Improvement of Heterologous Soluble Expression of L-amino Acid Oxidase Using Logistic Regression.

Successful implementation of enzymes in practical application hinges on the development of efficient mass production techniques. However, in a heterologous expression system, the protein is often unable to fold correctly and, thus, forms inclusion bodies, resulting in the loss of its original activity. In this study, we present a new and more accurate model for predicting amino acids associated with an increased L-amino acid oxidase (LAO) solubility. Expressing LAO from Rhizoctonia solani in Escherichia coli and combining random mutagenesis and statistical logistic regression, we modified 108 amino acid residues by substituting hydrophobic amino acids with serine and hydrophilic amino acids with alanine. Our results indicated that specific mutations in Euclidean distance, glycine, methionine, and secondary structure increased LAO expression. Furthermore, repeated mutations were performed for LAO based on logistic regression models. The mutated LAO displayed a significantly increased solubility, with the 6-point and 58-point mutants showing a 2.64- and 4.22-fold increase, respectively, compared with WT-LAO. Ultimately, using recombinant LAO in the biotransformation of α-keto acids indicates its great potential as a biocatalyst in industrial production.

Expression and characterization of C-terminal truncated mutants of γ-glutamyltranspeptidase II (PaGGTII) from Pseudomonas aeruginosa PAO1.

The gene encoding γ-glutamyltranspeptidase II (PaGGTII) from Pseudomonas aeruginosa PAO1 was cloned in Escherichia coli. Recombinant PaGGTII showed a weak activity (0.0332 U/mg), and it can be easily inactivated. Multiple alignment of microbial GGTs showed the redundancy of the C-terminal of the small subunit of PaGGTII in length. The truncation of eight amino acid residues at the C-terminal of PaGGTII remarkably improved the activity and stability of the enzyme (PaGGTIIΔ8; 0.388 U/mg). Further truncation at the C-terminal also provided the enzyme relatively higher activity (PaGGTIIΔ9, -Δ10, -Δ11, and -Δ12). Among C-terminal truncated mutants, we focused on PaGGTIIΔ8 and examined the effect of C-terminal amino acid residues on the properties of PaGGTIIΔ8 because the activity of PaGGTII was found to be greatly improved when 8 amino acid residues were truncated. Various mutant enzymes with different C-terminal amino acid residues were constructed. They were expressed in E. coli and purified to homogeneity by ion-exchange chromatography. The properties of PaGGTIIΔ8 and the mutants obtained from mutation at E569 were characterized. Km and kcat of PaGGTIIΔ8 for γ-glutamyl-p-nitroanilide (γ-GpNA) were 8.05 mM and 15.49 s-1, respectively. PaGGTIIΔ8E569Y showed the highest catalytic efficiency for γ-GpNA with a kcat/Km of 12.55 mM-1 s-1. Mg2+, Ca2+, and Mn2+ exhibited positive effects on the catalytic activity for PaGGTIIΔ8 and its ten E569 mutants.

Nigero-oligosaccharide production by enzymatic hydrolysis from alkaline-pretreated α-1,3-glucan.

Nigero-oligosaccharides are α-1,3-linked oligomers of glucose. Glycoside hydrolase 87 type α-1,3-glucanase Agl-KA from Bacillus circulans KA304 is an endo-lytic enzyme that releases nigero-oligosaccharides (tetra-, tri-, and di-saccharide) from α-1,3-glucan. α-1,3-Glucan is insoluble under natural conditions, thus the efficiency of enzymatic hydrolysis is low and only 5 mM of reducing sugars were released from 1% glucan by Agl-KA. To improve hydrolytic efficiency, α-1,3-glucan was solubilized by 1 M NaOH and alkaline-solubilized glucan was adjusted to approximately pH 8. As a result, glucan maintained a solubilized state. This alkaline-pretreated α-1,3-glucan (1%) was hydrolyzed by Agl-KA (0.64 nmol/mL) and approximately 11.6 mM of reducing sugars were released at 240 min of reaction. When 0.016, 0.032, and 0.13 nmol/mL enzyme were added, reducing sugar reached approximately 5.1, 7.5, and 9.8 mM, respectively, and reaction mixtures containing 0.016 and 0.032 nmol/mL enzyme gradually became cloudy. Our findings suggest α-1,3-glucan cannot maintain its solubilized state and gradually becomes insoluble. For deletion enzyme of α-1,3-glucan binding domains from Agl-KA (AglΔDCD-UCD) on glucan hydrolysis (2%), reducing sugar concentrations released by AglΔDCD-UCD were almost the same as Agl-KA. These findings suggest that alkaline-pretreated α-1,3-glucan maintains a soluble state during a short time period and that glucan is efficiently hydrolyzed even by α-1,3-glucanase without α-1,3-glucan binding domains.

