Characterisation of a novel Bacillus sp. SJ-10 β-1,3–1,4-glucanase isolated from jeotgal, a traditional Korean fermented fish

A novel β-1,3–1,4-glucanase gene was identified in Bacillus sp. SJ-10 (KCCM 90078) isolated from jeotgal, a traditional Korean fermented fish. We analysed the β-1,3–1,4-glucanase gene sequence and examined the recombinant enzyme. The open reading frame of the gene encoded 244 amino acids. The sequence was not identical to any β-glucanases deposited in GenBank. The gene was cloned into pET22b(+) and expressed in Escherichia coli BL21. Purification of recombinant β-1,3–1,4-glucanase was conducted by affinity chromatography using a Ni-NTA column. Enzyme specificity of β-1,3–1,4-glucanase was confirmed based on substrate specificity. The optimal temperature and pH of the purified enzyme towards barley β-glucan were 50 °C and pH 6, respectively. More than 80 % of activity was retained at temperatures of 30–70 °C and pH values of 4–9, which differed from all other bacterial β-1,3–1,4-glucanases. The degradation products of barley β-glucan by β-1,3–1,4-glucanase were analysed using thin-layer chromatography, and ultimately glucose was produced by treatment with cellobiase.     

Cloning and characterization of a new manganese superoxide dismutase from deep-sea thermophile Geobacillus sp. EPT3

A new gene encoding a superoxide dismutase (SOD) was identified from a thermophile Geobacillus sp. EPT3 isolated from a deep-sea hydrothermal field in east Pacific. The open reading frame of this gene encoded 437 amino acid residues. It was cloned, overexpressed in Escherichia coli (DE3), and the recombinant protein was purified to homogeneity. Geobacillus sp. EPT3 SOD was of the manganese-containing SOD type, as judged by the insensitivity of the recombinant enzyme to both KCN and H₂O₂, and the activity analysis of Fe or Mn reconstituted SODs by polyacrylamide gel electrophoresis. The recombinant SOD was determined to be a homodimer with monomeric molecular mass of 59.0 kDa. In comparison with other Mn–SODs, the manganese-binding sites are conserved in the sequence (His260, His308, Asp392, His396). The recombinant enzyme had high thermostability at 50 °C. It retained 57 % residual activity after incubation at 90 °C for 1 h, which indicated that this SOD was thermostable. The enzyme also showed striking stability over a wide range of pH 5.0–11.0. At tested conditions, the recombinant SOD from Geobacillus sp. EPT3 showed a relatively good tolerance to some inhibitors, detergents, and denaturants, such as β-mercaptoethanol, dithiothreitol, phenylmethylsulfonyl fluoride, Chaps, Triton X-100, urea, and guanidine hydrochloride.     

Purification, Characterization of GH11 Endo-β-1,4-xylanase from Thermotolerant Streptomyces sp. SWU10 and Overexpression in Pichia pastoris KM71H

We have previously described two forms of an endo-β-1,4-xylanase (XynSW2A and XynSW2B) synthesized by thermotolerant Streptomyces sp. SWU10. Here, we describe another xylanolytic enzyme, designated XynSW1. The enzyme was purified to homogeneity from 2 L of culture filtrate. Its apparent molecular mass was 24 kDa. The optimal pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was stable in a wide pH ranges (pH 1–11), more than 80 % of initial activity remained at pH 2–11 after 16 h of incubation at 4 °C and stable up to 50 °C for 1 h. Xylobiose and xylotriose were the major xylooligosaccharides released from oat spelt xylan by the action of XynSW1, indicating of endo-type xylanase. The complete xynSW1 gene contains 1,011 bp in length and encode a polypeptide of 336 with 41 amino acids of signal peptide. The amino acid sequence analysis revealed that it belongs to glycoside hydrolase family 11 (GH11). The mature xynSW1 gene without signal peptide sequence was overexpressed in Pichia pastoris KM71H. The recombinant XynSW1 protein showed higher molecular mass due to the differences in glycosylation levels at the six N-glycosylation sites in the amino acid sequence and exhibited better physicochemical properties than those of the native enzyme including higher optimal temperature (60 °C), and specific activity, but lower optimal pH (4.0). Because of their stability in a wide pH ranges, both of native and recombinant enzymes of XynSW1, may have potential application in several industries including food, textile, biofuel, and also waste treatment.     

