RESUMO
GtfB-type α-glucanotransferase enzymes from glycoside hydrolase family 70 (GH70) convert starch substrates into α-glucans that are of interest as food ingredients with a low glycemic index. Characterization of several GtfBs showed that they differ in product- and substrate specificity, especially with regard to branching, but structural information is limited to a single GtfB, preferring mostly linear starches and featuring a tunneled binding groove. Here, we present the second crystal structure of a 4,6-α-glucanotransferase (Limosilactobacillus reuteri NCC 2613) and an improved homology model of a 4,3-α-glucanotransferase GtfB (L. fermentum NCC 2970) and show that they are able to convert both linear and branched starch substrates. Compared to the previously described GtfB structure, these two enzymes feature a much more open binding groove, reminiscent of and evolutionary closer to starch-converting GH13 α-amylases. Sequence analysis of 287 putative GtfBs suggests that only 20% of them are similarly "open" and thus suitable as broad-specificity starch-converting enzymes.
Assuntos
Sistema da Enzima Desramificadora do Glicogênio , Amido , Glucanos , Sistema da Enzima Desramificadora do Glicogênio/genética , Glicosídeo HidrolasesRESUMO
Starch-acting α-glucanotransferase enzymes are of great interest for applications in the food industry. In previous work, we have characterized various 4,6- and 4,3-α-glucanotransferases of the glycosyl hydrolase (GH) family 70 (subfamily GtfB), synthesizing linear or branched α-glucans. Thus far, GtfB enzymes have only been identified in mesophilic Lactobacilli. Database searches showed that related GtfC enzymes occur in Gram-positive bacteria of the genera Exiguobacterium, Bacillus, and Geobacillus, adapted to growth at more extreme temperatures. Here, we report characteristics of the Geobacillus sp. 12AMOR1 GtfC enzyme, with an optimal reaction temperature of 60 °C and a melting temperature of 68 °C, allowing starch conversions at relatively high temperatures. This thermostable 4,6-α-glucanotransferase has a novel product specificity, cleaving off predominantly maltose units from amylose, attaching them with an (α1 â 6)-linkage to acceptor substrates. In fact, this GtfC represents a novel maltogenic α-amylase. Detailed structural characterization of its starch-derived α-glucan products revealed that it yielded a unique polymer with alternating (α1 â 6)/(α1 â 4)-linked glucose units but without branches. Notably, this Geobacillus sp. 12AMOR1 GtfC enzyme showed clear antistaling effects in bread bakery products.
Assuntos
Geobacillus , Sistema da Enzima Desramificadora do Glicogênio , Pão , Geobacillus/genética , Glucanos , Sistema da Enzima Desramificadora do Glicogênio/genéticaRESUMO
The glycemic carbohydrates we consume are currently viewed in an unfavorable light in both the consumer and medical research worlds. In significant part, these carbohydrates, mainly starch and sucrose, are looked upon negatively due to their rapid and abrupt glucose delivery to the body which causes a high glycemic response. However, dietary carbohydrates which are digested and release glucose in a slow manner are recognized as providing health benefits. Slow digestion of glycemic carbohydrates can be caused by several factors, including food matrix effect which impedes α-amylase access to substrate, or partial inhibition by plant secondary metabolites such as phenolic compounds. Differences in digestion rate of these carbohydrates may also be due to their specific structures (e.g. variations in degree of branching and/or glycosidic linkages present). In recent years, much has been learned about the synthesis and digestion kinetics of novel α-glucans (i.e. small oligosaccharides or larger polysaccharides based on glucose units linked in different positions by α-bonds). It is the synthesis and digestion of such structures that is the subject of this review.
