Uptake of α-Ketoglutarate by Citrate Transporter CitP Drives Transamination in Lactococcus lactis

Transamination is the first step in the conversion of amino acids into aroma compounds by lactic acid bacteria (LAB) used in food fermentations. The process is limited by the availability of α-ketoglutarate, which is the best α-keto donor for transaminases in LAB. Here, uptake of α-ketoglutarate by the citrate transporter CitP is reported. Cells of Lactococcus lactis IL1403 expressing CitP showed significant levels of transamination activity in the presence of α-ketoglutarate and one of the amino acids Ile, Leu, Val, Phe, or Met, while the same cells lacking CitP showed transamination activity only after permeabilization of the cell membrane. Moreover, the transamination activity of the cells followed the levels of CitP in a controlled expression system. The involvement of CitP in the uptake of the α-keto donor was further demonstrated by the increased consumption rate in the presence of l-lactate, which drives CitP in the fast exchange mode of transport. Transamination is the only active pathway for the conversion of α-ketoglutarate in IL1403; a stoichiometric conversion to glutamate and the corresponding α-keto acid from the amino acids was observed. The transamination activity by both the cells and the cytoplasmic fraction showed a remarkably flat pH profile over the range from pH 5 to pH 8, especially with the branched-chain amino acids. Further metabolism of the produced α-keto acids into α-hydroxy acids and other flavor compounds required the coupling of transamination to glycolysis. The results suggest a much broader role of the citrate transporter CitP in LAB than citrate uptake in the citrate fermentation pathway alone.     

Effects of cultivation conditions on the production of heterologous α-galactosidase by Kluyveromyces lactis

Growth conditions relevant for the large-scale production of heterologous proteins with yeasts were studied on a laboratory scale. A strain of Kluyveromyces lactis, containing 15 copies of an expression cassette encoding guar -galactosidase integrated into its ribosomal DNA, was used as a model. By using urea as a nitrogen source, it was possible to produce active extracellular -galactosidase in shake-flask cultures grown on a defined mineral medium. Inclusion of urea instead of ammonium sulphate prevented unwanted acidification of cultures. With urea-containing mineral medium, enzyme production in shake flasks was comparable to that in complex media containing peptone. In contrast, the presence of peptone was required to achieve high productivity in chemostat cultures. The low productivity in chemostat cultures growing on mineral media was not due to loss oft the expression cassette, since addition of peptone to such cultures resulted in an immediate high rate of -galactosidase production. The discrepancy between the behaviour of shake-flask and chemostat cultures during growth on mineral medium illustrates the necessity of physiological studies for the scalling-up of heterologous protein production from laboratory to production scale.     

Production of α-galactosidase by Monascus grown on soybean and sugarcane wastes

Extracts of both sugarcane and soybean wastes supported the growth of Monascus but sugarcane waste was superior for the production of -galactosidase. An aqueous extract prepared from 5% (w/v) soybean waste and 7% (w/v) sugarcane waste gave the best result and was superior to the standard peptone/glucose/yeast extract medium. Liquid-solid mixtures were slightly less effective. Enzyme production could be enhanced by adding raffinose. Enzymatic hydrolysis of p-nitrophenyl--D-galactoside was optimal at pH 4.5. Raffinose and stachyose were hydrolysed to sucrose and galactose.     

Quantification of extracellular α-acetolactate oxidative decarboxylation in diacetyl production by an α-acetolactate overproducing strain of Lactococcus lactis sp. lactis bv. diacetylactis

The quantification of -acetolactate (AAL) extracellular oxidative decarboxylation during an AAL overproducing strain culture shows that this reaction is at the origin of about 90% of the diacetyl production and that only a small proportion of extracellular AAL is readily transformed to diacetyl. These results, compared with previous ones obtained with a non AAL accumulating strain, allow research options to be put forward for the improvement of microbiological diacetyl production.     

Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min^–1 mg^–1, respectively.     