Characterization of three γ-glutamyltranspeptidases from Pseudomonas aeruginosa PAO1.

The Pseudomonas aeruginosa strain, PAO1, has three putative γ-glutamyltranspeptidase (GGT) genes: ggtI, ggtII, and ggtIII. In this study, the expression of each of these genes in P. aeruginosa PAO1 was analyzed, and the properties of the corresponding GGT proteins were investigated. This is the first report on biochemical characterization of GGT paralogs from Pseudomonas species. The crude extracts prepared from P. aeruginosa PAO1 exhibited hydrolysis and transpeptidation activities of 17.3 and 65.0 mU/mg, respectively, and the transcription of each gene to mRNA was confirmed by RT-PCR. All genes were cloned, and the expression plasmids constructed were introduced into an Escherichia coli expression system. Enzyme activity of the expressed protein of ggtI (PaGGTI) was not detected in the system, while the enzyme activities of the expressed proteins derived from ggtII and ggtIII (PaGGTII and PaGGTIII, respectively) were detected. However, the enzyme activity of PaGGTII was very low and easily decreased. PaGGTII with C-terminal his-tag (PaGGTII25aa) showed increased activity and stability, and the purified enzyme consisted of a large subunit of 40 kDa and a small subunit of 28 kDa. PaGGTIII consisted of a large subunit of 37 kDa and a small subunit of 24 kDa. The maximum hydrolysis and transpeptidation activities of PaGGTII25aa were obtained at 40ºC-50ºC, and the maximum hydrolysis and transpeptidation activities of PaGGTIII were obtained at 50ºC-60ºC. These enzymes retained approximately 80% of their hydrolysis and transpeptidation activities after incubation at 50ºC for 10 min, reflecting good stability. Both PaGGTII25aa and PaGGTIII showed higher activities of hydrolysis and transpeptidation in the alkali range than in the acidic range. However, they were highly stable at a wide pH range (5-10.5).

Optimization of protease production by Bacillus cereus HMRSC30 for simultaneous extraction of chitin from shrimp shell with value-added recovered products.

Chitin extraction from shrimp shell powder (SSP) using protease-producing microbes is an attractive approach for valorizing shrimp shell waste because it is simple and environmentally friendly. In this study, the protease production and chitin extraction from SSP by Bacillus cereus HMRSC30 were simultaneously optimized using statistical approaches. As a result, fermentation in medium composed of 30 g/L SSP, 0.2 g/L MgSO4 · 7H2O, 3 g/L (NH4)2SO4, 0.5 g/L K2HPO4, and 1.5 g/L KH2PO4 (pH 6.5) for 7 days maximized protease production (197.75 ± 0.33 U/mL) to approximately 1.64-fold compared to unoptimized condition (126.8 ± 0.047 U/mL). This level of enzyme production was enough to achieve 97.42 ± 0.28% deproteinization (DP) but low demineralization (DM) of 53.76 ± 0.21%. The high DM of 90% could be easily accomplished with the post-treatment using 0.4 M HCl and acetic acid. In addition, the study evaluated the possible roadmap to maximize the value of generated products and obtain additional profits from this microbial process. The observation showed the possibility of serving crude chitin as a bio-adsorbent with the highest removal capacity against Coomassie brilliant blue (97.99%), followed by methylene blue (74.42%). The recovered protease exhibited the function to remove egg yolk stain, indicating its potential for use as a detergent in de-staining. The results corroborated the benefits of microbial fermentation by B. cereus HMRSC30 as green process for comprehensive utilization of shrimp shell waste as well as minimizing waste generation along the established process.

Identification, characterization, and cloning of a novel aminoacylase, L-pipecolic acid acylase from Pseudomonas species.

