Expression and secretion of wheat α-amylase in Kluyveromyces lactis

The plasmid pCR1 has been constructed to express a wheat -amylase enzyme in Kluyveromyces lactis strains. The contruct is based on the vector pCXJ-kan1, which has been derived from pDK1, a native plasmid of K. lactis var. drosophilarum containing the essential regions for plasmid replication and stability. Contruct pCR1 produces an -amylase by DNA isolated from a wheat cDNA clone and is controlled by a Saccharomyces cerevisia PGK promoter. Correspondence to: C. Russell     

Expression and characterization of Kluyveromyces lactis β-galactosidase in Escherichia coli

Kluyveromyces lactis -galactosidase gene, LAC4, was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the -galactosidase was the major fraction but had a lower activity than those of the multimeric forms. The purified enzyme required Mn²⁺ for activity and was inactivated irreversibly by imidazole above 50 mM. The activity was optimal at 37 and 40 °C for o-nitrophenyl--d-galactopyranoside (oNPG) and lactose, respectively. The optimum pH value is 7. The K _m and V _max values of the purified enzyme for oNPG were 1.5 mM and 560 mol min^–1 mg^–1, and for lactose 20 mM and 570 mol min^–1 mg^–1, respectively.     

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.     

Growth and β-galactosidase synthesis in aerobic chemostat cultures of Kluyveromyces lactis

Growth and β-galactosidase (β-gal) expression were characterized in the yeast Kluyveromyces lactis strain NRRL Y-1118 growing in aerobic chemostat cultures under carbon, nitrogen or phosphate limitation. In lactose or galactose-limited cultures, β-gal accumulated in amounts equivalent to 10–12% of the total cell protein. The induced β-gal expression was repressed when cells were grown under N- or P-limitation. In lactose medium, enzyme levels were 4–8 times lower than those expressed in C-limited cultures. A similar response was observed when galactose was the carbon source. These results suggest that a galactose-dependent signal (in addition to glucose) may have limited induction when cells were grown in carbon-sufficient cultures. Constitutive β-gal expression was highest in lactate-limited and lowest in glucose-limited media and was also repressed in glucose-sufficient cultures. Other K. lactis strains (NRRL Y-1140 and CBS 2360) also showed glucose repression (although with different sensitivity) under non-inducing conditions. We infer that these strains share a common mechanism of glucose repression independent of the induction pathway. The kinetics of β-gal induction observed in C-limited cultures confirms that β-gal induction is a short-term enzyme adaptation process. Applying a lactose pulse to a lactose-limited chemostat culture resulted in ‘substrate-accelerated death’. Immediately after the pulse, growth was arrested and β-gal was progressively inactivated. Yeast metabolism in C-limited cultures was typically oxidative with the substrate being metabolized solely to biomass and CO₂. Cells grown under P- or N-limitation, either with glucose or lactose, exhibited higher rates of sugar consumption than C-limited cells, accumulated intracellular reserve carbohydrates and secreted metabolic products derived from the glycolytic pathway, mainly glycerol and ethanol. Received 16 October 1997/ Accepted in revised form 17 April 1998     

Induction of β galactosidase in the yeast Kluyveromyces lactis

Summary The inductive effect of lactose, -methyl-thio-D-galactopyranoside, (TMG) and glucose on galactosidase synthesis in Kluyveromyces lactis has been studied. Whereas TMG gave a five fold stimulation of the rate of -galactosidase synthesis, lactose only gave a small stimulation. Glucose caused represssion at levels above 10^-3M but stimulated -galactosidase synthesis when added at lower concentrations.     

State of fungal lipases of Rhizopus microsporus,Penicillium sp. and Oospora lactis in border layers water—solid phase and factors affecting catalytic properties of Enzymes

Applied Biochemistry and Microbiology - We demonstrated that a change in the catalytic activity of fungal lipases synthesized by Rhizopus-microsporus, Penicillium sp. and Oospora lactis and their...     