Secretion of human interferon-β 1b by recombinant Lactococcus lactis

Interferon-beta has anti-viral, anti-proliferation and multifunctional immunomodulatory activities and shows promising clinical effects for treatment of inflammatory disorders. The recombinant human interferon-beta (huIFN-beta) 1b was expressed in the food-grade lactic acid bacterium, Lactococcus lactis, using a nisin-controlled gene expression system. huIFN-beta production from recombinant strains (with and without LEISSTCDA propeptide) was approximately 21 and 7 mug l(-1), respectively. Moreover, 95% (former strain) and 88% (latter strain) of total recombinant proteins were secreted into the culture medium. The biological activities of huIFN-beta from recombinant strains revealed similar antiviral activities of 10(7) I.U. mg(-1). These results demonstrate the potential application of recombinant strains as a food grade vehicle to deliver bioactive huIFN-beta in vivo.     

Characterization of α-galacto-oligosaccharides formed via heterologous expression of α-galactosidases from Lactobacillus reuteri in Lactococcus lactis

α-Galacto-oligosaccharides (α-GOS) are produced by transgalactosylation reactions of α-galactosidase (α-Gal) or by conversion of raffinose family oligosaccharides by levansucrase. Similarly to β-GOS, α-GOS have the potential to mimic glycan receptors on eukaryotic cells and act as molecular decoys to prevent bacterial infection; however, data on transgalactosylation reactions of α-Gal remain scarce. The α-Gal gene sequence from Lactobacillus reuteri was cloned into an α-Gal negative strain of Lactococcus lactis. Transgalactosylation reactions were achieved using crude cell extracts with melibiose or raffinose as galactosyl donor and fucose, N-acetylglucosamine or lactose as galactosyl acceptor. The composition, sequence and most linkage types of α-GOS formed with acceptors saccharides were determined by liquid chromatography-tandem mass spectrometry. α-Gal of Lactobacillus reuteri formed (1?→?3)-, (1?→?4)- or (1?→?6)-linked α-GOS but exhibited a preference for formation of (1?→?6)-linkages. Fucose, N-acetylglucosamine and lactose were suitable galactosyl acceptors for α-Gal of L. reuteri, resulting in formation of (1?→?3)-, (1?→?4)- or (1?→?6)-linked hetero-oligosaccharides. By determining the structural specificity of α-Gal and increasing the variation of oligosaccharides produced by introducing alternative acceptor sugars, this work supports further studies to assess α-GOS pathogen adhesion prevention in mammalian hosts.     

Mg2+ ligands (Glu-384, Glu-429) of β-galactosidase from Lactococcus lactis ssp. lactis 7962

The secondary and the tertiary structures of -galactosidase from Lactococcus lactis ssp. lactis 7962 were designed by Nnpredict and Sybyl Version 6.3. Structural modeling of -galactosidase has shown that Glu-384 and Glu-429 are ligands for Mg²⁺ and Mg²⁺ is required for maximum activity. To confirm this prediction, we generated seven site specific mutants: Glu-384-Gln; Glu-384-Val; His-386-Phe; Asn-428-Asp; Glu-429-Gln; 384Gln-429Gln and 384Val-429Gln. The -galactosidases substituted at Glu-384 or Glu-429 had < 1% of the activity of the native enzyme with ONPG as substrate. The substitution of Glu-384 or Glu-429, which removed only one of the coordinating ligand for Mg²⁺, was still affected by Mg²⁺, but the mutants 384Gln-429Gln or 384Val-429Gln, which had been modified both Mg²⁺-binding sites, were not affected by Mg²⁺. Thus, Glu-384 and Glu-429 are probably ligands of Mg²⁺ and the three dimensional disposition of Mg²⁺ and its neighborhood interactions (Glu-384, Glu-429, Asp-428 or His-386) are important in the maintenance of –galactosidase activity.     

