Alpha-acetolactate Decarboxylase Research Articles

Immobilization of Alpha Acetolactate Decarboxylase in Hybrid Gelatin/Alginate Support for Application to Reduce Diacetyl Off-Flavor in Beer

Beer production is the largest among alcoholic beverages. Its production process is complex and demands several steps. Lager beers commonly present an off-flavor of butter that is due to the presence of diacetyl, and to avoid such a problem, a long period of maturation (3–5 weeks) is required. Another way is the application of (α-acetolactate decarboxylase) ALDC to accelerate the process. The objectives of the present work were to develop a low-cost support using gelatin, a residue from capsules from the nutraceutical industry, to immobilize the ALDC enzyme. For this, the yield, efficiency and activity recovered, and the stability of free and immobilized enzymes at different temperatures and pH were evaluated. To evaluate the capacity of immobilized enzymes when applied directly to beer and their operational stability, three concentrations of glutaraldehyde (1%, 2.5% and 5%) were tested in distilled water as a cross-linking agent. The best results obtained were 95.6%, 27.0% and 23.6%, respectively, for yield, efficiency and activity recovery. Immobilization provided a high activity over a wide pH range. The immobilized enzyme showed greater stability at temperatures of 50 and 60 °C. The immobilized derivative showed adequate reuse capacity, and its dehydrated form had excellent activity after long periods of storage.

2-Ketogluconic acid production by Klebsiella pneumoniae CGMCC 1.6366

Klebsiella pneumoniae CGMCC 1.6366 is a bacterium isolated for 1,3-propanediol or 2,3-butanediol production previously. K. pneumoniae ΔbudA, a 2,3-butanediol synthesis pathway truncated mutant with the gene deletion of budA which encodes alpha-acetolactate decarboxylase, was found to execrate an unknown chemical at a high titer when grown in the broth using glucose as carbon source. Later this chemical was identified to be 2-ketogluconic acid, which was formed through the glucose oxidation pathway in K. pneumoniae. It was found that 2-ketogluconic can also be produced by the wild strain. The fermentation studies showed that the production of this metabolite is strictly pH dependent, when the fermenting broth was maintained at pH 6-7, the main metabolite produced by K. pneumoniae CGMCC 1.6366 was 2,3-butanediol, or some organic acids in the budA mutated strain. However, if the cells were fermented at pH 4.7, 2-ketogluconic acid was formed, and the secretion of all other organic acids or 2,3-butanediol were limited. In the 5L bioreactors, a final level of 38.2 and 30.2g/L 2-ketogluconic acid were accumulated by the wild type and the budA mutant K. pneumoniae, respectively, in 26 and 56h; and the conversion ratios of glucose to 2-ketogluconic acid reached 0.86 and 0.91mol/mol for the wild and the budA mutant, respectively.

Development of Industrial‐Medium‐Required Elimination of the 2,3‐Butanediol Fermentation Pathway To Maintain Ethanol Yield in an Ethanologenic Strain of Klebsiellaoxytoca

Fermentation efficiency and nutrient costs are both significant factors in process economics for the microbial conversion of cellulosic biomass to commodity chemicals such as ethanol. In this study, we have developed a more industrial medium (OUM1) composed of 0.5% corn steep liquor (dry weight basis) supplemented with mineral salts (0.2%), urea (0.06%), and glucose (9%). Although the growth of strain P2 was vigorous in this medium, approximately 14% of substrate carbon was diverted into 2,3-butanediol and acetoin under the low pH conditions needed for optimal cellulase activity during simultaneous saccharification. Deleting the central region of the budAB genes encoding alpha-acetolactate synthase and alpha-acetolactate decarboxylase eliminated the butanediol and acetoin coproducts and increased ethanol yields by 12%. In OUM1 medium at pH 5.2, strain BW21 produced over 4% ethanol in 48 h (0.47 g ethanol per g glucose). Average productivity (48 h), ethanol titer, and ethanol yield for BW21 in OUM1 medium (pH 5.2) exceeded that of the parent (strain P2) in rich laboratory medium (Luria broth).

