Oleate Hydratase in Lactobacillus delbrueckii subsp. bulgaricus LBP UFSC 2230 Catalyzes the Reversible Conversion between Linoleic Acid and Ricinoleic Acid.

Conjugated linoleic acid (CLA) has been the subject of numerous studies in recent decades because of its associated health benefits. CLA is an intermediate product of the biohydrogenation pathway of linoleic acid (LA) in bacteria. Several bacterial species capable of efficiently converting LA into CLA have been widely reported in the literature, among them Lactobacillus delbrueckii subsp. bulgaricus LBP UFSC 2230. Over the last few years, a multicomponent enzymatic system consisting of three enzymes involved in the biohydrogenation process of LA has been proposed. Sequencing the genome of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 revealed only one gene capable of encoding an oleate hydratase (OleH), unlike the presence of multiple genes typically found in similar strains. This study investigated the biological effect of the OleH enzyme of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 on the hydration of LA and dehydration of ricinoleic acid (RA) and its possible role in the production of CLA. The OleH was cloned, expressed, purified, and characterized. Fatty acid measurements were made by an internal standard method using a gas chromatography-coupled flame ionization detector (GC-FID) system. It was found that the enzyme is a hydratase/dehydratase, leading to a reversible transformation between LA and RA. In addition, the results showed that L. delbrueckii subsp. bulgaricus LBP UFSC 2230 OleH protein plays a role in stress tolerance in Escherichia coli. In conclusion, the OleH of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 catalyzes the initial stage of saturation metabolism of LA, although it has not converted the substrates directly into CLA. IMPORTANCE This study provides insight into the enzymatic mechanism of CLA synthesis in L. delbrueckii subsp. bulgaricus and broadens our understanding of the bioconversion of LA and RA by OleH. The impact of OleH on the production of the c9, t11 CLA isomer and stress tolerance by E. coli has been assisted. The results provide an understanding of the factors which influence OleH activity. L. delbrueckii subsp. bulgaricus LBP UFSC 2230 OleH presented two putative fatty acid-binding sites. Recombinant OleH catalyzed both LA hydration and RA dehydration. OleH was shown to play a role in bacterial growth performance in the presence of LA.

Enzymatic characterization of D-lactate dehydrogenase and application in alanine aminotransferase activity assay kit.

D-lactate dehydrogenase (D-LDH) is widely used for the clinical detection of alanine aminotransferase (ALT) activity. It is a key enzyme in ALT detection kits, and its enzymatic properties directly determine sensitivity and accuracy of such kits. In this study, D-lactate dehydrogenase (WP_011543503, ldLDH) coding sequence derived from Lactobacillus delbrueckii was obtained from the NCBI database by gene mining. LdLDH was expressed and purified in Escherichia coli, and its enzyme activity, kinetic parameters, optimum temperature, and pH were characterized. Furthermore, stabilizers, including sugars, polyols, amino acids, certain salts, proteins, and polymers, were screened to improve stability of ldLDH during freeze-drying and storage. Finally, a kit based on ldLDH was tested to determine whether the enzyme had potential clinical applications. The results showed that ldLDH had a specific activity of 1,864 U/mg, Km value of 1.34 mM, optimal reaction temperature of 55°C, and an optimal pH between 7.0 and 7.5. When sucrose or asparagine was used as a stabilizer, freeze-dried ldLDH remained stable at 37°C for > 2 months without significant loss of enzymatic activity. These results indicated that ldLDH possesses high activity and stability. Test results using the ALT assay kit prepared with ldLDH were consistent with those of commercial kits, with a relative deviation <5%. These results indicated that ldLDH met the primary requirements for ALT assays, laying a foundation for the development of new ALT kits with potential clinical applications.

Imbalance between peptidoglycan synthases and hydrolases regulated lysis of Lactobacillus bulgaricus in batch culture.