Assuntos
Digestão , Trato Gastrointestinal/metabolismo , Glucanos/farmacologia , Glucose/metabolismo , Carboidratos da Dieta/metabolismo , Humanos , AmidoRESUMO
The fructophilic bacterium Lactobacillus kunkeei has promising applications as probiotics promoting the health of both honey bees and humans. Here, we report the synthesis of a highly branched dextran by L. kunkeei DSM 12361 and biochemical characterization of a GH70 enzyme (GtfZ). Sequence analysis revealed that GtfZ harbors two separate catalytic cores (CD1 and CD2), predicted to have glucansucrase and branching sucrase specificity, respectively. GtfZ-CD1 was not characterized biochemically due to its unsuccessful expression. With only sucrose as substrate, GtfZ-CD2 was found to mainly catalyze sucrose hydrolysis and leucrose synthesis. When dextran was available as acceptor substrate, GtfZ-CD2 displayed an efficient transglycosidase activity with sucrose as donor substrate. Kinetic analysis showed that the GtfZ-CD2-catalyzed transglycosylation reaction follows a Ping Pong Bi Bi mechanism, indicating the in-turn binding of donor and acceptor substrates in the active site. Structural characterization of the products revealed that GtfZ-CD2 catalyzes the synthesis of single glucosyl (α1 â 3) linked branches onto dextran, resulting in the production of highly branched comb-like α-glucan products. These (α1 â 3) branches can be formed on adjacent positions, as shown when isomaltotriose was used as acceptor substrate. Homology modeling of the GtfZ-CD1 and GtfZ-CD2 protein structure strongly suggests that amino acid differences in conserved motifs II, III, and IV in the catalytic domain contribute to product specificity. Our present study highlights the ability of beneficial lactic acid bacteria to produce structurally complex α-glucans and provides novel insights into the molecular mechanism of an (α1 â 3) branching sucrase.
Assuntos
Glicosiltransferases/química , Sacarase/química , Biocatálise , Domínio Catalítico , Dextranos/metabolismo , Glucanos/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Cinética , Lactobacillus/química , Lactobacillus/enzimologia , Lactobacillus/genética , Sacarase/genética , Sacarase/metabolismoRESUMO
Nine GtfB-like 4,6-α-glucanotransferases (4,6-α-GTs) (represented by GtfX of L. aviarius subsp. aviarius DSM 20655) were identified to show distinct characteristics in conserved motifs I-IV. In particular, the "fingerprint" Tyr in motif III of these nine GtfB-type 4,6-α-GTs was found to be replaced by a Trp. In L. aviarius subsp. aviarius DSM20655, a second GtfB-like protein (GtfY), containing the canonical GtfB Tyr residue in motif III, was located directly upstream of GtfX. Biochemical characterization revealed that both GtfX and GtfY showed GtfB-like 4,6-α-GT activity, cleaving (α1â4) linkages and catalyzing the synthesis of (α1â6) linkages. Nonetheless, they differ in product specificity; GtfY only synthesizes consecutive (α1â6) linkages, yielding linear α-glucan products, but GtfX catalyzes the synthesis of (α1â6) linkages predominantly at branch points (22%) rather than in linear segments (10%). The highly branched α-glucan produced by GtfX from amylose V is resistant to digestion by α-amylase, offering great potential as dietary fibers.
Assuntos
Sistema da Enzima Desramificadora do Glicogênio/química , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Lactobacillus/enzimologia , Motivos de Aminoácidos , Amilose/química , Amilose/metabolismo , Biocatálise , Fibras na Dieta , Digestão , Glucanos/química , Glucanos/metabolismo , Especificidade por SubstratoRESUMO
Transglucosidases belonging to the glycoside hydrolase (GH) family 70 are promising enzymatic tools for the synthesis of α-glucans with defined structures from renewable sucrose and starch substrates. Depending on the GH70 enzyme specificity, α-glucans with different structures and physicochemical properties are produced, which have found diverse (potential) commercial applications, e.g. in food, health and as biomaterials. Originally, the GH70 family was established only for glucansucrase enzymes of lactic acid bacteria that catalyze the synthesis of α-glucan polymers from sucrose. In recent years, we have identified 3 novel subfamilies of GH70 enzymes (designated GtfB, GtfC and GtfD), inactive on sucrose but converting starch/maltodextrin substrates into novel α-glucans. These novel starch-acting enzymes considerably enlarge the panel of α-glucans that can be produced. They also represent very interesting evolutionary intermediates between sucrose-acting GH70 glucansucrases and starch-acting GH13 α-amylases. Here we provide an overview of the repertoire of GH70 enzymes currently available with focus on these novel starch-acting GH70 enzymes and their biotechnological potential. Moreover, we discuss key developments in the understanding of structure-function relationships of GH70 enzymes in the light of available three-dimensional structures, and the protein engineering strategies that were recently applied to expand their natural product specificities.