Degradácia alfa-acetolaktátu imobilizovanou alfa-acetolaktátdekarboxylázou.

Overovali sme účinnosĹĽ degradĂĄcie α-acetolaktĂĄtu ako prekurzora diacetylu enzĂ˝mom α-acetolaktĂĄtdekarboxylĂĄza, ktorĂ˝ bol rĂ´znymi spĂ´sobmi imobilizovanĂ˝ do alginĂĄtu vĂĄpenatĂŠho, kvĂ´li moĹžnosti opakovanĂŠho pouĹžitia v nĂĄslednĂ˝ch fermentĂĄciĂĄch. Pri prvej fermentĂĄcii spĂ´sob imobilizĂĄcie enzĂ˝movĂŠho prĂ­pravku nemal vplyv na jeho stabilitu, koncentrĂĄcia diacetylu v mladom pive bola nulovĂĄ. Pri opakovanom pouĹžitĂ­ toho istĂŠho imobilizĂĄtu sa koncentrĂĄcia diacetylu pohybovala v rozmedzĂ­ od 0 do 0,11 mg/l v druhom cykle a v rozmedzĂ­ od 0,04 do 0,16 mg/l v treĹĽom cykle, v zĂĄvislosti od spĂ´sobu imobilizĂĄcie enzĂ˝movĂŠho prĂ­pravku. EnzĂ˝m bol najstabilnejĹĄĂ­, ak bol najprv naadsorbovanĂ˝ na povrch DEAE celulĂłzy a nĂĄsledne spolu s kvasinkami uzavretĂ˝ do alginĂĄtovĂŠho gĂŠlu, čím bolo znĂ­ĹženĂŠ jeho vyplavovanie z nosiča.

Purification, crystallization and preliminary X-ray crystallographic studies on acetolactate decarboxylase.

Acetolactate decarboxylase has the unique ability to decarboxylate both enantiomers of acetolactate to give a single enantiomer of the decarboxylation product, (R)-acetoin. A gene coding for alpha-acetolactate decarboxylase from Bacillus brevis (ATCC 11031) was cloned and overexpressed in B. subtilis. The enzyme was purified in two steps to homogeneity prior to crystallization. Three different diffraction-quality crystal forms were obtained by the hanging-drop vapour-diffusion method using a number of screening conditions. The best crystal form is suitable for structural studies and was grown from solutions containing 20% PEG 2000 MME, 10 mM cadmium chloride and 0.1 M Tris-HCl pH 7.0. They grew to a maximum dimension of approximately 0.4 mm and belong to the trigonal space group P3(1,2)21, with unit-cell parameters a = 47.0, c = 198.9 A. A complete data set was collected to 2 A from a single native crystal using synchrotron radiation.

Regulation of branched-chain amino acid biosynthesis by alpha-acetolactate decarboxylase in Streptococcus thermophilus.

To demonstrate the presence of an active alpha-acetolactate decarboxylase in Streptococcus thermophilus and to investigate its physiological function. Streptococcus thermophilus CNRZ385 contains a gene encoding an alpha-acetolactate decarboxylase. Comparison of the production of alpha-acetolactate and its decarboxylation products, by the parent strain and an alpha-acetolactate decarboxylase-deficient mutant, demonstrated the presence of a control of the pool of alpha-acetolactate by valine, leucine and isoleucine. This control occurs via an allosteric activation of the alpha-acetolactate decarboxylase. Cell-free extracts of S. thermophilus were not able to decarboxylate the isoleucine precursor alpha-acetohydroxybutyrate. These results strongly suggest that one of the physiological functions of the alpha-acetolactate decarboxylase in S. thermophilus is to regulate leucine and valine biosynthesis by diverting the flux of alpha-acetolactate towards acetoin when the branched-chain amino acids are present at a high concentration. Regulation of branched-chain amino acid biosynthesis by alpha-acetolactate decarboxylase may occur in several other micro-organisms and explain some of their growth properties.