Molecular and biochemical characterization of a new alkaline active multidomain xylanase from alkaline wastewater sludge

A xylanase gene, xyn-b39, coding for a multidomain glycoside hydrolase (GH) family 10 protein was cloned from the genomic DNA of the alkaline wastewater sludge of a paper mill. Its deduced amino acid sequence of 1,481 residues included two carbohydrate-binding modules (CBM) of family CBM_4_9, one catalytic domain of GH 10, one family 9 CBM and three S-layer homology (SLH) domains. xyn-b39 was expressed heterologously in Escherichia coli, and the recombinant enzyme was purified and characterized. Xyn-b39 exhibited maximum activity at pH 7.0 and 60 °C, and remained highly active under alkaline conditions (more than 80 % activity at pH 9.0 and 40 % activity at pH 10.0). The enzyme was thermostable at 55 °C, retaining more than 90 % of the initial activity after 2 h pre-incubation. Xyn-b39 had wide substrate specificity and hydrolyzed soluble substrates (birchwood xylan, beechwood xylan, oat spelt xylan, wheat arabinoxylan) and insoluble substrates (oat spelt xylan and wheat arabinoxylan). Hydrolysis product analysis indicated that Xyn-b39 was an endo-type xylanase. The K _m and V _max values of Xyn-b39 for birchwood xylan were 1.01 mg/mL and 73.53 U/min/mg, respectively. At the charge of 10 U/g reed pulp for 1 h, Xyn-b39 significantly reduced the Kappa number (P < 0.05) with low consumption of chlorine dioxide alone.     

Optimized expression of prolyl aminopeptidase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its characteristics after glycosylation

Prolyl aminopeptidases are specific exopeptidases that catalyze the hydrolysis of the N-terminus proline residue of peptides and proteins. In the present study, the prolyl aminopeptidase gene (pap) from Aspergillus oryzae JN-412 was optimized through the codon usage of Pichia pastoris. Both the native and optimized pap genes were inserted into the expression vector pPIC9 K and were successfully expressed in P. pastoris. Additionally, the activity of the intracellular enzyme expressed by the recombinant optimized pap gene reached 61.26 U mL^?1, an activity that is 2.1-fold higher than that of the native gene. The recombinant enzyme was purified by one-step elution through Ni-affinity chromatography. The optimal temperature and pH of the purified PAP were 60 °C and 7.5, respectively. Additionally, the recombinant PAP was recovered at a yield greater than 65 % at an extremely broad range of pH values from 6 to 10 after treatment at 50 °C for 6 h. The molecular weight of the recombinant PAP decreased from 50 kDa to 48 kDa after treatment with a deglycosylation enzyme, indicating that the recombinant PAP was completely glycosylated. The glycosylated PAP exhibited high thermo-stability. Half of the activity remained after incubation at 50 °C for 50 h, whereas the remaining activity of PAP expressed in E. coli was only 10 % after incubation at 50 °C for 1 h. PAP could be activated by the appropriate salt concentration and exhibited salt tolerance against NaCl at a concentration up to 5 mol L^?1.     