L-Pipecolic acid is utilized as a vital component of specific chemical compounds, such as immunosuppressive drugs, anticancer reagents, and anesthetic reagents. We isolated and characterized a novel L-aminoacylase, N-acetyl-L-pipecolic acid-specific aminoacylase (LpipACY), from Pseudomonas sp. AK2. The subunit molecular mass of LpipACY was 45 kDa and was assumed to be a homooctamer in solution. The enzyme exhibited high substrate specificity toward N-acetyl-L-pipecolic acid and a high activity for N-acetyl-L-pipecolic acid and N-acetyl-L-proline. This enzyme was stable at a high temperature (60°C for 10 min) and under an alkaline pH (6.0-11.5). The N-terminal and internal amino acid sequences of the purified enzyme were STTANTLILRNG and IMASGGV, respectively. These sequences are highly consistent with those of uncharacterized proteins from Pseudomonas species, such as amidohydrolase and peptidase. We also cloned and overexpressed the gene coding LpipACY in Escherichia coli. Moreover, the recombinant LpipACY exhibited properties similar to native enzyme. Our results suggest that LpipACY is a potential enzyme for the enzymatic synthesis of L-pipecolic acid. This study provides the first description of the enzymatic characterization of L-pipecolic acid specific amino acid acylase.

l-tryptophan-histidine synthesis by Pseudomonas serine peptidase, an amino acid ester hydrolase of the peptidase family S9.

Pseudomonas sp. KM1 produces an amino acid ester hydrolase (KM1AEH) that catalyzes peptide bond formation by acting on carboxylic ester bonds. The KM1AEH gene was cloned from genomic DNA and expressed in Escherichia coli. The recombinant enzyme (rKM1AEH) was purified, and gel filtration showed that it is a 68 kDa monomeric protein. rKM1AEH can synthesize the vasoactive dipeptide tryptophan-histidine from tryptophan methyl ester and histidine as acyl donor and acceptor, respectively. The enzyme showed maximum activity at pH 9.5 and 45 °C and was specifically inhibited by silver (Ag+). Mutation of the catalytic Ser459 residue in the active site of rKM1AEH with Ala, Cys, or Thr eliminated all catalytic activity. The enzyme is a novel ester hydrolase that belongs to the peptidase family S9 based on the phylogenetic analysis.

Functional analysis of α-1,3-glucanase domain structure from Streptomyces thermodiastaticus HF3-3.

α-1,3-Glucanase from Streptomyces thermodiastaticus HF3-3 (Agl-ST) has been classified in the glycoside hydrolase (GH) family 87. Agl-ST is a multi-modular domain consisting of an N-terminal ß-sandwich domain (ß-SW), a catalytic domain, an uncharacterized domain (UC), and a C-terminal discoidin domain (DS). Although Agl-ST did not hydrolyze α-1,4-glycosidic bonds, its amino acid sequence is more similar to GH87 mycodextranase than to α-1,3-glucanase. It might be categorized into a new subfamily of GH87. In this study, we investigated the function of the domains. Several fusion proteins of domains with green fluorescence protein (GFP) were constructed to clarify the function of each domain. The results showed that ß-SW and DS domains played a role in binding α-1,3-glucan and enhancing the hydrolysis of α-1,3-glucan. The binding domains, ß-SW and DS, also showed binding activity toward xylan, although it was lower than that for α-1,3-glucan. The combination of ß-SW and DS domains demonstrated high binding and hydrolysis activities of Agl-ST toward α-1,3-glucan, whereas the catalytic domain showed only a catalytic function. The binding domains also achieved effective binding and hydrolysis of α-1,3-glucan in the cell wall complex of Schizophyllum commune.

Mutagenesis and structure-based analysis of the role of Tryptophan525 of γ-glutamyltranspeptidase from Pseudomonas nitroreducens.

