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The GDS(L)-like lipase from the Basidiomycota Pleurotus sapidus (PSA_Lip) was heterologously expressed using Trichoderma reesei with an activity of 350 U L-1. The isoelectric point of 5.0 was determined by isoelectric focusing. The novel PSA_Lip showed only 23.8-25.1%, 25.5%, 26.6% and 28.4% identity to the previously characterized GDSL-like enzymes phospholipase, plant lipase, acetylcholinesterase and acetylxylan esterase, from the carbohydrate esterase family 16, respectively. Therefore, the enzyme was purified from the culture supernatant and the catalytic properties and the substrate specificity of the enzyme were investigated using different assays to reveal its potential function. While no phospholipase, acetylcholinesterase and acetylxylan esterase activities were detected, studies on the hydrolysis of ferulic acid methyl ester (~ 8.3%) and feruloylated carbohydrate 5-O-transferuloyl-arabino-furanose (~ 0.8%) showed low conversions of these substrates. By investigating the hydrolytic activity towards p-nitrophenyl-(pNP)-esters with various chain-lengths, the highest activity was determined for medium chain-length pNP-octanoate at 65 °C and a pH value of 8, while almost no activity was detected for pNP-hexanoate. The enzyme is highly stable when stored at pH 10 and 4 °C for at least 7 days. Moreover, using consensus sequence analysis and homology modeling, we could demonstrate that the PSA_Lip does not contain the usual SGNH residues in the actives site, which are usually present in GDS(L)-like enzymes.
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BACKGROUND: Various neutral and alkaline peptidases are commercially available for use in protein hydrolysis under neutral to alkaline conditions. However, the hydrolysis of proteins under acidic conditions by applying fungal aspartic peptidases (FAPs) has not been investigated in depth so far. The aim of this study, thus, was to purify a FAP from the commercial enzyme preparation, ROHALASE® BXL, determine its biochemical characteristics, and investigate its application for the hydrolysis of food and animal feed proteins under acidic conditions. RESULTS: A Trichoderma reesei derived FAP, with an apparent molecular mass of 45.8 kDa (sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SDS-PAGE) was purified 13.8-fold with a yield of 37% from ROHALASE® BXL. The FAP was identified as an aspartate protease (UniProt ID: G0R8T0) by inhibition and nano-LC-ESI-MS/MS studies. The FAP showed the highest activity at 50°C and pH 4.0. Monovalent cations, organic solvents, and reducing agents were tolerated well by the FAP. The FAP underwent an apparent competitive product inhibition by soy protein hydrolysate and whey protein hydrolysate with apparent Ki -values of 1.75 and 30.2 mg*mL-1 , respectively. The FAP showed promising results in food (soy protein isolate and whey protein isolate) and animal feed protein hydrolyses. For the latter, an increase in the soluble protein content of 109% was noted after 30 min. CONCLUSION: Our results demonstrate the applicability of fungal aspartic endopeptidases in the food and animal feed industry. Efficient protein hydrolysis of industrially relevant substrates such as acidic whey or animal feed proteins could be conducted by applying fungal aspartic peptidases. © 2022 Society of Chemical Industry.
Assuntos
Ácido Aspártico Proteases , Trichoderma , Ração Animal , Animais , Ácido Aspártico Proteases/metabolismo , Hidrólise , Hypocreales , Hidrolisados de Proteína/química , Proteínas de Soja/metabolismo , Espectrometria de Massas em TandemRESUMO
Calcium- and protein-rich fermented milk products, such as concentrated yoghurts and fresh cheeses, may contain undesired bitter peptides, which are generated by the proteolytic cleavage of casein. Up to now, it is not clear whether this process is caused by endogenous milk enzymes, such as plasmin and cathepsin D, or whether proteolytic enzymes from applied starter cultures, such as the lactococcal cell-envelope peptidase PrtP, are involved. A sensory analysis of fresh cheese products made from milk concentrates fermented with prtP-negative and -positive Lactococcus lactis strains revealed bitterness in the products fermented with prtP-positive L. lactis strains. Two prtP-positive strains, LTH 7122 and LTH 7123, were selected to investigate the effect of increased calcium concentrations (additional 5 mM and 50 mM CaCl2) at neutral (pH 6.6) and acidic (pH 5.5) pH-values on the transcription of the prtP gene and its corresponding PrtP peptidase activity in milk citrate broth (MCB). For both strains, it was shown that prtP transcription was upregulated only under slightly elevated calcium conditions (5 mM CaCl2) after 5 h of growth. In concordance with these findings, PrtP peptidase activity also increased. When higher concentrations of calcium were used (50 mM), prtP expression of both strains decreased strongly by more than 50%. Moreover, PrtP peptidase activity of strain LTH 7123 decreased by 15%, but enzymatic activity of strain LTH 7122 increased slightly during growth under elevated calcium concentrations (50 mM CaCl2). Fermentations of reconstituted casein medium with 3.4% (w/v) and 8.5% (w/v) protein and different calcium concentrations using strain LTH 7122 revealed no clear relationship between prtP transcription and calcium or protein concentration. However, an increase in PrtP peptidase activity under elevated protein and calcium conditions was observed. The activity increase was accompanied by increased levels of bitter peptides derived from different casein fractions. These findings could be a possible explanation for the bitterness in fermented milk concentrates that was detected by a trained bitter panel.