Characterization of ergothionase from Burkholderia sp. HME13 and its application to enzymatic quantification of ergothioneine

We identified ergothionase, which catalyzes conversion of ergothioneine to thiolurocanic acid and trimethylamine, in a newly isolated ergothioneine-utilizing strain, Burkholderia sp. HME13. The enzyme was purified and its N-terminal amino acid sequence was determined. Based on the amino acid sequence, the gene encoding the enzyme was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity and characterized. The enzyme consisted of four identical 55-kDa subunits. The enzyme showed maximum activity at pH 8.0 and 65 °C and was stable between pH 7.0 and pH 10.0 and up to 60 °C. The enzyme acted on ergothioneine (K _m: 19 μM, V _max: 270 μmol/min/mg), but not d-histidine, l-histidine, d-tyrosine, l-tyrosine, d-phenylalanine, or l-phenylalanine. The enzyme was activated by BaCl₂ and strongly inhibited by CuSO₄, ZnSO₄, and HgCl₂. The amino acid sequence of ergothionase showed 23 % similarity to histidine ammonia-lyase (HAL) from Pseudomonas putida and 17 % similarity to phenylalanine ammonia-lyase (PAL) from parsley. However, the tripeptide sequence, Ala-Ser-Gly, which is important for catalysis in both HAL and PAL, was not conserved in ergothionase. The application of ergothionase for the quantification of ergothioneine contained in practical food and blood samples was investigated by performing a recovery test. Satisfactory recovery data (98.7–104 %) were obtained when ergothioneine was added to extract of tamogitake and hemolysis blood.     

Overproduction, purification, and characterization of extracellular lipoxygenase of Pseudomonas aeruginosa in Escherichia coli

Lipoxygenase (LOX; EC 1.13.11.12,) is an enzyme that is widely used in food industry to improve aroma, rheological, or baking properties of foods. In this study, we described the expression and characterization of Pseudomonas aeruginosa LOX in Escherichia coli. The recombinant LOX was successfully expressed and secreted by E. coli using its endogenous signal peptide. When induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (final concentration) at 20 °C for 47 h, the titer of the recombinant enzyme reached 3.89 U/mL. In order to characterize the catalytic properties, the recombinant LOX was purified to homogeneity on Q High Performance and Mono Q5/50GL sequentially. The molecular weight of the LOX was estimated as 70 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The K _m and V _max of the recombinant enzyme were 48.9 μM and 0.226 μmol/min, respectively. The purified enzyme exhibited a maximum activity at 25 °C and pH 7.5. High-performance liquid chromatography analysis of the linoleic acid hydroperoxides produced by recombinant LOX revealed that the LOX from P. aeruginosa falls into linoleic acid 13(S)-LOX. To the best of our knowledge, this is the first report on the overexpression of extracellular LOX in microorganisms, and the achieved LOX yield is the highest ever reported.     

Purification,characterization, and overexpression of an endo-1,4-β-mannanase from thermotolerant <Emphasis Type="Italic">Bacillus</Emphasis> sp. SWU60

Endo-β-1,4-mannanases are important catalytic agents in several industries. The enzymes randomly cleave the β-1,4-linkage in the mannan backbone and release short β-1,4-mannooligosaccharides and mannose. In the present study, mannanase (ManS2) from thermotolerant Bacillus sp. SWU60 was purified, characterized, and its gene was cloned and overexpressed in Escherichia coli. ManS2 was purified from culture filtrate (300 ml) by using hydrophobic, ion-exchange, and size-exclusive liquid chromatography. The apparent molecular mass was 38 kDa. Optimal pH and temperature for enzyme activity were 6.0 and 60?°C, respectively. The enzyme was stable up to 60?°C for 1 h and at pH 5–9 at 4?°C for 16 h. Its enzyme activity was inhibited by Hg²⁺. The full-length mans2 gene was 1,008 bp, encoding a protein of 336 amino acids. Amino acid sequence analysis revealed that it belonged to glycoside hydrolase family 26. Konjac glucomannan was a favorable substrate for recombinant ManS2 (rManS2). rManS2 also degraded galactomannan from locust bean gum, indicating its potential for production of glucomanno- and galactomanno-oligosaccharides. Both native and recombinant ManS2 from Bacillus sp. SWU60 can be applied in several industries especially food and feed.     