γ-Glutamyltranspeptidase (GGT) is a ubiquitous enzyme that catalyzes the hydrolysis of the γ-glutamyl linkage of γ-glutamyl compounds and the transfer of their γ-glutamyl moiety to acceptor substrates. Pseudomonas nitroreducens GGT (PnGGT) is used for the industrial synthesis of theanine, thus it is important to determine the structural basis of hydrolysis and transfer reactions and identify the acceptor site of PnGGT to improve the efficient of theanine synthesis. Our previous structural studies of PnGGT have revealed that crucial interactions between three amino acid residues, Trp385, Phe417, and Trp525, distinguish PnGGT from other GGTs. Here we report the role of Trp525 in PnGGT based on site-directed mutagenesis and structural analyses. Seven mutant variants of Trp525 were produced (W525F, W525V, W525A, W525G, W525S, W525D, and W525K), with substitution of Trp525 by nonaromatic residues resulting in dramatically reduced hydrolysis activity. All Trp525 mutants exhibited significantly increased transfer activity toward hydroxylamine with hardly any effect on acceptor substrate preference. The crystal structure of PnGGT in complex with the glutamine antagonist, 6-diazo-5-oxo-l-norleucine, revealed that Trp525 is a key residue limiting the movement of water molecules within the PnGGT active site.

Crystal structure of the catalytic unit of thermostable GH87 α-1,3-glucanase from Streptomyces thermodiastaticus strain HF3-3.

α-1,3-Glucan is a homopolymer composed of D-glucose (Glc) and it is an extracellular polysaccharide found in dental plaque due to Streptococcus species. α-1,3-Glucanase from Streptomyces thermodiastaticus strain HF3-3 (Agl-ST) has been identified as a thermostable α-1,3-glucanase, which is classified into glycoside hydrolase family 87 (GH87) and specifically hydrolyzes α-1,3-glucan with an endo-action. The enzyme has a potential to inhibit the production of dental plaque and to be used for biotechnological applications. Here we show the structure of the catalytic unit of Agl-ST determined at 1.16 Å resolution using X-ray crystallography. The catalytic unit is composed of two modules, a ß-sandwich fold module, and a right-handed ß-helix fold module, which resembles other structural characterized GH87 enzymes from Bacillus circulans str. KA-304 and Paenibacillus glycanilyticus str. FH11, with moderate sequence identities between each other (approximately 27% between the catalytic units). However, Agl-ST is smaller in size and more thermally stable than the others. A disulfide bond that anchors the C-terminal coil of the ß-helix fold, which is expected to contribute to thermal stability only exists in the catalytic unit of Agl-ST.

Prevention of oral biofilm formation and degradation of biofilm by recombinant α-1,3-glucanases from Streptomyces thermodiastaticus HF3-3.

The genes encoding α-1,3-glucanases (Agls; AglST1 and AglST2) from Streptomyces thermodiastaticus HF3-3 were cloned and were then expressed in Escherichia coli Rosetta-gami B (DE3). We purified the resultant histidine (His)-tagged α-1,3-glucanases (recombinant enzymes, rAglST1 and rAglST2). Both the recombinant enzymes were similar to the wild-type enzymes. We examined the effects of rAglST1 and rAglST2 on the formation and degradation of biofilms on glass plates with Streptococcus mutans NRBC 13955 by evaluating the biofilm content (%), release of reducing sugar (mM), release of S. mutans (log CFU/mL), and the biofilm structure using laser scanning microscopy (LSM). The results showed that after incubation for 16 h, rAglST1 and rAglST2 reduced the formation of biofilm to 52% and 49% of the control, respectively. The result may reflect the fact that the concentration of the reducing sugar and the number of S. mutans cells in the rAglATs-added medium were higher than in the control medium. After an 8-h treatment with rAglST1 and rAglST2, biofilms decreased to less than 60% of the control. The number of S. mutans cells in the reaction mixture gradually increased during the incubation period. The enzymes can degrade the biofilms that were pre-formed on the glass plate by more than 50% after a 30-min incubation in the presence of toothpaste ingredients (1% w/v of sodium fluoride, benzethonium chloride, and sodium dodecyl sulfate) at 50°C. Our study showed that rAglST1 and rAglST2 have advantageous properties for dental care applications.

Structural insights into substrate recognition and catalysis by glycoside hydrolase family 87 α-1,3-glucanase from Paenibacillus glycanilyticus FH11.