Lactobacillus bulgaricus is an important starter culture in the dairy industry, cell lysis is negative to the high density of this strain. This work describes the response of peptidoglycan synthases and hydrolases in Lactobacillus bulgaricus sp1.1 when pH decreasing in batch culture. First, the cell lysis was investigated by measuring the cytosolic lactate dehydrogenase released to the fermentation broth, a continuous increase in extracellular lactate dehydrogenase was observed after the lag phase in batch culture. Then, the peptidoglycan hydrolases profile analyzed using the zymogram method showed that eight proteins have the ability of peptidoglycan hydrolysis, three of the eight proteins were considered to contribute lysis of L. bulgaricus sp1.1 according to the changes and extents of peptidoglycan hydrolysis. In silico analysis showed that three putative peptidoglycan hydrolases, including N-acetylmuramyl-L-Ala amidase (protein ID: ALT46642.1), amidase (protein ID: ALT46641.1), and N-acetylmuramidase (protein ID: WP_013439201.1) were compatible with these proteins. Finally, the transcription of the three putative peptidoglycan hydrolases was upregulated in batch culture, in contrast, the expression of four peptidoglycan synthases was downregulated. These observations suggested the imbalance between peptidoglycan synthases and hydrolases involved in the lysis of Lactobacillus bulgaricus sp1.1.

Green Production of Cladribine by Using Immobilized 2'-Deoxyribosyltransferase from Lactobacillus delbrueckii Stabilized through a Double Covalent/Entrapment Technology.

Nowadays, enzyme-mediated processes offer an eco-friendly and efficient alternative to the traditional multistep and environmentally harmful chemical processes. Herein we report the enzymatic synthesis of cladribine by a novel 2'-deoxyribosyltransferase (NDT)-based combined biocatalyst. To this end, Lactobacillus delbrueckii NDT (LdNDT) was successfully immobilized through a two-step immobilization methodology, including a covalent immobilization onto glutaraldehyde-activated biomimetic silica nanoparticles followed by biocatalyst entrapment in calcium alginate. The resulting immobilized derivative, SiGPEI 25000-LdNDT-Alg, displayed 98% retained activity and was shown to be active and stable in a broad range of pH (5-9) and temperature (30-60 °C), but also displayed an extremely high reusability (up to 2100 reuses without negligible loss of activity) in the enzymatic production of cladribine. Finally, as a proof of concept, SiGPEI 25000-LdNDT-Alg was successfully employed in the green production of cladribine at mg scale.

Heterologous Expression and Application of Multicopper Oxidases from Enterococcus spp. for Degradation of Biogenic Amines.

BACKGROUND: Biogenic amines are harmful to human health at a certain extent. As a kind of biogenic amine oxidase, multicopper oxidase can be used to degrade them. Currently, the literature about enzyme from Enterococcus spp. are limited, and recombinant multicopper oxidase might be an effective way to degrade biogenic amines. OBJECTIVE: (i) Select and identify strains that can degrade biogenic amines, (ii) overexpress enzyme from Enterococcus spp., (iii) measure gene expression and probe amine-degradation differences among strains (native, E. coli DH5α, and L. delbruckii), and (iv) examine the biochemical properties of recombinant multicopper oxidase, (v) apply the recombinant enzyme into smoked horsemeat sausage. METHODS: Reverse transcription PCR and high-performance liquid chromatography were performed to examine gene expression and amine degradation rate. RESULTS: The results demonstrated that target enzymes were successfully overexpressed, accompanied by increased amine-degrading activity (P <0.05). Gene from E. faecalis M5B was expressed in L. delbrueckii resulted in degradation rates for phenylethylamine, putrescine, histamine and tyramine of 54%, 52%, 70% and 40%, respectively, significantly higher than achieved by other recombinant strains. CONCLUSION: In this work, gene expression levels were higher in recombinant M5B than recombinant M2B, regardless of host. E. coli is more stable to express multicopper oxidase. Besides, the amine-degrading ability was markedly increased in the two recombinant strains. After prolonged incubation, the recombinant enzyme could degrade three amines, and it displayed high alkali resistance and thermostability.

Hydrogen peroxide-producing pyruvate oxidase from Lactobacillus delbrueckii is catalytically activated by phosphotidylethanolamine.