Improved performances and control of beer fermentation using encapsulated alpha-acetolactate decarboxylase and modeling.

The use of the enzyme alpha-acetolactate decarboxylase allows the acceleration of beer fermentation/maturation because it shunts diacetyl formation, whose elimination is the rate-limiting step of the process. To obtain a cost reduction by using this exogenous enzyme, we propose a new process involving recoverable encapsulated alpha-acetolactate decarboxylase. The performance of traditional and new processes was investigated by a modeling approach. A simple model, focused on alpha-acetolactate and diacetyl profiles during beer fermentation, was set up. The simulated profiles are consistent with literature data. This study shows also that encapsulated alpha-acetolactate decarboxylase allows the acceleration of beer fermentation as efficiently as free alpha-acetolactate decarboxylase. The advantage of immobilized alpha-acetolactate decarboxylase versus free enzyme is that it is recoverable and reusable, which means a process cost reduction.

Transcriptional and translational regulation of alpha-acetolactate decarboxylase of Lactococcus lactis subsp. lactis.

The alpha-acetolactate decarboxylase (ALDC) gene, aldB, is the penultimate gene of the leu-ilv-ald operon, which encodes the three branched-chain amino acid (BCAA) biosynthesis genes in Lactococcus lactis. Its product plays a dual role in the cell: (i) it catalyzes the second step of the acetoin pathway, and (ii) it controls the pool of alpha-acetolactate during leucine and valine synthesis. It can be transcribed from the two promoters present upstream of the leu and ilv genes (P1 and P2) or independently under the control of its own promoter (P3). In this paper we show that the production of ALDC is limited by two mechanisms. First, the strength of P3 decreases greatly during starvation for BCAAs and under other conditions that generally provoke the stringent response. Second, although aldB is actively transcribed from P1 and P2 during BCAA starvation, ALDC is not significantly produced from these transcripts. The aldB ribosome binding site (RBS) appears to be entrapped in a stem-loop, which is itself part of a more complex RNA folding structure. The function of the structure was studied by mutagenesis, using translational fusions with luciferase genes to assess its activity. The presence of the single stem-loop entrapping the aldB RBS was responsible for a 100-fold decrease in the level of aldB translation. The presence of a supplementary secondary structure upstream of the stem-loop led to an additional fivefold decrease of aldB translation. Finally, the translation of the ilvA gene terminating in the latter structure decreased the level of translation of aldB fivefold more, leading to the complete extinction of the reporter gene activity. Since three leucines and one valine are present among the last six amino acids of the ilvA product, we propose that pausing of the ribosomes during translation could modulate the folding of the messenger, as a function of BCAA availability. The purpose of the structure-dependent regulation could be to ensure the minimal production of ALDC required for the control of the acetolactate pool during BCAA synthesis but to avoid its overproduction, which would dissipate acetolactate. Large amounts of ALDC, necessary for operation of the acetoin pathway, could be produced under favorable conditions from the P3 transcripts, which do not contain the secondary structures.

The production of D‐acetoin by a transgenic Escherichia coli

A 6 kbp SphI fragment encoding genes for the enzymes alpha-acetolactate synthase (ALS), alpha-acetolactate decarboxylase (ALDC) and meso-2,3-butanediol dehydrogenase (meso-BDH), involved in the formation of meso-2,3-butanediol (meso-BD) from pyruvic acid, was cloned into plasmid pUC118. When derivatives of this plasmid were introduced into Escherichia coli JM109, the transformants were able to produce D-acetoin (D-AC) without contamination with L-acetoin (L-AC).