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Heat-stable endopeptidases in raw milk, especially the alkaline metallopeptidase AprX secreted by Pseudomonas spp., are a well-known challenge for the dairy industry. They can withstand UHT treatment and may cause quality defects over the shelf life of milk products. Therefore, we established an indirect ELISA for the detection of Pseudomonas AprX in milk. We developed a 2-step sample treatment for milk contaminated with AprX to avoid the interference of milk proteins with the detection system. First, casein micelles were destabilized by the detraction of Ca2+ using trisodium citrate; then, AprX was concentrated 10-fold using hydrophobic interaction chromatography. The recovery of AprX in spiked milk samples after the 2-step treatment was 43 ± 0.1%. Specific antibodies for purified AprX from Pseudomonas lactis were produced to establish the ELISA. Western blot experiments showed that the binding affinity of these antibodies depended on the sequence homology of the AprX from P. lactis and several other Pseudomonas spp. The indirect ELISA, which was completed in 6 to 7 h, had a limit of detection of 21.0 ng mL-1 and a limit of quantification of 25.7 ng mL-1. Milk proteins or milk endogenous peptidases were not detected by the antibodies. The ELISA had high precision, with a CV between 0.2 and 0.8% measured on the same day (intraday) and 5.6 and 6.8% measured on 5 separate days (interday). Milk samples were spiked with different AprX activity levels [7.5-150 nkat Na-caseinate/o-phthalaldehyde (OPA) mL-1] and evaluated by ELISA. The recovery of the ELISA was 92.3 ± 1.6 to 105 ± 4.7%. The lowest AprX activity quantifiable in the spiked milk samples was 500 pkat Na-caseinate/OPA mL-1. The proof of concept to detect heat-stable Pseudomonas AprX in milk by ELISA was established.
Assuntos
Endopeptidases , Temperatura Alta , Animais , Ensaio de Imunoadsorção Enzimática/veterinária , PseudomonasRESUMO
Lactic acid bacteria (LAB) are capable of producing a variety of exopolysaccharide α-glucans, such as dextran, mutan, reuteran, and alternan. Their structural diversity allows LAB-derived α-glucans to hold vast commercial value and application potential in the food, cosmetic, medical, and biotechnology fields, garnering much attention in recent years. Glycoside Hydrolase 70 family (GH70) enzymes are efficient tools for the biosynthesis of α-glucans with various sizes, linkage compositions, and degrees of branching, using renewable and low-cost sucrose and starch as substrates. To date, plenty of various LAB-derived GH70 glucansucrases (especially dextransucrase) have been biochemically characterized to synthesize α-glucans from sucrose with a variety of structural organizations. This review mainly aimed at the biotechnological synthesis of α-glucans using GH70 family enzymes and their diverse (potential) applications. The purification, structural analysis and physicochemical properties of α-glucan polysaccharides were reviewed in detail. Synchronously, some new insights and future perspectives of LAB-derived α-glucans enzymatic synthesis and applications were also discussed. To expand the range of applications, the physicochemical properties and bioactivities of LAB-derived α-glucans, other than dextran, should be further explored. Additionally, screening novel GH70 subfamily starch-acting enzymes is conducive to expanding the repertoire of α-glucans.