BACKGROUND: Pyruvate oxidase (Pox) is an important enzyme in bacterial metabolism for increasing ATP production and providing a fitness advantage via hydrogen peroxide production. However, few Pox enzymes have been characterized from bacterial species. The tetrameric non-hydrogen-peroxide producing Pox from E. coli is activated by phospholipids, which is important for its function in vivo. RESULTS: We characterized the hydrogenperoxide-producing Pox from L. delbrueckii strain STYM1 and showed it is specifically activated by phosphotidylethanolamine (16:0-18:1), but not by phosphotidylcholine or phosphotidylglycerol. This activation is a mixture of K- and V-type activation as both km and enzyme turnover are altered. Furthermore, we demonstrated that the L. delbrueckii Pox forms pentamers and either decamers or dimers of pentamers in solution, which is different from other characterized Pox enzymes. Lastly, we generated a C-terminal truncation mutant that was only weakly activated by phosphotidylethanolamine, which suggests the C-terminus is important for lipid activation. CONCLUSIONS: To our knowledge this is the first known hydrogenperoxide-producing Pox enzyme that is activated by phospholipids. Our results suggest that there are substantial differences between Pox enzymes from different bacterial species, which could be important for their role in biological systems as well as in the development of Pox-based biosensors.

A comparative study of extraction techniques for maximum recovery of ß-galactosidase from the yogurt bacterium Lactobacillus delbrueckii ssp. bulgaricus.

The study reported in this research communication evaluates the chemical (solvents) and mechanical (sonication, bead-beater) extraction methods to determine the maximum recovery of ß-galactosidase from L. bulgaricus spp. Among all extraction techniques, sonication-assisted extraction yielded the highest amounts of enzyme activity (between 1892-2156 Miller Units) in cell-free extract (supernatant). Interestingly, solvent extracted enzyme activities were found to be very low (between 83-153 Miller Units) in supernatant. SDS-polyacrylamide gel electrophoresis and the total protein determination showed that mechanical methods can completely lyse the cells. Our results thus demonstrated that the mechanical extraction method of sonication is the best one for recovering the maximum amount of lactase from L. bulgaricus strains.

Proteolytic activity of Lactobacillus strains isolated from Mongolian traditional dairy products: A multiparametric analysis.

The aim of our study was to characterize the proteolytic activity of 170 Lactobacillus strains isolated from traditional Mongolian dairy products (yogurt and fermented milk), and to investigate their capacity to generate bioactive peptides during milk fermentation. All isolates were screened for proteolytic activity using skim milk agar-well diffusion test. Fifteen strains (9 Lactobacillus helveticus and 6 Lactobacillus delbrueckii subsp. bulgaricus) were then selected and further evaluated using an original strategy based on multiparametric analysis, taking into account growth rate, acidification capacity, proteolytic activity, cell envelope associated peptidase (CEP) profile and LC-MS/MS analysis of peptides. All parameters were analyzed using principal component analysis (PCA). Results showed that strain growth and acidification correlate with peptide production and that Mongolian L. helveticus strains differ from Western strains in terms of CEP distribution. The PCA revealed that CEP profiles are major determinants of ß-casein hydrolysis patterns. Strains with distinctive proteolytic activities were identified.

Enzymatic Synthesis of Nucleoside Triphosphates and Deoxynucleoside Triphosphates by Surface-Displayed Kinases.

Nucleoside triphosphates and deoxynucleoside triphosphates are important biochemical molecules. In this study, recombinant Escherichia coli that could display nucleotide kinases (INP-N-NMKases) and acetate kinase (INP-N-ACKase) on the cell surface were constructed by fusing an enzyme (NMKase/ACKase) to the N-terminus of ice nucleation protein (INP-N). By using intact recombinant bacteria cells as a catalyst coupled with an ACKase-catalyzed adenosine-5'-triphosphate (ATP) regeneration system, nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) could be synthesized efficiently. In a reaction system with 5 mmol/l substrate, the conversion rates of cytidine-5'-triphosphate (CTP) and deoxycytidine-5'-triphosphate (dCTP) were 96% and 93%, respectively, the conversion rate of ATP and deoxyadenosine-5'-triphosphate (dATP) was 96%, the conversion rate of deoxythymidine-5'-triphosphate (dTTP) was 91%, and the conversion rate of uridine-5'-triphosphate (UTP) was 80%. There was no obvious degradation. At 37 °C, the stability of the surface-displayed fusion protein, especially in the presence of the substrate, was significantly improved. Each whole cell could be reused more than 8 times.

Interspecies Inhibition of Porphyromonas gingivalis by Yogurt-Derived Lactobacillus delbrueckii Requires Active Pyruvate Oxidase.