Assuntos
Glicosídeo Hidrolases , Amido/metabolismo , Sacarose/metabolismo , Biotecnologia , Glicosiltransferases , Lactobacillus , Engenharia de Proteínas , Proteínas RecombinantesRESUMO
The Glycoside hydrolase (GH) family 70 originally was established for glucansucrases of lactic acid bacteria (LAB) converting sucrose into α-glucan polymers. In recent years we have identified 3 subfamilies of GH70 enzymes (designated GtfB, GtfC and GtfD) as 4,6-α-glucanotransferases, cleaving (α1 â 4)-linkages in maltodextrins/starch and synthesizing new (α1 â 6)-linkages. In this work, 106 putative GtfBs were identified in the Nestlé Culture Collection genome database with ~2700 genomes, and the L. reuteri NCC 2613 one was selected for further characterization based on variations in its conserved motifs. Using amylose the L. reuteri NCC 2613 GtfB synthesizes a low-molecular-mass reuteran-like polymer consisting of linear (α1 â 4) sequences interspersed with (α1 â 6) linkages, and (α1 â 4,6) branching points. This product specificity is novel within the GtfB subfamily, mostly comprising 4,6-α-glucanotransferases synthesizing consecutive (α1 â 6)-linkages. Instead, its activity resembles that of the GtfD 4,6-α-glucanotransferases identified in non-LAB strains. This study demonstrates the potential of large-scale genome sequence data for the discovery of enzymes of interest for the food industry. The L. reuteri NCC 2613 GtfB is a valuable addition to the starch-converting GH70 enzyme toolbox. It represents a new evolutionary intermediate between families GH13 and GH70, and provides further insights into the structure-function relationships of the GtfB subfamily enzymes.
Assuntos
Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Limosilactobacillus reuteri/enzimologia , Limosilactobacillus reuteri/genética , Amido/metabolismo , Mineração de Dados , Bases de Dados de Ácidos Nucleicos , Glicosídeo Hidrolases/isolamento & purificação , HidróliseRESUMO
Previously we have reported that the Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 uses the 4,6-α-glucanotransferase GtfD to convert maltodextrins and starch into a reuteran-like polymer consisting of (α1â4) glucan chains connected by alternating (α1â4)/(α1â6) linkages and (α1â4,6) branching points. This enzyme constituted the single evidence for this reaction and product specificity in the GH70 family, mostly containing glucansucrases encoded by lactic acid bacteria (http://www.CAZy.org). In this work, 4 additional GtfD-like proteins were identified in taxonomically diverse plant-associated bacteria forming a new GH70 subfamily with intermediate characteristics between the evolutionary related GH13 and GH70 families. The GtfD enzyme encoded by Paenibacillus beijingensis DSM 24997 was characterized providing the first example of a reuteran-like polymer synthesizing 4,6-α-glucanotransferase in a Gram-positive bacterium. Whereas the A. chroococcum GtfD activity on amylose resulted in the synthesis of a high molecular polymer, in addition to maltose and other small oligosaccharides, two reuteran-like polymer distributions are produced by P. beijingensis GtfD: a high-molecular mass polymer and a low-molecular mass polymer with an average Mw of 27 MDa and 19 kDa, respectively. Compared to the A. chroooccum GtfD product, both P. beijingensis GtfD polymers contain longer linear (α1â4) sequences in their structure reflecting a preference for transfer of even longer glucan chains by this enzyme. Overall, this study provides new insights into the evolutionary history of GH70 enzymes, and enlarges the diversity of natural enzymes that can be applied for modification of the starch present in food into less and/or more slowly digestible carbohydrate structures.