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Lactobacillales , Glucanos , Glicosídeo Hidrolases , Polissacarídeos , AmidoRESUMO
Cellobiose 2-epimerase (CE) is a promising industrial enzyme that can catalyze bioconversion of lactose to its high-value derivatives, namely epilactose and lactulose. A need exists in the dairy industry to catalyze lactose bioconversions at low temperatures to avoid microbial growth. We focused on the discovery of cold-active CE in this study. A genome mining method based on computational prediction was used to screen the potential genes encoding cold-active enzymes. The CE-encoding gene from Roseburia intestinalis, with a predicted high structural flexibility, was expressed heterologously in Escherichia coli. The catalytic property of the recombinant enzyme was extensively studied. The optimum temperature and pH of the enzyme were 45°C and 7.0, respectively. The specific activity of this enzyme to catalyze conversion of lactose to epilactose was measured to be 77.3 ± 1.6 U/mg. The kinetic parameters, including turnover number (kcat), Michaelis constant (Km), and catalytic efficiency (kcat/Km) using lactose as a substrate were 117.0 ± 7.7 s-1, 429.9 ± 57.3 mM, and 0.27 mM-1s-1, respectively. In situ production of epilactose was carried out at 8°C: 20.9% of 68.4 g/L lactose was converted into epilactose in 4 h using 0.02 mg/mL (1.5 U/mL, measured at 45°C) of recombinant enzyme. The enzyme discovered by this in silico method is suitable for low-temperature applications.
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Carboidratos Epimerases/análise , Clostridiales/enzimologia , Metodologias Computacionais , Celobiose/metabolismo , Clostridiales/genética , Temperatura Baixa , Mineração de Dados , Dissacarídeos/metabolismo , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Cinética , Lactulose/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Deamidation is a promising tool to improve solubility and other functional properties of food proteins. One possibility of protein deamidation is the use of a protein glutaminase (PG; EC 3.5.1.44), an enzyme that catalyzes the deamidation of internal glutamine residues in proteins to glutamic acid residues. The PG from Chryseobacterium proteolyticum is the only one described in literature to date and is commercially available (Amano Enzyme Inc., Japan; PGA). Based on a similarity search, we discovered a predicted, uncharacterized protein from Bacteroides helcogenes and this protein was verified as a PG. After recombinant production and purification, the novel PG (BH-PG) was biochemically characterized and compared with PGA. Some advantageous characteristics for potential application of BH-PG compared with PGA were the higher temperature stability (residual activity after 24 h of incubation at 50 °C was 87% for BH-PG and 2% for PGA), an optimum pH value at acidic conditions (pH 5.5) and less product inhibition by ammonia that is released during the deamidation of proteins (residual activity after adding 40 mM ammonia was 77% for BH-PG and 27% for PGA). Finally, the applicability of BH-PG and PGA was compared by gluten deamidation experiments. Consequently, the final solubility of the nearly insoluble food protein gluten was 94% after BH-PG treatment, whereas the solubility was around 66% when using PGA.
Assuntos
Bacteroides/enzimologia , Glutaminase/genética , Glutaminase/isolamento & purificação , Bacteroides/genética , Escherichia coli/genética , Glutamina/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/isolamento & purificação , TemperaturaRESUMO
BACKGROUND: One possible way to modify the emulsifying properties of whey proteins is by enzymatic hydrolysis. However, most studies covering the influence of the hydrolysis on whey proteins used a heating step (>65 °C) to inactivate the enzyme. This leads to irreversible product changes, like protein denaturation and increased viscosity. Here, the objective was to investigate the single effect of hydrolysis on the emulsifying properties of whey proteins under conditions without a temperature step for enzyme inactivation. Therefore, two acidic peptidase preparations (Maxipro AFP, Protease AP-30L) differing in their peptidase composition were investigated and applied at 45 °C and pH 2.75. The enzyme inactivation was realized by a simple shift to pH 7.0. RESULTS: After the pH shift, no activity or further hydrolysis was measurable. For the products, no differences (assuming P > 0.05) regarding the emulsifying properties were detected between the two peptidase preparations used. The emulsifying properties of the whey protein isolate hydrolysates produced increased (i.e. half-life >71%) until a degree of hydrolysis of 1.1%. This indicated that the endopeptidase (aspergillopepsin I) present in both preparations was determining the emulsifying properties. As a plus, the presence of exopeptidases in Protease AP-30L compared with Maxipro AFP reduced the bitterness of the hydrolysate (-50%). CONCLUSION: The application of acidic endo- and exopeptidases enables the production of emulsifying whey protein isolate hydrolysates at high protein concentrations (≥10%) without a commonly used heat inactivation step. The presence of exopeptidases in acidic peptidase preparations is favorable, due to the improved taste. © 2019 Society of Chemical Industry.