Despite a growing interest in using probiotic microorganisms to prevent disease, the mechanisms by which probiotics exert their action require further investigation. Porphyromonas gingivalis is an important pathogen implicated in the development of periodontitis. We isolated several strains of Lactobacillus delbrueckii from dairy products and examined their ability to inhibit P. gingivalis growth in vitro We observed strain-specific inhibition of P. gingivalis growth in vitro Whole-genome sequencing of inhibitory and noninhibitory strains of L. delbrueckii revealed significant genetic differences supporting the strain specificity of the interaction. Extracts of the L. delbrueckii STYM1 inhibitory strain contain inhibitory activity that is abolished by treatment with heat, proteinase K, catalase, and sodium sulfite. We purified the inhibitory protein(s) from L. delbrueckii STYM1 extracts using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration chromatography. Pyruvate oxidase was highly enriched in the purified samples. Lastly, we showed that purified, catalytically active, recombinant pyruvate oxidase is sufficient to inhibit P. gingivalis growth in vitro without the addition of cofactors. Further, using a saturated transposon library, we isolated transposon mutants of P. gingivalis in the feoB2 (PG_1294) gene that are resistant to killing by inhibitory L. delbrueckii, consistent with a mechanism of hydrogen peroxide production by pyruvate oxidase. Our results support the current understanding of the importance of strain selection, not simply species selection, in microbial interactions. Specific L. delbrueckii strains or their products may be effective in the treatment and prevention of P. gingivalis-associated periodontal disease.IMPORTANCEP. gingivalis is implicated in the onset and progression of periodontal disease and associated with some systemic diseases. Probiotic bacteria represent an attractive preventative therapy for periodontal disease. However, the efficacy of probiotic bacteria can be variable between studies. Our data support the known importance of selecting particular strains of bacteria for probiotic use, not simply a single species. Specifically, in the context of probiotic intervention of periodontitis, our data suggest that high-level expression of pyruvate oxidase with hydrogen peroxide production in L. delbrueckii could be an important characteristic for the design of a probiotic supplement or a microbial therapeutic.

Soluble Lactobacillus delbrueckii subsp. bulgaricus 92059 PrtB proteinase derivatives for production of bioactive peptide hydrolysates from casein.

The proteinase-encoding prtB gene of Lactobacillus (Lb.) delbrueckii (d.) subsp. bulgaricus 92059 was cloned and sequenced. Two soluble, secreted, C-terminally His-tagged derivatives were constructed and expressed in Lactococcus lactis by means of the NICE® Expression System. In both obtained derivatives PrtBb and PrtB2, the C-terminal, cell wall-binding domain was deleted. In addition, in derivative PrtB2, the C-terminal part of the B domain was deleted and the signal sequence was replaced by a lactococcal export signal. The affinity-purified derivatives were both proteolytically active. Peptide hydrolysates produced from casein with each of the derivatives showed identical peptide composition, as determined by liquid chromatography-mass spectrometry. Comparison of the peptides generated to those generated with living Lb. d. subsp. bulgaricus 92059 cells (Kliche et al. Appl Microbiol Biotechnol 101:7621-7633, 2017) showed that ß-casein was the casein fraction most susceptible to hydrolysis and that some significant differences were observed between the products obtained by either the derivatives or living Lb. d. subsp. bulgaricus 92059 cells. When tested for biological activity, the hydrolysate obtained with PrtBb showed 50% inhibition of angiotensin-converting enzyme at a concentration of 0.5 mg/ml and immunomodulation/anti-inflammation in an in vitro assay of TNF-α induced NFκB activation at concentrations of 5 and 2.5 mg/ml, respectively. The enzymatically obtained hydrolysate did not show any pro-inflammatory or cytotoxic activity.

Biosynthesis of (deoxy)guanosine-5'-triphosphate by GMP kinase and acetate kinase fixed on the surface of E. coli.