Cloning and nucleotide sequence of the β-galactosidase gene from Lactococcus lactis ssp. Lactis ATCC7962

The gene encoding -galactosidase of Lactococcus lactis ssp. lactis ATCC7962 was cloned and its nucleotide sequence was determined. The -galactosidase of L. lactis was expressed in Escherichia coli and transformants containing this gene fragment appeared as blue colonies on LB plates containing X-gal. The -galactosidase activity of E. coli transformant was thirty times higher than that of L. lactis. The gene for the 115 kDa -galactosidase has a 2991-bp open reading frame preceded by a putative ribosome binding site. The deduced amino acid sequence show a high degree of homology to the -galactosidase of E. coli, and the putative active site residues are conserved (Glu-429 and Tyr-475)     

Heterologous Production of Methionine-γ-Lyase from Brevibacterium linens in Lactococcus lactis and Formation of Volatile Sulfur Compounds

The conversion of methionine to volatile sulfur compounds (VSCs) is of great importance in flavor formation during cheese ripening and is the focus of biotechnological approaches toward flavor improvement. A synthetic mgl gene encoding methionine-γ-lyase (MGL) from Brevibacterium linens BL2 was cloned into a Lactococcus lactis expression plasmid under the control of the nisin-inducible promoter PnisA. When expressed in L. lactis and purified as a recombinant protein, MGL was shown to degrade l-methionine as well as other sulfur-containing compounds such as l-cysteine, l-cystathionine, and l-cystine. Overproduction of MGL in recombinant L. lactis also resulted in an increase in the degradation of these compounds compared to the wild-type strain. Importantly, gas chromatography-mass spectrometry analysis identified considerably higher formation of methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) in reactions containing either purified protein, whole cells, or cell extracts from the heterologous L. lactis strain. This is the first report of production of MGL from B. linens in L. lactis. Given their significance in cheese flavor development, the use of lactic acid bacteria with enhanced VSC-producing abilities could be an efficient way to enhance cheese flavor development.Methionine (Met) catabolism plays a major role in cheese flavor development. Met is believed to be the precursor of numerous diverse and quantitatively minor volatile sulfur compounds (VSCs) (38) which make important contributions to the overall flavor that are typical to different cheeses (7). Most of these compounds are derived from the degradation of Met to methanethiol (MTL), giving rise to a variety of compounds such as dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), and S-methylthioesters (41).The Met biosynthetic and catabolic pathways leading to MTL vary among bacteria (36), as do the enzymes involved and the amount of MTL produced during cheese ripening (15). The most direct pathway occurs via l-methionine γ-elimination of Met to MTL, α-ketobutyrate, and ammonia. This l-methionine γ-elimination activity has been shown to be quite efficient in Brevibacterium linens (21), while its presence has been suggested in several other cheese surface bacteria such as Micrococcus luteus, Arthrobacter sp., Corynebacterium glutamicum, and Staphylococcus equorum (9). In B. linens, this activity is catalyzed by an l-methionine-γ-lyase (MGL), which has been previously purified and characterized (16). Moreover, disruption of the mgl gene encoding the enzyme has been shown to almost eliminate this strain''s considerable capacity to produce VSCs (2). In lactococci, which produce only limited amounts of VSCs (15), this reaction is catalyzed by cystathionine lyases (β- or γ-) which are responsible for the simultaneous deamination and demethylthiolation of Met to MTL (1, 11, 18, 22); recently, a new C-S lyase (YtjE) with α,γ-elimination activity that degrades Met into MTL has been characterized in our laboratory (30). Unfortunately, C-S lyases display relatively low activities toward Met, limiting their capacities to produce VSCs. Another route for Met catabolism involving a two-step mechanism initiated by an aminotransferase has also been identified in lactococci (8, 23) and other cheese-ripening bacteria (3, 9), which leads to the formation of α-keto-γ-methylthiobutyric acid which is subsequently converted to MTL; this route, however, produces only limited amounts of MTL.In recent years, numerous studies have pursued the control and/or diversification of VSCs primarily by means of using selected cheese-ripening microorganisms or combinations of them (4, 7, 25). A few studies have focused on engineering lactic acid bacteria (LAB) with enhanced VSC-producing abilities by increasing cystathionine lyase activities (22, 27). In this respect, a Lactococcus lactis strain engineered to overproduce cystathionine β-lyase was shown to produce larger quantities of VSCs with Met as a substrate compared to the wild-type strain (22). However, this study also showed no significant difference in VSC production between the wild-type strain and a cystathionine β-lyase-knockout variant, implying that other enzymes may play a more prominent role in the conversion of Met.The aim of this study was to develop LAB with improved capability to produce MTL and other related VSCs as a more efficient strategy to enhance cheese flavor development. We describe the bioengineering of food-grade L. lactis strains to produce MGL, which has been reported to play a major role in the catabolism of Met to MTL in B. linens, a good producer of VSCs (2). The enzyme activity of the recombinant MGL was confirmed, and its role in the production of VSCs by recombinant L. lactis was also investigated.     