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Endopeptidases/química , Manipulação de Alimentos/métodos , Peptídeo Hidrolases/química , Hidrolisados de Proteína/química , Proteínas do Soro do Leite/química , Soro do Leite/enzimologia , Emulsões/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Hidrólise , Desnaturação Proteica , Viscosidade , Soro do Leite/químicaRESUMO
In the current study, the extracellular endopeptidases from Pseudomonas lundensis and Pseudomonas proteolytica were investigated. The amino acid sequence identity between both endopeptidases is 68%. Both endopeptidases were purified to homogeneity and partially characterized. They were classified as metallopeptidases with a maximum activity at pH 10.0 ( P. lundensis) or 8.5 ( P. proteolytica) at 35 °C. Both remained active in skim milk with 39.7 ± 2.4% and 24.5 ± 3.3%, respectively, of the initial enzyme activity after UHT processing (138 °C for 20 s), indicating the relevance for milk destabilization. The transition points in buffer were determined at 50 °C ( P. lundensis) and 43 °C ( P. proteolytica) using circular dichroism spectroscopy. The loss of the secondary structure at different temperatures was correlated with residual peptidase activities after heat treatment. The ability to destabilize UHT milk was proven by supplementation of skim milk with endopeptidase and storage for 4 weeks.
Assuntos
Proteínas de Bactérias/química , Endopeptidases/química , Aditivos Alimentares/química , Leite/química , Pseudomonas/enzimologia , Animais , Proteínas de Bactérias/metabolismo , Bovinos , Endopeptidases/metabolismo , Estabilidade Enzimática , Armazenamento de Alimentos , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Pseudomonas/químicaRESUMO
Trehalose is a non-reducing disaccharide with beneficial physiological properties and commercial potential. Trehalose synthase (EC 5.4.99.16) catalyzes the reversible conversion between maltose and trehalose. A recombinant trehalose synthase from Arthrobacter chlorophenolicus SK 33.001 (ACTS) was cloned, expressed, and characterized. The recombinant enzyme encoded a protein of 598 amino acids with a molecular mass of 66 kDa. Gel filtration showed that ACTS is a tetramer in sodium phosphate buffer. The enzyme was metal ion independent and exhibited maximal activity in sodium phosphate buffer (pH 7.5) at 30 °C. The kinetic investigations resulted in a KM value of 120.5 ± 4.5 mM for maltose and a KM value of 343.1 ± 13.8 mM for trehalose. The catalytic efficiency (Vmax/KM) for maltose and trehalose were 0.2 and 0.15 U mg-1 mM-1, respectively. In addition, a cooperative substrate binding was found displayed by the determined Hill coefficients (nH) of 2.8 for maltose and 2.1 for trehalose as a substrate, respectively. The final trehalose yield of various maltose concentrations (50-1000 mM) was constant between 58 and 59%, implying that substrate concentration had no inhibitory influence on ACTS activity.
Assuntos
Arthrobacter/enzimologia , Glucosiltransferases/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Arthrobacter/genética , Clonagem Molecular , Glucose/metabolismo , Glucosiltransferases/química , Glucosiltransferases/genética , Concentração de Íons de Hidrogênio , Cinética , Metais/farmacologia , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato , Temperatura , Trealose/metabolismoRESUMO
Enzymatic hydrolysis with endopeptidases can be used to modify the colloidal properties of food proteins. In this study, sodium caseinate was hydrolyzed with Sternzym BP 25201, containing a thermolysin-like endopeptidase from Geobacillus stearothermophilus as the only peptidase, to a DH of 2.3 ± 1%. The hydrolysate (pre-hydrolysate) obtained was increased in its foam (+35%) and emulsion stability (+200%) compared to untreated sodium caseinate but showed a bitter taste. This hydrolysate was further treated with the exopeptidases PepN, PepX or PepA, acting on the N-terminus of peptides. Depending on the specificity of the exopeptidase used, changes regarding the hydrolysate properties (hydrophobicity, size), colloidal behavior (emulsions, foams) and taste were observed. No changes regarding the bitterness but further improvements regarding the colloidal stability (foam: +69%, emulsion: +29%) were determined after the application of PepA, which is specific for the hydrophilic amino acids Asp, Glu and Ser. By contrast, treatment with the general aminopeptidase PepN resulted in a non-bitter product, with no significant changes regarding the colloidal properties compared to the pre-hydrolysate (p < 0.05). Similar results to those for PepN (reduced bitterness compared to the pre-hydrolysate, enhanced colloidal stability compared to sodium caseinate) were also obtained using commercial Flavourzyme, which was reduced in its endopeptidase activity (exo-flavourzyme). In conclusion, the modifications obtained with the applied exopeptidases offer a potent tool for researchers and the industry to produce non-bitter protein hydrolysates with increased colloidal properties.