Biochemical characterization and high-level production of oxidized polyvinyl alcohol hydrolase from Sphingopyxis sp. 113P3 expressed in methylotrophic Pichia pastoris

The Sphingopyxis sp. 113P3 gene oph, encoding oxidized polyvinyl alcohol hydrolase (OPH), was optimized with the preferred codons of Pichia pastoris and ligated into the pPIC9K vector behind the α-factor signal sequence. The vector was then transfected into P. pastoris GS115 and genomic integration was confirmed. Large-scale production of recombinant protein was performed by induction with 14.4 g/L methanol at 22 °C in a 3-L bioreactor. The maximal OPH activity obtained was 68.4 U/mL, which is the highest activity reported. The optimal pH and temperature of recombinant OPH were 8.0 and 45 °C, respectively. OPH activity was stable over a pH range of 5.0–8.5, and at a maximal temperature of 45 °C. The K _cat /K _m of recombinant OPH was 598 mM^?1 s^?1, which was 4.27-fold higher than that of recombinant OPH derived from Escherichia coli. The improved catalytic efficiency of OPH expressed in recombinant P. pastoris makes it favorable for industrial applications.     

Gene cloning and soluble expression of Aspergillus niger phytase in E. coli cytosol via chaperone co-expression

A phytase gene from Aspergillus niger was isolated and two Escherichia coli expression systems, based on T7 RNA polymerase promoter and tac promoter, were used for its recombinant expression. Co-expression of molecular chaperone, GroES/EL, aided functional cytosolic expression of the phytase in E. coli BL21 (DE3). Untagged and maltose-binding protein-tagged recombinant phytase showed an activity band of ~49 and 92 kDa, respectively, on a zymogram. Heterologously-expressed phytase was fractionated from endogenous E. coli phytase by (NH₄)₂SO₄ precipitation. The enzyme had optimum activity at 50 °C and pH 6.5.     

Expression of a keratinase (kerA) gene from Bacillus licheniformis in Escherichia coli and characterization of the recombinant enzymes

The gene kerA (1,047 bp) encoding the main keratinase from Bacillus licheniformis was cloned into two conventional vectors, pET30α and pET32α, and expressed in Escherichia coli. From SDS-PAGE analysis, the recombinant keratinases were 45 and 55 kDa. They had different optimal pH values (7.5 and 8.5) but the same optimum temperature of 50 °C. The recombinant keratinase produced in E. coli pET30α-kerA was more stable than that produced in E. coli pET32α-kerA, and retained approx. 70 % of its total enzyme activity after 30 min at 70 °C.     

A novel cold-adapted β-galactosidase isolated from Halomonas sp. S62: gene cloning, purification and enzymatic characterization

A novel 1,170 bp β-galactosidase gene sequence from Halomonas sp. S62 (BGalH) was identified through whole genome sequencing and was submitted to GenBank (Accession No. JQ337961). The BGalH gene was heterologously expressed in Escherichia coli BL21(DE3) cells, and the enzymatic properties of recombinant BGalH were studied. According to the polyacrylamide gel electrophoresis results and the sequence alignment analysis, BGalH is a dimeric protein and cannot be classified into one of the known β-galactosidase families (GH1, GH2, GH35, GH42). The optimal pH and temperature were determined to be 7.0 and 45 °C, respectively; the K _m and K _cat were 2.9 mM and 390.3 s^?1, respectively, for the reaction with the substrate ortho-nitrophenyl-β-d-galactopyranoside. At 0–20 °C, BGalH exhibited 50–70 % activity relative to its activity under the optimal conditions. BGalH was stable over a wide range of pHs (6.0–8.5) after a 1 h incubation (>93 % relative activity) and was thermostable at 50 °C and below (>60 % relative activity). The enzyme hydrolyzes lactose completely in milk over 24 h at 7 °C. The characteristics of this novel β-galactosidase suggest that BGalH may be a good candidate for medical researches and food industry applications.     