Diacetyl production mechanism by a strain ofLactococcus lactis spp.lactis bv.diacetylactis

Diacetyl production via alpha-acetolactic acid (ALA) extracellular decarboxylation in Lactococcus lactis spp. lactis bv. diacetylactis SD 933 cultures has been assessed under anaerobiosis both in batch and continuous fermentations at pH 5.5 and 8.0 by studying the effects of alpha-acetolactate decarboxylase (ADC) addition in the culture broth. This enzyme, favoring the formation of acetoin instead of diacetyl, was added extracellularly and did not disturb diacetyl production. Moreover, oxidation experiments on extracellular culture media did not reveal any increase in diacetyl amount caused by extracellular ALA oxidative decarboxylation. These observations confirm previous assertions concerning the mechanism and localization of diacetyl synthesis by the SD 933 strain.

Dual role of alpha-acetolactate decarboxylase in Lactococcus lactis subsp. lactis.

The alpha-acetolactate decarboxylase gene aldB is clustered with the genes for the branched-chain amino acids (BCAA) in Lactococcus lactis subsp. lactis. It can be transcribed with BCAA genes under isoleucine regulation or independently of BCAA synthesis under the control of its own promoter. The product of aldB is responsible for leucine sensibility under valine starvation. In the presence of more than 10 microM leucine, the alpha-acetolactate produced by the biosynthetic acetohydroxy acid synthase IlvBN is transformed to acetoin by AldB and, consequently, is not available for valine synthesis. AldB is also involved in acetoin formation in the 2,3-butanediol pathway, initiated by the catabolic acetolactate synthase, AlsS. The differences in the genetic organization, the expression, and the kinetics parameters of these enzymes between L. lactis and Klebsiella terrigena, Bacillus subtilis, or Leuconostoc oenos suggest that this pathway plays a different role in the metabolism in these bacteria. Thus, the alpha-acetolactate decarboxylase from L. lactis plays a dual role in the cell: (i) as key regulator of valine and leucine biosynthesis, by controlling the acetolactate flux by a shift to catabolism; and (ii) as an enzyme catalyzing the second step of the 2,3-butanediol pathway.

Cloning and sequencing of the gene encoding alpha-acetolactate decarboxylase from Leuconostoc oenos.

The alsD gene encoding alpha-acetolactate decarboxylase was isolated from a genomic library of Leuconostoc oenos, using a screening procedure developed on microtiter plates. The nucleotide sequence of alsD encodes a putative protein of 239 amino acids showing significant similarity with other bacterial alpha-acetolactate decarboxylases. Upstream from alsD lies an open reading frame (alsS) which is highly similar to bacterial genes coding for catabolic alpha-acetolactate synthases. Northern (RNA) blotting analyses indicated the presence of a 2.4-kb dicistronic transcript of alsS and alsD. This suggests that the alsS and alsD genes are organized in a single operon.

Genetic manipulation of the pathway for diacetyl metabolism in Lactococcus lactis.

Diacetyl is an important food flavor compound produced by certain strains of citrate-metabolizing lactic acid bacteria. Citrate is converted to pyruvate, from which diacetyl is produced via intermediate alpha-acetolactate. This paper reports the cloning and analysis of the gene (aldB) encoding alpha-acetolactate decarboxylase from Lactococcus lactis MG1363. Deletion of the MG1363 chromosomal aldB gene was achieved by double crossover homologous recombination. The mutant strain was found to produce diacetyl; alpha-acetolactate decarboxylase activity was eliminated. Overexpression of the cloned ilvBN genes (encoding an alpha-acetolactate synthase) in the aldB deletion strain produced even higher levels of alpha-acetolactate, acetoin, and diacetyl.

Imbalance of leucine flux in Lactococcus lactis and its use for the isolation of diacetyl-overproducing strains.

Diacetyl is a by-product of pyruvate metabolism in Lactococcus lactis, where pyruvate is first converted to alpha-acetolactate, which is slowly decarboxylated to diacetyl in the presence of oxygen. L. lactis usually converts alpha-acetolactate to acetoin enzymatically, by alpha-acetolactate decarboxylase encoded by the aldB gene. We took advantage of the fact that this enzyme also has a central role in the regulation of branched-chain amino acids, to select spontaneous aldB mutants in an unbalanced concentration of leucine versus those of valine and isoleucine in the medium. Industrial dairy strains of L. lactis subsp. lactis biovar diacetylactis containing point mutations and deletions of aldB were isolated and characterized. Their growth in milk was not affected, but they rapidly accumulated a large amount of alpha-acetolactate instead of acetoin from citrate in milk. Under aerated condition, strains devoid of AldB produced about 10 times more diacetyl than did the parental strains.

Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes

The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena genes budB and budC, coding for alpha-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long alpha-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic alpha-ALS enzymes encoded by the genes ilvB, ilvG, and ilvI. The K. terrigena alpha-ALS is also shown to complement an anabolic alpha-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.

5043276 DNA strand coding for alpha-acetolactate decarboxylase and yeast transformed with the DNA strand: Shigeyuk Yamano, Junichi Tanaka, Takashi Inoue, Takasaki, Japan assigned to Kirin Beer Kabushiki Kaisha

5043276 DNA strand coding for alpha-acetolactate decarboxylase and yeast transformed with the DNA strand: Shigeyuk Yamano, Junichi Tanaka, Takashi Inoue, Takasaki, Japan assigned to Kirin Beer Kabushiki Kaisha

5043276 DNA strand coding for alpha-acetolactate decarboxylase and yeast transformed with the DNA strand: Shigeyuk Yamano, Junichi Tanaka, Takashi Inoue, Takasaki, Japan assigned to Kirin Beer Kabushiki Kaisha

5043276 DNA strand coding for alpha-acetolactate decarboxylase and yeast transformed with the DNA strand: Shigeyuk Yamano, Junichi Tanaka, Takashi Inoue, Takasaki, Japan assigned to Kirin Beer Kabushiki Kaisha

Cloning of aldB, which encodes alpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevis

A gene for alpha-acetolactate decarboxylase (ALDC) was cloned from Bacillus brevis in Escherichia coli and in Bacillus subtilis. The 1.3-kilobase-pair nucleotide sequence of the gene, aldB, encoding ALDC and its flanking regions was determined. An open reading frame of 285 amino acids included a typical N-terminal signal peptide of 24 or 27 amino acids. A B. subtilis strain harboring the aldB gene on a recombinant plasmid processed and secreted ALDC. In contrast, a similar enzyme from Enterobacter aerogenes is intracellular.

Application of a ribosomal DNA integration vector in the construction of a brewer's yeast having alpha-acetolactate decarboxylase activity

An integration plasmid, pIARL28, containing the ribosomal DNA gene as a homologous recombination sequence was constructed for introduction of the alpha-acetolactate decarboxylase gene into brewer's yeast. The transformation efficiency of pIARL28 was 20- to 50-fold higher than those of the other YIp vectors, as yeast cells had approximately 140 copies of the ribosomal DNA gene. All transformants showed very high alpha-acetolactate decarboxylase activity due to the multiple integrated copies of the plasmid. The transformants were grown in nonselective conditions, and segregants which had maintained the alpha-acetolactate decarboxylase expression cassette but no other vector sequences were isolated. Southern analysis showed that these marker-excised segregants contained more than 20 copies of the alpha-acetolactate decarboxylase gene and were stably maintained under nonselective conditions. Fermentation tests confirmed that the diacetyl concentration was considerably reduced in wort fermented by these marker-excised segregants. The degree of reduction was related to the copy number of the alpha-acetolactate decarboxylase gene.

4895802 DNA strand coding for alpha-acetolactate decarboxylase and yeast transformed with the DNA strand: Hidetaka Sone, Junichi Tanaka, Takashi Inoue, Takasaki, Japan assigned to Kirin Beer Kabushiki Kaisha

4895802 DNA strand coding for alpha-acetolactate decarboxylase and yeast transformed with the DNA strand: Hidetaka Sone, Junichi Tanaka, Takashi Inoue, Takasaki, Japan assigned to Kirin Beer Kabushiki Kaisha