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Caseínas/química , Exopeptidases/metabolismo , Hidrolisados de Proteína/química , Paladar , Adulto , Aminopeptidases/metabolismo , Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Pessoa de Meia-Idade , Peso Molecular , Peptídeo Hidrolases/metabolismo , Peptídeos , Adulto JovemRESUMO
A combined approach of sequence-based screening from metagenomic soil DNA and subsequent in silico screening was established to identify novel trehalose synthases (TS, EC 5.4.99.16). Metagenomic DNA was isolated from diverse soil samples and used as template for PCR-based screening targeted against conserved regions of trehalose synthases. This resulted in four metagenomic TS-like fragments with broad sequence diversity (41-67% identity to each other). The encoded open reading frames were used as templates for further in silico screening. Two trehalose synthases were discovered using this novel approach and their enzymatic properties were further investigated. The trehalose synthase from Micrococcus terreus MtTS exhibited a broad pH optimum between 6.5 and 7.5 with highest reaction velocity at 35⯰C and a protruding stability at this temperature (t1/2â¯=â¯50â¯h). Characteristic of enzymes from thermophilic organisms, the trehalose synthase from Thermobaculum terrenum had a distinct temperature optimum at 50⯰C, exhibiting also a prominent half time with t1/2â¯=â¯45â¯h at pH 6.5. Both bioconversions resulted in final trehalose levels of 60%, whereas TtTS produced reduced amounts of the byproduct glucose (10%) compared with MtTS (15%), which is favorable for trehalose production. This combined screening approach intended to circumvent the bottleneck of metagenomic enzyme mining, regarding time and cost of intensive screening procedures for industrial relevant biocatalysts such as trehalose synthases.
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Bactérias/enzimologia , Simulação por Computador , Glucosiltransferases/genética , Glucosiltransferases/isolamento & purificação , Trealose/metabolismo , Sequência de Aminoácidos , Bactérias/classificação , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Glucosiltransferases/metabolismo , Metagenômica , Fases de Leitura Aberta , Alinhamento de Sequência , Análise de Sequência de DNA/métodos , Solo/químicaRESUMO
The incorporation of novel plant-based proteins into foods is often challenging due to an unacceptable bitter sensation. Typically, a combination of electrostatic and hydrophobic forces contributes to the proteins' bitterness. The current study therefore focuses on the development of electrical properties on cationic plant proteins to reduce their overall bitterness in order to improve the perceived sensorial acceptance. As such, we utilized a simple mixing process to induce complex coacervation of oppositely charged biopolymers under acidic conditions. Pea and potato protein stock solutions were mixed with apple pectin (DE 71%) solutions at various biopolymer ratios to modulate the electrical, rheological, and sensorial properties of the complexes. Whey protein hydrolyzate was used as a control sample. Surface charge measurements revealed a transition from positive to negative values as the pectin concentration was increased regardless of the plant protein, whereas stable dispersions without sedimentation were observed above a critical pectin : protein ratio of 1. Low and intermediate biopolymer ratios (<1) promoted aggregation and led to rapid sedimentation. Sensory evaluation showed that bitterness scores depended on protein type and decreased from pea protein > potato protein > whey protein. Moreover, bitter off-notes were increasingly reduced with increasing pectin : protein ratios; however, high dispersion viscosities above 0.05 Pa s led to undesirable texture and mouthfeel of the biopolymer dispersions. Our results might have important implications for the utilization of novel plant proteins in food and beverage applications.