(Deoxy)guanosine-5'-triphosphate (5'-(d)GTP), the precursor for synthesizing DNA or RNA in vivo, is an important raw material for various modern biotechnologies based on PCR. In this study, we investigated the application of whole-cell catalysts constructed by bacterial cell surface display in biosynthetic reactions of 5'-(d)GTP from (deoxy)guanosine-5'-monophosphate (5'-(d)GMP). By N-terminal or N- and C-terminal fusion of the ice nucleation protein, we successfully displayed the GMP kinase of Lactobacillus bulgaricus and the acetate kinase of E. coli on the surface of E. coli cells. A large amount of soluble target protein was obtained upon induction with 0.2 mM IPTG at 25 °C for 30 h. The conversion of dGMP was up to 91% when catalysed by the surface-displayed enzymes at 37 °C for 4 h. Up to 95% of the GMP was converted after 3 h of reaction. The stability of the whole-cell catalyst at 37 °C was very good. The enzyme activity was maintained above 50% after 9 rounds of recovery. Our research showed that only one-twentieth of the initial substrate concentration of added ATP was sufficient to meet the reaction requirements.

The critical role of urease in yogurt fermentation with various combinations of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus.

The protocooperation between Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus relies on metabolite exchanges that accelerate acidification during yogurt fermentation. Conflicting results have been obtained in terms of the effect of the Strep. thermophilus urease and the NH3 and CO2 that it generates on the rate of acidification in yogurt fermentation. It is difficult to perform a systematic study of the effects of urease on protocooperation because it is necessary to distinguish among the direct, indirect, and strain-specific effects resulting from the combination of the strains of both species. To evaluate the direct effects of urease on protocooperation, we generated 3 urease-deficient mutants (ΔureC) of fast- and slow-acidifying Strep. thermophilus strains and observed the effects of NH3 or CO2 supplementation on acidification by the ΔureC strains. Further, we examined 5 combinations of 3 urease-deficient ΔureC strains with 2 CO2-responsive or CO2-unresponsive strains of L. bulgaricus. Urease deficiency induced a shortage of ammonia nitrogen and CO2 for the fast- and slow-acidifying Strep. thermophilus and for the CO2-responsive L. bulgaricus, respectively. Notably, the shortage of ammonia nitrogen had more severe effects than that of CO2 on yogurt fermentation, even if coculture with L. bulgaricus masked the effect of urease deficiency. Our work established (1) that urease deficiency inhibits the fermentative acceleration of protocooperation regardless of the Strep. thermophilus and L. bulgaricus strain combinations, and (2) that urease is an essential factor for effective yogurt acidification.

Enhanced Inulin Saccharification by Self-Produced Inulinase from a Newly Isolated Penicillium sp. and its Application in D-Lactic Acid Production.

In order to find an alternative for commercial inulinase, a strain XL01 identified as Penicillium sp. was screened for inulinase production. The broth after cultivated was centrifuged, filtered, and used as crude enzyme for the following saccharification. At pH 5.0 and 50 °C, the crude enzyme released 84.9 g/L fructose and 20.7 g/L glucose from 120 g/L inulin in 72 h. In addition, simultaneous saccharification and fermentation of chicory flour for D-lactic acid production was carried out using the self-produced crude inulinase and Lactobacillus bulgaricus CGMCC 1.6970. A high D-lactic acid titer and productivity of 122.0 g/L and 1.69 g/(L h) was achieved from 120 g/L chicory flour in 72 h. The simplicity for inulinase production and the high efficiency for D-lactic acid fermentation provide a perspective and profitable industrial biotechnology for utilization of the inulin-rich biomass.

From gene to structure: Lactobacillus bulgaricus D-lactate dehydrogenase from yogurt as an integrated curriculum model for undergraduate molecular biology and biochemistry laboratory courses.

We have developed an integrated, project-oriented curriculum for undergraduate molecular biology and biochemistry laboratory courses spanning two semesters that is organized around the ldhA gene from the yogurt-fermenting bacterium Lactobacillus bulgaricus, which encodes the enzyme d-lactate dehydrogenase. The molecular biology module, which consists of nine experiments carried out over eleven sessions, begins with the isolation of genomic DNA from L. bulgaricus in yogurt and guides students through the process of cloning the ldhA gene into a prokaryotic expression vector, followed by mRNA isolation and characterization of recombinant gene expression levels using RT-PCR. The biochemistry module, which consists of nine experiments carried out over eight sessions, begins with overexpression of the cloned ldhA gene and guides students through the process of affinity purification, biochemical characterization of the purified LdhA protein, and analysis of enzyme kinetics using various substrates and an inhibitor, concluding with a guided inquiry investigation of structure-function relationships in the three-dimensional structure of LdhA using molecular visualization software. Students conclude by writing a paper describing their work on the project, formatted as a manuscript to be submitted for publication in a scientific journal. Overall, this curriculum, with its emphasis on experiential learning, provides hands-on training with a variety of common laboratory techniques in molecular biology and biochemistry and builds experience with the process of scientific reasoning, along with reinforcement of essential transferrable skills such as critical thinking, information literacy, and written communication, all within the framework of an extended project having the look and feel of a research experience. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(3):270-278, 2018.