Assuntos
Proteínas de Bactérias/metabolismo , Glucanos/metabolismo , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Paenibacillus/enzimologia , Amilose/metabolismo , Animais , Azotobacter/enzimologia , Azotobacter/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Cromatografia , Escherichia coli , Evolução Molecular , Sistema da Enzima Desramificadora do Glicogênio/química , Sistema da Enzima Desramificadora do Glicogênio/genética , Sistema da Enzima Desramificadora do Glicogênio/isolamento & purificação , Humanos , Espectroscopia de Ressonância Magnética , Metilação , Paenibacillus/genética , Filogenia , Domínios Proteicos , Ratos , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The probiotic bacterium Lactobacillus reuteri 121 is a well-known producer of diverse homoexopolysaccharides (α-glucans and ß-fructans) from sucrose and maltodextrins/starches of interest for food applications. Here, we report the draft genome sequence of this strain, with a focus on carbohydrate-active enzymes.
RESUMO
Lactic acid bacteria possess a diversity of glucansucrase (GS) enzymes that belong to glycoside hydrolase family 70 (GH70) and convert sucrose into α-glucan polysaccharides with (α1 â 2)-, (α1 â 3)-, (α1 â 4)- and/or (α1 â 6)-glycosidic bonds. In recent years 3 novel subfamilies of GH70 enzymes, inactive on sucrose but using maltodextrins/starch as substrates, have been established (e.g. GtfB of Lactobacillus reuteri 121). Compared to the broad linkage specificity found in GSs, all GH70 starch-acting enzymes characterized so far possess 4,6-α-glucanotransferase activity, cleaving (α1 â 4)-linkages and synthesizing new (α1 â 6)-linkages. In this work a gene encoding a putative GH70 family enzyme was identified in the genome of Lactobacillus fermentum NCC 2970, displaying high sequence identity with L. reuteri 121 GtfB 4,6-α-glucanotransferase, but also with unique variations in some substrate-binding residues of GSs. Characterization of this L. fermentum GtfB and its products revealed that it acts as a 4,3-α-glucanotransferase, converting amylose into a new type of α-glucan with alternating (α1 â 3)/(α 1 â 4)-linkages and with (α1 â 3,4) branching points. The discovery of this novel reaction specificity in GH70 family and clan GH-H expands the range of α-glucans that can be synthesized and allows the identification of key positions governing the linkage specificity within the active site of the GtfB-like GH70 subfamily of enzymes.
Assuntos
Proteínas de Bactérias/metabolismo , Glucosiltransferases/metabolismo , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Limosilactobacillus fermentum/enzimologia , Limosilactobacillus reuteri/enzimologia , Proteínas de Bactérias/química , Evolução Molecular , Glucanos/química , Glucanos/metabolismo , Glucosiltransferases/química , Sistema da Enzima Desramificadora do Glicogênio/química , Glicosídeos/química , Glicosídeos/metabolismo , Filogenia , Polissacarídeos/química , Polissacarídeos/metabolismo , Especificidade por Substrato , Sacarose/química , Sacarose/metabolismoRESUMO
Food processing and refining has dramatically changed the human diet, but little is known about whether this affected the evolution of enzymes in human microbiota. We present evidence that glycoside hydrolase family 70 (GH70) glucansucrases from lactobacilli, synthesizing α-glucan-type extracellular polysaccharides from sucrose, likely evolved from GH13 starch-acting α-amylases, via GH70 4,6-α-glucanotransferases. The crystal structure of a 4,6-α-glucanotransferase explains the mode of action and unique product specificity of these enzymes. While the α-amylase substrate-binding scaffold is retained, active-site loops adapted to favor transglycosylation over hydrolysis; the structure also gives clues as to how 4,6-α-glucanotransferases may have evolved further toward sucrose utilization instead of starch. Further supported by genomic, phylogenetic, and in vivo studies, we propose that dietary changes involving starch (and starch derivatives) and sucrose intake were critical factors during the evolution of 4,6-α-GTs and glucansucrases from α-amylases, allowing oral bacteria to produce extracellular polymers that contribute to biofilm formation from different substrates.