Cloning and nucleotide sequence of the α-galactosidase cDNA from Cyamopsis tetragonoloba (guar)

Polyadenylated mRNA was purified from the aleurone cells of Cyamopsis tetragonoloba (guar) seeds germinated for 18 h and used for the construction of a cDNA library. Clones with the -galactosidase encoding gene were identified using oligo-nucleotide mixed probes based on the NH₂ terminal amino acid sequence and on the sequence of an internal peptide. The nucleotide sequence of the cDNA clone showed that the enzyme is synthesized as a precursor with a 47 amino acid NH₂ terminal extension. This pre-sequence most likely functions to target the protein outside the aleurone cells into the endosperm. Based upon structural features, it is proposed to divide the precursor into a pre-(signal sequence) part and a glycosylated pro-part comparable with those of the yeast mat A/ factor and killer factor. A comparison of the derived amino acid sequence of this -galactosidase from plant origin revealed significant stretches of homology with respect to the amino acid sequences of the enzymes from Saccharomyces cerevisiae and from human origin but only to a minor extent compared with the -galactosidase from Escherichia coli.     

Composition of α-amylase secreted by aleurone layers of grains of himalaya barley

α-Amylases secreted by the aleurone layer of whole barley grains were relatively rich in histidine and relatively poor in glutamate/glutamine and serine when compared to other eukaryotic proteins. The secreted α-amylases had an estimated 0.5 residues each of glucose, mannose and N-acetylglucosamine per molecule of protein (MW 41 400 daltons), and gave positive staining reactions for carbohydrate on sodium dodecylsulfate polyacrylamide gels. Because the average α-amylase molecule had less than one sugar residue per enzyme molecule, it was concluded that secreted α-amylases were heterogeneous with respect to glycosylation. A second protein co-purified with α-amylase, but the amino acid composition of this protein was different from that of barley or wheat α-amylase. This protein was composed of two 21 500 dalton polypeptides. No significant amounts of L-leucine (¹⁴C-U) were incorporated into this second protein in isolated aleurone tissue during incubation with gibberellic acid, perhaps because much of it was already present in the starchy endosperm at the time of hormone addition.     

Synthesis of α-galacto-oligosaccharides by a cloned α-galactosidase from Bifidobacterium adolescentis

A genomic library of Bifidobacterium adolescentis was constructed in Escherichia coli and a gene encoding an -galactosidase was isolated. The identified open reading frame showed high similarity and identity with bacterial -galactosidases, which belong to Family 36 of the glycosyl hydrolases. For the purification of the enzyme from the medium a single chromatography step was sufficient. The yield of the recombinant enzyme was 100 times higher than from B. adolescentis itself. In addition to hydrolytic activity the -galactosidase showed transglycosylation activity and can be used for the production of -galacto-oligosaccharides.     