The α-1,3-glucanase from Paenibacillus glycanilyticus FH11 (Agl-FH1), a member of the glycoside hydrolase family 87 (GH87), hydrolyzes α-1,3-glucan with an endo-action. GH87 enzymes are known to degrade dental plaque produced by oral pathogenic Streptococcus species. In this study, the kinetic analyses revealed that this enzyme hydrolyzed α-1,3-tetraglucan into glucose and α-1,3-triglucan with ß-configuration at the reducing end by an inverting mechanism. The crystal structures of the catalytic domain (CatAgl-FH1) complexed with or without oligosaccharides at 1.4-2.5 or 1.6 Å resolutions, respectively, are also presented. The initial crystal structure of CatAgl-FH1 was determined by native single-wavelength anomalous diffraction. The catalytic domain was composed of two modules, a ß-sandwich fold module, and a right-handed ß-helix fold module. The structure of the ß-sandwich was similar to those of the carbohydrate-binding module family 35 members. The glycerol or nigerose enzyme complex structures demonstrated that this ß-sandwich fold module is a novel carbohydrate-binding module with the capabilities to bind saccharides and to promote the degradation of polysaccharides. The structures of the inactive mutant in complexes with oligosaccharide showed that at least eight subsites for glucose binding were located in the active cleft of the ß-helix fold and the architecture of the active cleft was suitable for the recognition and hydrolysis of α-1,3-glucan by the inverting mechanism. The structural similarity to GH28 and GH49 enzymes and the results of site-directed mutagenesis indicated that three Asp residues, Asp1045, Asp1068, and Asp1069, are the most likely candidates for the catalytic residues of Agl-FH1. DATABASE: Structural data are available in RCSB Protein Data Bank under the accession numbers 6K0M (CatAgl-FH1), 6K0N (WT/nigerose), 6K0P (D1045A/nigerose), 6K0Q (D1068A/nigerose), 6K0S (D1069A/ nigerose), 6K0U (D1068A/oligo), and 6K0V (D1069A/oligo). ENZYMES: Agl-FH1, α-1,3-glucanase (EC3.2.1.59) from Paenibacillus glycanilyticus FH11.

Crystal structure of the catalytic unit of GH 87-type α-1,3-glucanase Agl-KA from Bacillus circulans.

Glycoside hydrolase (GH) 87-type α-1,3-glucanase hydrolyses the α-1,3-glucoside linkages of α-1,3-glucan, which is found in fungal cell walls and extracellular polysaccharides produced by oral Streptococci. In this study, we report on the molecular structure of the catalytic unit of GH 87-type α-1,3-glucanase, Agl-KA, from Bacillus circulans, as determined by x-ray crystallography at a resolution of 1.82 Å. The catalytic unit constitutes a complex structure of two tandemly connected domains-the N-terminal galactose-binding-like domain and the C-terminal right-handed ß-helix domain. While the ß-helix domain is widely found among polysaccharide-processing enzymes, complex formation with the galactose-binding-like domain was observed for the first time. Biochemical assays showed that Asp1067, Asp1090 and Asp1091 are important for catalysis, and these residues are indeed located at the putative substrate-binding cleft, which forms a closed end and explains the product specificity.

Construction of Cellulose Binding Domain Fusion FMN-Dependent NADH-Azoreductase and Glucose 1-Dehydrogenase for the Development of Flow Injection Analysis with Fusion Enzymes Immobilized on Cellulose.

The cellulose binding domain (CBD) of cellulosome-integrating protein A from Clostridium thermocellum NBRC 103400 was genetically fused to FMN-dependent NADH-azoreductase (AZR) and glucose 1-dehydrogenase (GDH) from Bacillus subtilis. The fusion enzymes, AZR-CBD and CBD-GDH, were expressed in Escherichia coli Rosetta-gami B (DE3). The enzymes were purified from cell-free extracts, and the specific activity of AZR-CBD was 15.1 U/mg and that of CBD-GDH was 22.6 U/mg. AZR-CBD and CBD-GDH bound strongly to 0.5 % swollen cellulose at approximately 95 and 98 % of the initial protein amounts, respectively. After immobilization onto the swollen cellulose, AZR-CBD and CBD-GDH retained their catalytic activity. Both enzymes bound weakly to 0.5 % microcrystalline cellulose, but the addition of a high concentration of microcrystalline cellulose (10 %) improved the binding rate of both enzymes. A reactor for flow injection analysis was filled with microcrystalline cellulose-immobilized AZR-CBD and CBD-GDH. This flow injection analysis system was successfully applied for the determination of glucose, and a linear calibration curve was observed in the range of approximately 0.16-2.5 mM glucose, with a correlation coefficient, r, of 0.998.