Assuntos
Proteínas de Bactérias/química , Sistema da Enzima Desramificadora do Glicogênio/química , Limosilactobacillus reuteri/genética , Amido/química , Sacarose/química , alfa-Amilases/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Carboidratos , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Dieta , Carboidratos da Dieta/administração & dosagem , Evolução Molecular , Microbioma Gastrointestinal/genética , Expressão Gênica , Sistema da Enzima Desramificadora do Glicogênio/genética , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Glicosilação , Humanos , Limosilactobacillus reuteri/classificação , Limosilactobacillus reuteri/enzimologia , Modelos Moleculares , Mutação , Filogenia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Amido/metabolismo , Especificidade por Substrato , Sacarose/metabolismo , alfa-Amilases/genética , alfa-Amilases/metabolismoRESUMO
Lactic acid bacteria (LAB) are known to produce large amounts of α-glucan exopolysaccharides. Family GH70 glucansucrase (GS) enzymes catalyze the synthesis of these α-glucans from sucrose. The elucidation of the crystal structures of representative GS enzymes has advanced our understanding of their reaction mechanism, especially structural features determining their linkage specificity. In addition, with the increase of genome sequencing, more and more GS enzymes are identified and characterized. Together, such knowledge may promote the synthesis of α-glucans with desired structures and properties from sucrose. In the meantime, two new GH70 subfamilies (GTFB- and GTFC-like) have been identified as 4,6-α-glucanotransferases (4,6-α-GTs) that represent novel evolutionary intermediates between the family GH13 and "classical GH70 enzymes". These enzymes are not active on sucrose; instead, they use (α1 â 4) glucans (i.e. malto-oligosaccharides and starch) as substrates to synthesize novel α-glucans by introducing linear chains of (α1 â 6) linkages. All these GH70 enzymes are very interesting biocatalysts and hold strong potential for applications in the food, medicine and cosmetic industries. In this review, we summarize the microbiological distribution and the structure-function relationships of family GH70 enzymes, introduce the two newly identified GH70 subfamilies, and discuss evolutionary relationships between family GH70 and GH13 enzymes.
Assuntos
Evolução Molecular , Sistema da Enzima Desramificadora do Glicogênio/química , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Bactérias/enzimologia , Biocatálise , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Originally the glycoside hydrolase (GH) family 70 only comprised glucansucrases of lactic acid bacteria which synthesize α-glucan polymers from sucrose. Recently we have identified 2 novel subfamilies of GH70 enzymes represented by the Lactobacillus reuteri 121 GtfB and the Exiguobacterium sibiricum 255-15 GtfC enzymes. Both enzymes catalyze the cleavage of (α1â4) linkages in maltodextrin/starch and the synthesis of consecutive (α1â6) linkages. Here we describe a novel GH70 enzyme from the nitrogen-fixing Gram-negative bacterium Azotobacter chroococcum, designated as GtfD. METHODS: The purified recombinant GtfD enzyme was biochemically characterized using the amylose-staining assay and its products were identified using profiling chromatographic techniques (TLC and HPAEC-PAD). Glucans produced by the GtfD enzyme were analyzed by HPSEC-MALLS-RI, methylation analysis, 1D/2D [1]H/[13]C NMR spectroscopy and enzymatic degradation studies. RESULTS: The A. chroococcum GtfD is closely related to GtfC enzymes, sharing the same non-permuted domain organization also found in GH13 enzymes and displaying 4,6-α-glucanotransferase activity. However, the GtfD enzyme is unable to synthesize consecutive (α1â6) glucosidic bonds. Instead, it forms a high molecular mass and branched α-glucan with alternating (α1â4) and (α1â6) linkages from amylose/starch, highly similar to the reuteran polymer synthesized by the L. reuteri GtfA glucansucrase from sucrose. CONCLUSIONS: In view of its origin and specificity, the GtfD enzyme represents a unique evolutionary intermediate between family GH13 (α-amylase) and GH70 (glucansucrase) enzymes. GENERAL SIGNIFICANCE: This study expands the natural repertoire of starch-converting enzymes providing the first characterization of an enzyme that converts starch into a reuteran-like α-glucan polymer, regarded as a health promoting food ingredient.