Structural basis of specificity in tetrameric Kluyveromyces lactis β-galactosidase

β-Galactosidase or lactase is a very important enzyme in the food industry, being that from the yeast Kluyveromyces lactis the most widely used. Here we report its three-dimensional structure both in the free state and complexed with the product galactose. The monomer folds into five domains in a pattern conserved with the prokaryote enzymes of the GH2 family, although two long insertions in domains 2 and 3 are unique and related to oligomerization and specificity. The tetrameric enzyme is a dimer of dimers, with higher dissociation energy for the dimers than for its assembly. Two active centers are located at the interface within each dimer in a narrow channel. The insertion at domain 3 protrudes into this channel and makes putative links with the aglycone moiety of docked lactose. In spite of common structural features related to function, the determinants of the reaction mechanism proposed for Escherichia coli β-galactosidase are not found in the active site of the K. lactis enzyme. This is the first X-ray crystal structure for a β-galactosidase used in food processing.     

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.     

Decarboxylation of α-Keto Acids by Streptococcus lactis var. maltigenes

Decarboxylation rates for a series of C-3 to C-6 α-keto acids were determined in the presence of resting cells and cell-free extracts of Streptococcus lactis var. maltigenes. The C-5 and C-6 acids branched at the penultimate carbon atom were converted most rapidly to the respective aldehydes in the manner described for α-carboxylases. Pyruvate and α-ketobutyrate did not behave as α-carboxylase substrates, in that O₂ was absorbed when they were reacted with resting cells. The same effect with pyruvate was noted in a nonmalty S. lactis, accounting for CO₂ produced by some “homofermentative” streptococci. Mixed substrate reactions indicated that the same enzyme was responsible for decarboxylation of α-ketoisocaproate and α-ketoisovalerate, but it appeared unlikely that this enzyme was responsible for the decarboxylation of pyruvate. Ultrasonic disruption of cells of the malty culture resulted in an extract inactive for decarboxylation of pyruvate in the absence of ferricyanide. Dialyzed cell-free extracts were inactive against all keto acids and could not be reactivated.     

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)     

Comparison of bovine β-casein hydrolysis by PI and PIII-type proteinases from Lactobacillus lactis subsp. cremoris

Summary The action of the cell-wall-associated proteinases from Lactococcus lactis subsp. cremoris strains H2 and SK112 on bovine -casein was compared. The proteinase from the H2 strain was characterised as a P_I-type proteinase since it did not hydrolyse _s1-casein and the initial trifluoroacetic acid-soluble products of -casein hydrolysis were identical to those previously identified as hydrolysis products of P_I-type lactococcal proteinase action. The time-course of product formation by the proteinase from the H2 strain indicated that the bonds Tyr₁₉₃-Gln₁₉₄ and Gln₁₈₂-Arg₁₈₃ were the first to be hydrolysed. Cleavage of the bonds Gln₁₇₅-Lys₁₇₆, Ser₁₆₈-Lys₁₆₉, Ser₁₆₆-Gln₁₆₇ and Leu₁₆₃-Ser₁₆₄ was also very rapid. Four of the five bonds in -casein most susceptible to hydrolysis by the P_III-type proteinase from strain SK112 were different from those cleaved by the P_I-type proteinase, initial hydrolysis being at the sites Tyr₁₉₃-Gln₁₉₄, Leu₁₉₂-Tyr₁₉₃, Asp₄₃-Glu₄₄, Gln₄₆-Asp₄₇ and Phe₅₂-Ala₅₃. Early hydrolysis at the three sites in the N-terminal region of -casein, leading to cleavage of the N-terminal phosphopeptide and rapid precipitation of the residual fragment, represents a marked contrast to the action of P_I-type proteinases where cleavage at sites in the N-terminal region occurs only very slowly. Offprint requests to: G. G. Pritchard     

A comparative study of the regioselectivity of the β-galactosidases from Kluyveromyces lactis and Bacillus circulans in the enzymatic synthesis of N-Acetyl-lactosamine in aqueous media