Enzymatic and molecular characterization of α-1,3-glucanase (AglST2) from Streptomyces thermodiastaticus HF3-3 and its relation with α-1,3-glucanase HF65 (AglST1).

Extracellular α-1,3-glucanase HF90 (AglST2), with a sodium dodecyl sulfate (SDS)-PAGE-estimated molecular mass of approximately 91 kDa, was homogenously purified from the culture filtrate of Streptomyces thermodiastaticus HF3-3. AglST2 showed a high homology with mycodextranase in an amino acid sequence and demonstrated specificity with an α-1,3-glycosidic linkage of homo α-1,3-glucan. It has been suggested that AglST2 may be a new type of α-1,3-glucanase. The optimum pH and temperature of AglST2 were pH 5.5 and 60°C, respectively. AglST2 action was significantly stimulated in the presence of 5-20% (w/v) NaCl, and 1 mM metal ions Mn2+ and Co2+. On the other hand, it was inhibited by 1 mM of Ag+, Cu2+, Fe2+ and Ni2+. Regarding the stability properties, AglST2 retained more than 80% of its maximum activity over a pH range of 5.0-7.0 at up to 60°C and in the presence of 0-20% (w/v) NaCl. Based on these results, the properties of AglST2 were comparable with those of AglST1, which had been previously purified and characterized from S. thermodiastaticus HF3-3 previously. The N-terminal amino acid sequence of AglST2 showed a good agreement with that of AglST1, suggesting that AglST1 was generated from AglST2 by proteolysis during cultivation. MALDI-TOF mass analysis suggested that AglST1 might be generated from AglST2 by the proteolytic removal of C-terminus polypeptide (approximately 20 kDa). Our investigation thus revealed the properties of AglST2, such as tolerance against high temperature, salts, and surfactants, which have promising industrial applications.

Crystal structure analysis and enzymatic characterization of γ-glutamyltranspeptidase from Pseudomonas nitroreducens.

Theanine (γ-glutamylethylamide) is an amino acid analog that reduces blood pressure and improves immune responses. The ϒ-glutamyltranspeptidase (GGT) from Pseudomonas nitroreducens IFO12694 (PnGGT) has a unique preference for primary amines as ϒ-glutamyl acceptors over standard L-amino acids and peptides. This characteristic is useful for the synthesis of theanine. We used X-ray crystallographic analysis to understand the structural basis of PnGGT's hydrolysis and transpeptidation reactions and to characterize its previously unidentified acceptor site. Structural studies of PnGGT have shown that key interactions between three residues (Trp385, Phe417, and Trp525) distinguish PnGGT from other GGTs. We studied the roles of these residues in the distinct biochemical properties of PnGGT using site-directed mutagenesis. All mutants showed a significant decrease in hydrolysis activity and an increase in transpeptidase activity, suggesting that the aromatic side chains of Trp385, Phe417, and Trp525 were involved in the recognition of acceptor substrates. Abbreviations: ϒ-glutamyl peptide, theanine, X-ray crystallography.

X-ray crystallographic analysis of the catalytic domain of α-1,3-glucanase FH1 from Paenibacillus glycanilyticus overexpressed in Brevibacillus choshinensis.

α-1,3-Glucanase hydrolyzes α-1,3-glucan, an insoluble linear α-1,3-linked homopolymer of glucose that is found in the extracellular polysaccharides produced by oral streptococci in dental plaque and in fungal cell walls. This enzyme could be of application in dental care and the development of fungal cell-wall lytic enzymes, but its three-dimensional structure has not been available to date. In this study, the recombinant catalytic domain of α-1,3-glucanase FH1 from Paenibacillus glycanilyticus FH11, which is classified into glycoside hydrolase family 87, was prepared using a Brevibacillus choshinensis expression system and purified in a soluble form. Crystals of the purified protein were produced by the sitting-drop vapor-diffusion method. Diffraction data were collected to a resolution of 1.6 Šusing synchrotron radiation. The crystals obtained belonged to the tetragonal space group P41212 or P43212, with unit-cell parameters a = b = 132.6, c = 76.1 Å. The space group and unit-cell parameters suggest that there is one molecule in the asymmetric unit.