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Aditivos Alimentares/química , Manipulação de Alimentos/métodos , Pisum sativum/química , Proteínas de Plantas/química , Solanum tuberosum/química , Biopolímeros/química , Humanos , Malus/química , Pectinas/química , Extratos Vegetais/química , Reologia , Paladar , ViscosidadeRESUMO
Trehalose (α-D-glucopyranosyl-(1 â 1)-α-D-glucopyranoside) is a non-reducing disaccharide composed of two glucose molecules linked by an α,α-1,1-glycosidic bond. It possesses physicochemical properties, which account for its biological roles in a variety of prokaryotic and eukaryotic organisms and invertebrates. Intensive studies of trehalose gradually uncovered its functions, and its applications in foods, cosmetics, and pharmaceuticals have increased every year. Currently, trehalose is industrially produced by the two-enzyme method, which was first developed in 1995 using maltooligosyltrehalose synthase (EC 5.4.99.15) and subsequently using maltooligosyltrehalose trehalohydrolase (EC 3.2.1.141), with starch as the substrate. This biotechnical method has lowered the price of trehalose and expanded its applications. However, when trehalose synthase (EC 5.4.99.16) was later discovered, this method for trehalose production using maltose as the substrate soon became a popular topic because of its simplicity and potential in industrial production. Since then, many trehalose synthases have been studied. This review summarizes the sources and characteristics of reported trehalose synthases, and the most recent advances on structural analysis of trehalose synthase, catalytic mechanism, molecular modification, and usage in industrial production processes.
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Glucosiltransferases/metabolismo , Microbiologia Industrial/tendências , Trealose/biossíntese , Bactérias/enzimologia , Bactérias/genética , Maltose/metabolismo , Amido/metabolismoRESUMO
Kluyveromyces lactis is a common fungal microorganism used for the production of enzyme preparations such as ß-galactosidases (native) or chymosin (recombinant). It is generally important that enzyme preparations have no unwanted side activities. In the case of ß-galactosidase preparations produced from K. lactis, an unwanted side activity could be the presence of arylsulfatase (EC 3.1.6.1). Due to the action of arylsulfatase, an unpleasant "cowshed-like" off-flavor would occur in the final product. The best choice to avoid this is to use a yeast strain without this activity. Interestingly, we found that certain natural K. lactis strains express arylsulfatases, which only differ in one amino acid at position 139. The result of this difference is that K. lactis DSM 70799 (expressing R139 variant) shows no arylsulfatase activity, unlike K. lactis GG799 (expressing S139 variant). After recombinant production of both variants in Escherichia coli, the R139 variant remains inactive, whereas the S139 variant showed full activity. Mass spectrometric analyses showed that the important posttranslational modification of C56 to formylglycine was not found in the R139 variant. By contrast, the C56 residue of the S139 variant was modified. We further investigated the packing and secondary structure of the arylsulfatase variants using optical spectroscopy, including fluorescence and circular dichroism. We found out that the inactive R139 variant exhibits a different structure regarding folding and packing compared to the active S139 variant. The importance of the amino acid residue 139 was documented further by the construction of 18 more variants, whereof only ten showed activity but always reduced compared to the native S139 variant.
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Arilsulfatases/genética , Arilsulfatases/metabolismo , Glicina/análogos & derivados , Kluyveromyces/enzimologia , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Arilsulfatases/química , Biotransformação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glicina/metabolismo , Kluyveromyces/genética , Espectrometria de Massas , Estrutura Secundária de Proteína , Análise EspectralRESUMO
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.
Assuntos
Glutamil Aminopeptidase/metabolismo , Lactobacillus delbrueckii/enzimologia , Metais/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Especificidade por Substrato , TemperaturaRESUMO
Lactose is a main by-product in the cheese industry. Many attempts have been made to convert the lactose to high value-added products, including epilactose. Epilactose is a valuable prebiotic and can be epimerized from lactose with cellobiose 2-epimerase (CEase). The objective of the present work was to construct a food-grade recombinant Bacillus subtilis that produces CEase from Thermoanaerobacterium saccharolyticum. The CEase was expressed in B. subtilis without antibiotic resistance genes. After fermentation, the maximum volumetric activity of the fermented broth was more than 7 U/mL. The activity of the recombinant B. subtilis was increased by up to 3.7 fold after ethanol permeabilization. Then, 66.9 ± 0.7 g/L of epilactose was produced from 300 g/L of whey powder solution in 1 h with 13.3 U/mL of permeabilized biocatalyst. In addition, an enzymatic route including degradation of the lactose, yeast fermentation, and cation exchange chromatography was described to further purify the produced epilactose from lactose. Finally, epilactose with a purity >98% was produced from 300 g/L of lactose with a yield of 24.0%. In conclusion, neither antibiotics nor pathogenic bacteria were used throughout the epilactose production and purification procedure.