The enzymatic synthesis of N-acetyl-lactosamine (LacNAc) was studied in aqueous media with high substrate concentrations using the transgalactosylation of N-acetyl-D-glucosamine (GlcNAc), starting from lactose as a galactosyl donor. The efficiency and regioselectivity of the β-galactosidases from Kluyveromyces lactis (KlβGal) and Bacillus circulans (BcβGal) were compared. The reaction was optimized by varying the experimental conditions (pH, catalytic activity concentration, and mass concentration ratio of the substrates), which enhanced the synthesis yields with both enzymes and especially with BcβGal. BcβGal catalyzed the formation of the maximal LacNAc concentration obtained (101 mM or 39 g L(-1), corresponding to a yield of 11% on the basis of GlcNAc conversion), after 5 h at pH 6.5 and for a substrate mass concentration ratio of 1. This enzyme also gave an optimal synthesis yield of about 17.5%. No change in regioselectivity was observed when using KlβGal, whereas the regioselectivity of BcβGal proved to be subject to variations, the 1-4 and 1-6 linkages being favored under kinetic and thermodynamic control conditions, respectively. Finally, it was demonstrated that the N-acetyl-allolactosamine synthesized during the GlcNAc transgalactosylation catalyzed by BcβGal was a thermodynamic product and did not result from a chemical and/or enzymatic isomerization of LacNAc.     

Effects of Roundup(®) and glyphosate on three food microorganisms: Geotrichum candidum, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus

Use of many pesticide products poses the problem of their effects on environment and health. Amongst them, the effects of glyphosate with its adjuvants and its by-products are regularly discussed. The aim of the present study was to shed light on the real impact on biodiversity and ecosystems of Roundup^?, a major herbicide used worldwide, and the glyphosate it contains, by the study of their effects on growth and viability of microbial models, namely, on three food microorganisms (Geotrichum candidum, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus) widely used as starters in traditional and industrial dairy technologies. The presented results evidence that Roundup^? has an inhibitory effect on microbial growth and a microbicide effect at lower concentrations than those recommended in agriculture. Interestingly, glyphosate at these levels has no significant effect on the three studied microorganisms. Our work is consistent with previous studies which demonstrated that the toxic effect of glyphosate was amplified by its formulation adjuvants on different human cells and other eukaryotic models. Moreover, these results should be considered in the understanding of the loss of microbiodiversity and microbial concentration observed in raw milk for many years.     

Simple one-step purification of nisin Z from unclarified culture broth of Lactococcus lactis subsp. lactis A164 using expanded bed ion exchange chromatography

A simple one-step purification method, using expanded bed, ion-exchange chromatography, for the fractionation of nisin Z produced by Lactococcus lactis subsp. lactis A164 was developed. The highest dynamic binding capacity (0.92) of the adsorbent was obtained at a superficial velocity of 367 cm h(-1), resulting in approx. 2.7-fold bed expansion. The range of pH for the maximum adsorption was 3-4. The isocratic elution with 0.15 M NaCl led to approx. >90% recovery. Single-step purification of nisin Z from unclarified A164 culture broth resulted in 31-fold purification with a 90% yield.     

DNA,RNA and protein synthesis in ØLL55-infected Lactobacillus lactis

Lactobacillus lactis cells were infected with the bacteriophage ØLL55. The changes in DNA, RNA and protein synthesis were studied by following a long-term (over 3 h) incorporation of radioactive precursors into acid-insoluble material. Stimulation of DNA synthesis caused by phage occurred 30–35 min after infection and thymidine incorporation continued for about 70 min ceasing 10–20 min before the cells started to lyse. Cumulative (¹⁴C)-uracil incorporation into RNA continued at the level of uninfected cells for 30–40 min before starting to slow up. Protein synthesis in the infected cells followed that of a control culture for 40–50 min before the further incorporation of (¹⁴C)-leucine began to decrease.The additions of antibiotic inhibitors of RNA and protein synthesis (rifampicin and chloramphenicol, respectively) at various times before or during the prereplicative period showed that rifampicin, added up to 15 min after infection and chloramphenicol, added as late as 20–25 min after infection completely prevented the initiation of phage-genome replication. The later addition of these drugs did not prevent the out-burst of thymidine up-take, but promoted, however, a deduction in the initiations of new replication cycles. The results indicate that certain genes of ØLL55 genome must be expressed at the early stages of infection to confirm a proper onset and continuation of phage DNA replication.Abbreviations Rif rifampicin - CAL chloramphenicol - TCA trichloroacetic acid - cpm counts per minute     

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.