Characterization and Thermal Denaturation Kinetic Analysis of Recombinant l-Amino Acid Ester Hydrolase from Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia HS1 exhibits l-amino acid ester hydrolase (SmAEH) activity, which can synthesize dipeptides such as Ile-Trp, Val-Gly, and Trp-His from the corresponding amino acid methyl esters and amino acids. The gene encoding SmAEH was cloned and expressed in Escherichia coli and was purified and characterized. SmAEH shared 77% sequence identity with a known amino acid ester hydrolase (AEH) from Xanthomonas citri, which belongs to a class of ß-lactam antibiotic acylases. The thermal stability of SmAEH was evaluated using various mathematical models to assess its industrial potential. First-order kinetics provided the best description for the inactivation of the enzyme over a temperature range of 35-50 °C. Decimal reduction time ranged from 212.76 to 3.44 min, with a z value of 8.06 °C, and the deactivation energy was 204.1 kJ mol-1.

Deletion of uncharacterized domain from α-1,3-glucanase of Bacillus circulans KA-304 enhances heterologous enzyme production in Escherichia coli.

α-1,3-Glucanase (Agl-KA) of Bacillus circulans KA-304 consists of an N-terminal discoidin domain (DS1), a carbohydrate binding module family 6 (CBM6), threonine and proline repeats (TP), a second discoidin domain (DS2), an uncharacterized conserved domain (UCD), and a C-terminal catalytic domain. Previously, we reported that DS1, CBM6, and DS2 have α-1,3-glucan-binding activity and contribute to α-1,3-glucan hydrolysis. In this study, UCD deletion mutant (AglΔUCD) was constructed, and its properties were compared with those of Agl-KA. α-1,3-Glucan hydrolyzing, α-1,3-glucan binding, and protoplast-forming activities of AglΔUCD were almost the same as those of Agl-KA. kcat/Km values of AgΔUCD and Agl-KA were 11.4 and 11.1 s-1 mg-1 mL, respectively. AglΔUCD and Agl-KA exhibited similar characteristics, such as optimal pH, pH stability, optimal temperature, and thermostability. These results suggest that UCD is not α-1,3-glucan-binding and flexible linker domain, and that deletion of UCD does not affect the affinity of N-terminal binding domains and the catalytic action of the C-terminal domain. Subsequently, heterologous UCenzyme productivity of AglΔD in Escherichia coli was compared with that of Agl-KA. The productivity of AglΔUCD was about 4-fold larger than that of Agl-KA after an 8-h induction at 30°C. In the case of induction at 20°C, the productivity of AglΔUCD was also larger than that of Agl-KA. These findings indicate that deletion of only UCD enhances the enzyme productivity in E. coli.

Enzymatic and molecular characterization of an acidic and thermostable chitinase 1 from Streptomyces thermodiastaticus HF 3-3.

Chitinase 1 (Chi1) is an acidic and thermostable hydrolytic enzyme capable of the breakdown of chitin, a resilient biopolymer that is the primary building block of fungi cell walls and marine exoskeletons. In this study, Chi1 was purified from the bacterium Streptomyces thermodiastaticus HF 3-3, and its properties were carefully characterized. The molecular mass of Chi1 was estimated to be approximately 46 kDa and, through sequencing, its N-terminal amino acid sequence was identified as ADSGKVKL. Although the optimal operating temperature and pH for Chi1 were determined to be 65°C and pH 5.5, respectively, the purified enzyme was stable over wide pH (1.5-9) and temperature ranges. Moreover, Chi1 retained 87% of its activity in the presence of 15% NaCl. While Chi1 activity was inhibited by Ag+ and Mn2+, other chemicals tested had no significant effect on its enzymatic activity. The Km and Vmax values of Chi1 for the substrate colloidal chitin were 1.23 ± 0.7 mg/mL and 6.33 ± 1.0 U/mg, respectively. Thin-layer chromatography analysis of the enzymatic reaction end products mainly detected diacetylchitobiose. We also cloned the Chi1 gene and purified the recombinant protein; the properties of the recombinant enzyme were nearly identical to those of the native enzyme. Therefore, Chi1 purified from S. thermodiastaticus HF 3-3 is unique, as it is highly stable under broad range of pH values, temperatures, and chemical exposures. Combined, these properties make this enzyme attractive for use in the industrial bioconversion of chitin.