Assuntos
Azotobacter/enzimologia , Glucanos/biossíntese , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Polissacarídeos/metabolismo , Amido/metabolismo , Sequência de Aminoácidos , Sistema da Enzima Desramificadora do Glicogênio/química , Dados de Sequência Molecular , Oligossacarídeos/biossíntese , Especificidade por SubstratoRESUMO
The glycoside hydrolase 70 (GH70) family originally was established for glucansucrase enzymes found solely in lactic acid bacteria synthesizing α-glucan polysaccharides from sucrose (e.g., GtfA). In recent years, we have characterized GtfB and related Lactobacillus enzymes as 4,6-α-glucanotransferase enzymes. These GtfB-type enzymes constitute the first GH70 subfamily of enzymes that are unable to act on sucrose as a substrate but are active with maltodextrins and starch, cleave α1â4 linkages, and synthesize linear α1â6-glucan chains. The GtfB disproportionating type of activity results in the conversion of malto-oligosaccharides into isomalto/malto-polysaccharides with a relatively high percentage of α1â6 linkages. This paper reports the identification of the members of a second GH70 subfamily (designated GtfC enzymes) and the characterization of the Exiguobacterium sibiricum 255-15 GtfC enzyme, which is also inactive with sucrose and displays 4,6-α-glucanotransferase activity with malto-oligosaccharides. GtfC differs from GtfB in synthesizing isomalto/malto-oligosaccharides. Biochemically, the GtfB- and GtfC-type enzymes are related, but phylogenetically, they clearly constitute different GH70 subfamilies, displaying only 30% sequence identity. Whereas the GtfB-type enzyme largely has the same domain order as glucansucrases (with α-amylase domains A, B, and C plus domains IV and V), this GtfC-type enzyme differs in the order of these domains and completely lacks domain V. In GtfC, the sequence of conserved regions I to IV of clan GH-H is identical to that in GH13 (I-II-III-IV) but different from that in GH70 (II-III-IV-I because of a circular permutation of the (ß/α)8 barrel. The GtfC 4,6-α-glucanotransferase enzymes thus represent structurally and functionally very interesting evolutionary intermediates between α-amylase and glucansucrase enzymes.