Influence of the metal ion on the enzyme activity and kinetics of PepA from Lactobacillus delbrueckii.

The aminopeptidase A (PepA; EC 3.4.11.7) belongs to the group of metallopeptidases with two bound metal ions per subunit (M1M2(PepA)) and is specific for the cleavage of N-terminal glutamic (Glu) and aspartic acid (Asp) and, in low amounts, serine (Ser) residues. Our group recently characterized the first PepA from a Lactobacillus strain. However, the characterization was performed using synthetic para-nitroaniline substrates and not original peptide substrates, as was done in the current study. Prior to the characterization using original peptide substrates, the PepA purified was converted to its inactive apo-form and eight different metal ions were tested to restore its activity. It was found that five of the metal ions were able to reactivate apo-PepA: Co2+, Cu2+, Mn2+, Ni2+ and Zn2+. Interestingly, depending on the metal ion used for reactivation, the activity and the pH and temperature profile differed. Exemplarily, MnMn(PepA), NiNi(PepA) and ZnZn(PepA) had an activity optimum using MES buffer (50mM, pH 6.0) and 60°C, whereas the activity optimum changed to Na/K-phosphate-buffer (50mM, pH 7.0) and 55°C for CuCu(PepA). However, more important than the changes in optimum pH and temperature, the kinetic properties of PepA were affected by the metal ion used. While all PepA variants could release N-terminal Glu or Asp, only CoCo(PepA), NiNi(PepA) and CuCu(PepA) could release Ser from the particular peptide substrate. In addition, it was found that the enzyme efficiency (Vmax/KM) and catalytic mechanism (positive cooperative binding (Hill coefficent; n), substrate inhibition (KIS)) were influenced by the metal ion. Exemplarily, a high cooperativity (n>2),KIS value >20mM and preference for N-terminal Glu were detected for CuCu(PepA). In summary, the results suggested that an exchange of the metal ion can be used for tailoring the properties of PepA for specific hydrolysis requirements.

Screening for proteolytically active lactic acid bacteria and bioactivity of peptide hydrolysates obtained with selected strains.

In a screening for proteolytically active lactic acid bacteria, three strains, Lactobacillus delbrueckii ssp. lactis 92202, Lactobacillus helveticus 92201, and Lactobacillus delbrueckii ssp. bulgaricus 92059, showed the highest activities following growth in milk. All three strains degraded α- and ß-casein, but did not hydrolyse κ-casein. HPLC analysis of skim milk fermentation revealed increasing amounts of peptides after 5 and 10 h with Lb. d. ssp. bulgaricus 92059. Hydrolysates obtained with Lb. d. ssp. lactis 92202 and Lb. d. ssp. bulgaricus 92059 revealed the highest angiotensin-converting enzyme-inhibitory effect. The effect was dose dependent. Almost no effect (<10%) was seen for Lb. helveticus 92201. For Lb. d. ssp. bulgaricus 92059, maximal inhibition of approx. 65% was reached after 25 h of fermentation. In an in vitro assay measuring potential immunomodulation, hydrolysates of the three strains yielded anti-inflammatory activities in the presence of TNF-α. However, the effects were more pronounced at lower hydrolysate concentrations. In the absence of TNF-α, slight pro-inflammatory effects were observed. The hydrolysate of Lb. d. ssp. bulgaricus 92059, when purified by means of solid-phase extraction, exhibited pro-inflammatory activity. Sour whey containing Lb. d. ssp. bulgaricus 92059 cells showed pro-inflammatory activity while cell-free sour whey was clearly anti-inflammatory. In the purified hydrolysate, 20 different α- and ß-casein (CN)-derived peptides could be identified by LC-MS. Most peptides originated from the central and C-terminal regions of ß-casein. Peptide length was between 9 (ß-CN(f 59-67)) and 22 amino acids (ß-CN(f 117-138)).