Assuntos
Bacillus subtilis/genética , Carboidratos Epimerases/genética , Dissacarídeos/biossíntese , Lactose/química , Thermoanaerobacterium/enzimologia , Bacillus subtilis/enzimologia , Carboidratos Epimerases/metabolismo , Clonagem Molecular , Dissacarídeos/isolamento & purificação , Fermentação , Prebióticos , Thermoanaerobacterium/genética , Soro do Leite/metabolismoRESUMO
Lactulose (4-O-ß-d-galactopyranosyl-d-fructofuranose) is a prebiotic sugar derived from the milk sugar lactose (4-O-ß-d-galactopyranosyl-d-glucopyranose). In our study we observed for the first time that known cellobiose 2-epimerases (CEs; EC 5.1.3.11) from mesophilic microorganisms were generally able to catalyze the isomerization reaction of lactose into lactulose. Commonly, CEs catalyze the C2-epimerization of d-glucose and d-mannose moieties at the reducing end of ß-1,4-glycosidic-linked oligosaccharides. Thus, epilactose (4-O-ß-d-galactopyranosyl-d-mannopyranose) is formed with lactose as substrate. So far, only four CEs, exclusively from thermophilic microorganisms, have been reported to additionally catalyze the isomerization reaction of lactose into lactulose. The specific isomerization activity of the seven CEs in this study ranged between 8.7 ± 0.1 and 1300 ± 37 pkat/mg. The results indicate that very likely all CEs are able to catalyze both the epimerization as well as the isomerization reaction, whereby the latter is performed at a comparatively much lower reaction rate.
Assuntos
Proteínas de Bactérias/química , Carboidratos Epimerases/química , Celobiose/metabolismo , Lactulose/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroides/enzimologia , Bacteroides/genética , Biocatálise , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Celobiose/química , Estabilidade Enzimática , Flavobacterium/enzimologia , Flavobacterium/genética , Lactose/metabolismo , Lactulose/químicaRESUMO
Five strains, designated WS 4672T, WS 4998, WS 4992T, WS 4997 and WS 5000, isolated from bovine raw milk formed two individual groups in a phylogenetic analysis. The most similar species on the basis of 16S rRNA gene sequences were Pseudomonas azotoformans IAM 1603T, Pseudomonas gessardii CIP 105469T and Pseudomonas libanensis CIP 105460T showing 99.7-99.6â% similarity. Using rpoD gene sequences Pseudomonas veronii LMG 17761T (93.3â%) was most closely related to strain WS 4672T and Pseudomonas libanensis CIP 105460T to strain WS 4992T (93.3â%). The five strains could be differentiated from their closest relatives and from each other by phenotypic and chemotaxonomic characterization and ANIb values calculated from draft genome assemblies. ANIb values of strains WS 4992T and WS4671T to the closest relatives are lower than 90â%. The major cellular polar lipids of both strains are phosphatidylethanolamine, phosphatidylglycerol, a phospholipid and diphosphatidylglycerol, and their major quinone is Q-9. The DNA G+C content of strains WS 4992T and WS 4672T were 60.0â and 59.7â mol%, respectively. Based on these genotypic and phenotypic traits two novel species of the genus Pseudomonas are proposed: Pseudomonas lactis sp. nov. [with type strain WS 4992T (=DSM 29167T=LMG 28435T) and the additional strains WS 4997 and WS 5000], and Pseudomonasparalactis sp. nov. [with type strain WS 4672T (=DSM 29164T=LMG 28439T) and additional strain WS 4998].
Assuntos
Leite/microbiologia , Filogenia , Pseudomonas/classificação , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , Bovinos , DNA Bacteriano/genética , Ácidos Graxos/química , Hibridização de Ácido Nucleico , Fosfolipídeos/química , Pseudomonas/genética , Pseudomonas/isolamento & purificação , Quinonas/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
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.