Assuntos
Bacillales/enzimologia , Proteínas de Bactérias/metabolismo , Glicosídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Bacillales/química , Bacillales/classificação , Bacillales/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Dados de Sequência Molecular , Família Multigênica , Filogenia , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
The extracellular medium-chain-length polyhydroxyalkanote (MCL-PHA) depolymerase from an isolate identified as Streptomyces venezuelae SO1 was purified to electrophoretic homogeneity and characterized. The molecular mass and pI of the purified enzyme were approximately 27 kDa and 5.9, respectively. The depolymerase showed its maximum activity in the alkaline pH range and 50 °C and retained more than 70 % of its initial activity after 8 h at 40 °C. The MCL-PHA depolymerase hydrolyzes various p-nitrophenyl-alkanoates and polycaprolactone but not polylactide, poly-3-hydroxybutyrate, and polyethylene succinate. The enzymatic activity was markedly enhanced by the presence of low concentrations of detergents and organic solvents, being inhibited by dithiothreitol and EDTA. The potential of using the enzyme to produce (R)-3-hydroxyoctanoate in aqueous media or to catalyze ester-forming reactions in anhydrous media was investigated. In this sense, the MCL-PHA depolymerase catalyzes the hydrolysis of poly-3-hydroxyoctanoate to monomeric units and the ring-opening polymerization of ß-butyrolactone and lactides, while ε-caprolactone and pentadecalactone were hardly polymerized.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Poliésteres/metabolismo , Streptomyces/enzimologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/isolamento & purificação , Ativadores de Enzimas/metabolismo , Inibidores Enzimáticos/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Ponto Isoelétrico , Peso Molecular , Especificidade por Substrato , TemperaturaRESUMO
Nineteen medium-chain-length (mcl) poly(3-hydroxyalkanoate) (PHA)-degrading microorganisms were isolated from natural sources. From them, seven Gram-positive and three Gram-negative bacteria were identified. The ability of these microorganisms to hydrolyze other biodegradable plastics, such as short-chain-length (scl) PHA, poly(ε-caprolactone) (PCL), poly(ethylene succinate) (PES), and poly(l-lactide) (PLA), has been studied. On the basis of the great ability to degrade different polyesters, Streptomyces roseolus SL3 was selected, and its extracellular depolymerase was biochemically characterized. The enzyme consisted of one polypeptide chain of 28 kDa with a pI value of 5.2. Its maximum activity was observed at pH 9.5 with chromogenic substrates. The purified enzyme hydrolyzed mcl PHA and PCL but not scl PHA, PES, and PLA. Moreover, the mcl PHA depolymerase can hydrolyze various substrates for esterases, such as tributyrin and p-nitrophenyl (pNP)-alkanoates, with its maximum activity being measured with pNP-octanoate. Interestingly, when poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate [11%]) was used as the substrate, the main hydrolysis product was the monomer (R)-3-hydroxyoctanoate. In addition, the genes of several Actinobacteria strains, including S. roseolus SL3, were identified on the basis of the peptide de novo sequencing of the Streptomyces venezuelae SO1 mcl PHA depolymerase by tandem mass spectrometry. These enzymes did not show significant similarity to mcl PHA depolymerases characterized previously. Our results suggest that these distinct enzymes might represent a new subgroup of mcl PHA depolymerases.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Poli-Hidroxialcanoatos/metabolismo , Streptomyces/enzimologia , Sequência de Aminoácidos , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Dados de Sequência Molecular , Peso Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Streptomyces/genética , Especificidade por SubstratoRESUMO
The extracellular medium-chain-length polyhydroxyalkanoate (MCL-PHA) depolymerase of Pseudomonas fluorescens GK13 catalyzes the hydrolysis of poly(3-hydroxyoctanoic acid) [P(3HO)]. Based on the strong tendency of the enzyme to interact with hydrophobic materials, a low-cost method which allows the rapid and easy purification and immobilization of the enzyme has been developed. Thus, the extracellular P(3HO) depolymerase present in the culture broth of cells of P. fluorescens GK13 grown on mineral medium supplemented with P(3HO) as the sole carbon and energy source has been tightly adsorbed onto a commercially available polypropylene support (Accurel MP-1000) with high yield and specificity. The activity of the pure enzyme was enhanced by the presence of detergents and organic solvents, and it was retained after treatment with an SDS-denaturing cocktail under both reducing and nonreducing conditions. The time course of the P(3HO) hydrolysis catalyzed by the soluble and immobilized enzyme has been assessed, and the resulting products have been identified. After 24 h of hydrolysis, the dimeric ester of 3-HO [(R)-3-HO-HO] was obtained as the main product of the soluble enzyme. However, the immobilized enzyme catalyzes almost the complete hydrolysis of P(3HO) polymer to (R)-3-HO monomers under the same conditions.