³ ions/cm². In addition, the initial screening methods were also studied, and it was found that the transparent halos method was unavailable, for some high yield strains of l-(+)-lactic acid were missed. Then a mutant strain which was named as N-2 was isolated, its optimum fermentation temperature was 40°C and the l-(+)-lactic acid yield was 136 g/l compared to the original strain whose optimum fermentation temperature was 34°C and l-(+)-lactic acid production was 98 g/l. Finally, High Performance Liquid Chromatography method was used to analyze the purity of l-(+)-lactic acid that was produced by the mutant N-2, and the result showed the main production of N-2 was l-(+)-lactic acid.