Production of β-galactosidase by Streptococcus salivarius subsp thermophilus 11F

The production of β-galactosidase by an autolytic strain of Streptococcus salivarius subsp thermophilus 11F was investigated in batch and fed-batch 2-L working volume stirred tank bioreactors. β-Galactosidase was released into the medium upon cell lysis within 1–2 h after the maximum biomass quantity was reached. In batch fermentations the highest β-galactosidase activity of 69 U ml⁻¹ was obtained when the temperature was increased to 42°C after a 4-h growth period at 30°C. In fed-batch experiments the highest β-galactosidase activity of 74 U ml⁻¹ was obtained at a constant 37°C. Received 18 December 1997/ Accepted in revised form 03 February 1998     

Production of galactooligosaccharide by β-galactosidase fromKluyveromyces maxianus varlactis OE-20

A galactooligosaccharide (GalOS)-producing yeast, OE-20 was selected from forty seven strains of yeast growing in Korean traditionalMeju (cooked soybean) and the yeast was tentatively identified asKluyveromyces maxianus varlactis by its morphology and fermentation profile. A maximum yield of 25.1%(w/w) GalOS, which corresponds to 25.1 g of GalOS per liter, was obtained from the reaction of 100 g per liter of lactose solution at 30°C, pH 7.0 for 18 h with an intracellular crude β-galactosidase. Glucose and galactose were found to inhibit GalOS formation. The GalOS that were purified by active carbon and celite 545 column chromatography were supplemented in MRS media and a stimulated growth was observed of some intestinal bacteria. In particular the growth rate ofBifidobacterium infantis in the GalOS containing MRS broth increased up to 12.5% compared to that of the MRS-glucose broth during a 48 h incubation period.     

Purification of secreted α-amylases by immunoaffinity chromatography with cross-reactive antibody

Two isozymes of rice -amylases expressed and secreted by recombinant yeast were purified by immunoaffinity chromatography by using cross-reactive antibody. Antibodies raised against partially purified barley -amylase adsorbed rice -amylases in fermentation broth by a cross-reaction. By use of these antibodies as ligands, rice -amylases were concentrated and purified to a high degree in one-step immunoaffinity chromatography. Because of the differences in the contaminating impurities between the barley -amylase (antigen) from barley malt and rice -amylases (target protein) secreted from yeast, the high purity of eluted -amylases was attained without the use of highly purified antigen for immunization. Utilization of cross-reactive antibodies in immunoaffinity chromatography is useful for the purification of recombinant proteins in the absence of a sufficient amount and high enough purity of the target proteins to be purified.     

Mechanism of action of α-galactosidase

The effect ofpH on K_m and V_max values of coconut α-galactosidase indicates the involvement of two ionizing groups with pK_a values of 3.5 and 6.5 in catalysis. Chemical modification has indicated the presence of two carboxyl groups, a tryptophan and a tyrosine, at or near the active site of α-galactosidase. Based on these facts a new mechanism of action for α-galactosidase is proposed in which the ionizing group with a pK_a of 3.5 is a carboxyl group involved in stabilizing a carbonium ion intermediate and the ionizing group with a pK_a of 6.5 is a carboxyl group perturbed due to the presence of a hydrophobic residues in its vicinity which donates a H⁺ ion in catalysis.     

Specificity of sweet-almond α-galactosidase

1. The specificity of purified sweet-almond alpha-galactosidase has been investigated with 17 substrates. 2. Some of them exhibited inhibition at high substrate concentrations but others did not. Both substrate types were bound and hydrolysed at the same site on the enzyme. 3. The enzyme is specific for alpha-d-galactosides and beta-l-arabinosides. It did not hydrolyse beta-d-galactosides or alpha-d-glucosides. 4. Among galactosides the order of decreasing rates of enzymic hydrolysis was: aryl alpha-galactosides; sugars; alkyl alpha-galactosides. 5. All substituents in the aryl moiety of aryl alpha-galactosides enhanced V(max.), the electron-releasing (-sigma) groups being more effective than the electron-withdrawing (+sigma) groups. The substituent groups did not alter K(m) appreciably. 6. Implications of these results are discussed from a mechanistic viewpoint.