Characterization of C-S lyase from Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 and its potential role in food flavour applications.

Volatile thiols have substantial impact on the aroma of many beverages and foods. Thus, the control of their formation, which has been linked to C-S lyase enzymatic activities, is of great significance in industrial applications involving food flavours. Herein, we have carried out a spectroscopic and functional characterization of a putative pyridoxal 5'-phosphate (PLP)-dependent C-S lyase from the lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365 (LDB C-S lyase). Recombinant LDB C-S lyase exists as a tetramer in solution and shows spectral properties of enzymes containing PLP as cofactor. The enzyme has a broad substrate specificity toward sulphur-containing amino acids with aminoethyl-L-cysteine and L-cystine being the most effective substrates over L-cysteine and L-cystathionine. Notably, the protein also reveals cysteine-S-conjugate ß-lyase activity in vitro, and is able to cleave a cysteinylated substrate precursor into the corresponding flavour-contributing thiol, with a catalytic efficiency higher than L-cystathionine. Contrary to similar enzymes of other lactic acid bacteria however, LDB C-S lyase is not capable of α,γ-elimination activity towards L-methionine to produce methanethiol, which is a significant compound in flavour development. Based on our results, future developments can be expected regarding the flavour-forming potential of Lactobacillus C-S lyase and its use in enhancing food flavours.

Simple purification method for a recombinantly expressed native His-tag-free aminopeptidase A from Lactobacillus delbrueckii.

The aminopeptidase A (PepA; EC 3.4.11.7) is an intracellular exopeptidase present in lactic acid bacteria. The PepA cleaves glutamyl/aspartyl residues from the N-terminal end of peptides and can, therefore, be applied for the production of protein hydrolysates with an increased amount of these amino acids, which results in a savory taste (umami). The first PepA from a lactobacilli strain was recombinantly expressed in Escherichia coli in a recently published study and harbored a C-terminal His6-tag for easier purification. Due to the fact that a His-tag might influence the properties of an enzyme, a simple purification method for the non-His-tagged PepA was required. Surprisingly, the PepA precipitated at a very low ammonium sulfate concentration of 5%. Unusual for a precipitating step, the purity of PepA was over 95% and the obtained activity yield was 110%. The high purity allows biochemical characterization and kinetic investigation. As a result, the optimum pH (6.0-6.5) and temperature (60-65 °C) were comparable to the His6-tag harboring PepA; the KM value was at 0.79 mM slightly lower compared to 1.21 mM, respectively. Since PepA is a homo dodecamer, it has a high molecular mass of approximately 480 kDa. Therefore, a subsequent preparative size-exclusion chromatography (SEC) step seemed promising. The PepA after SEC was purified to homogeneity. In summary, the simple two-step purification method presented can be applied to purify high amounts of PepA that will allow the performance of experiments in the future to crystalize PepA for the first time.

Engineering a d-lactate dehydrogenase that can super-efficiently utilize NADPH and NADH as cofactors.

Engineering the cofactor specificity of a natural enzyme often results in a significant decrease in its activity on original cofactor. Here we report that a NADH-dependent dehydrogenase (d-LDH) from Lactobacillus delbrueckii 11842 can be rationally engineered to efficiently use both NADH and NADPH as cofactors. Point mutations on three amino acids (D176S, I177R, F178T) predicted by computational analysis resulted in a modified enzyme designated as d-LDH*. The Kcat/Km of the purified d-LDH* on NADPH increased approximately 184-fold while the Kcat/Km on NADH also significantly increased, showing for the first time that a rationally engineered d-LDH could exhibit comparable activity on both NADPH and NADH. Further kinetic analysis revealed that the enhanced affinity with NADH or NADPH and the significant increased Kcat of d-LDH* resulted in the significant increase of d-LDH* activity on both NADPH and NADH. This study thus demonstrated that the cofactor specificity of dehydrogenase can be broadened by using targeted engineering approach, and the engineered enzyme can efficiently function in NADH-rich, or NADPH-rich, or NADH and NADPH-rich environment.