Molecular cloning, characterization, and heterologous expression of a new κ-carrageenase gene from marine bacterium Zobellia sp. ZM-2

κ-Carrageenases exhibit apparent distinctions in gene sequence, molecular weight, enzyme properties, and posttranslational processes. In this study, a new κ-carrageenase gene named cgkZ was cloned from the marine bacterium Zobellia sp. ZM-2. The gene comprised an open reading frame of 1,638 bp and encoded 545 amino acids. The natural signal peptide of κ-carrageenase was used successfully for the secretory production of the recombinant enzyme in Escherichia coli. A posttranslational process that removes an amino acid sequence of about 20 kDa from the C-terminal end of κ-carrageenase was first discovered in E. coli. An increase in enzyme activity by 167.3 % in the presence of 5 mM DTT was discovered, and Na⁺ at a certain concentration range was positively correlated with enzyme activity. The κ-carrageenase production of E. coli was 9.0 times higher than that of ZM-2. These results indicate the potential use of the enzyme in the biotechnological industry.     

Cold-Adapted RTX Lipase from Antarctic Pseudomonas sp. Strain AMS8: Isolation, Molecular Modeling and Heterologous Expression

A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S²⁰⁷, D²⁵⁵ and H³¹³, based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 °C and retained almost 50 % of its activity at 10 °C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 °C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5.     

Recombinant expression and characterization of a novel endoglucanase from Bacillus subtilis in Escherichia coli

The goal of this work was to produce high levels of endoglucanase in Escherichia coli for its potential usage in different industrial applications. Endoglucanase gene was amplified from genomic DNA of Bacillus subtilis JS2004 by PCR. The isolated putative endoglucanase gene consisted of an open reading frame of 1,701 nucleotides and encoded a protein of 567 amino acids with a molecular mass of 63-kDa. The gene was cloned into pET-28a(+) and expressed in E. coli BL21 (DE3). Optimum temperature and pH of the recombinant endoglucanase were 50 °C and 9, respectively which makes it very attractive for using in bio-bleaching and pulp industry. It had a K _M of 1.76 μmol and V _max 0.20 μmol/min with carboxymethylcellulose as substrate. The activity of recombinant endoglucanse was enhanced by Mg²⁺, Ca²⁺, isopropanol and Tween 20 and inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Ni²⁺ and SDS. The activity of this recombinant endoglucanase was significantly higher than wild type. Therefore, this recombinant enzyme has potential for many industrial applications involving biomass conversions, due to characteristic of broad pH and higher temperature stability.     

Expression and Biochemical Characterization of a Thermophilic Organic Solvent-Tolerant Lipase from Bacillus sp. DR90

The objective of the present study was the isolation, molecular cloning and biochemical characterization of a thermophilic organic solvent-resistant lipase from Bacillus sp. DR90. The lipase gene was expressed in Escherichia coli BL21(DE3) using pET-28a(+) vector. The purification of recombinant lipase was conducted by nickel affinity chromatography and its biochemical properties were determined. The lipase sequence with an ORF of 639 bp contains the conserved pentapeptide Ala-His-Ser-Met-Gly. His-tagged recombinant lipase had a specific activity of 1,126 U/mg with a molecular mass of 26.8 kDa. The cloned lipase was optimally active at pH 8.0 and 75 °C representing high stability in broad ranges of temperature and pH. High performance liquid chromatography was used to determine the major compounds released during the lipase-catalyzed reaction of p-nitrophenyl derivatives as well as the substrate specificity. The purified lipase showed high compatibility towards various organic solvents, surfactants and commercial solid/liquid detergents; therefore the recombinant DR90 lipase could be considered as a probable candidate for future applications, predominantly in detergent processing industries.     

Molecular Cloning and Characterization of the Gene Coding for the Aerobic Azoreductase from Xenophilus azovorans KF46F

The gene coding for an aerobic azoreductase was cloned from Xenophilus azovorans KF46F (formerly Pseudomonas sp. strain KF46F), which was previously shown to grow with the carboxylated azo compound 1-(4′-carboxyphenylazo)-2-naphthol (carboxy-Orange II) as the sole source of carbon and energy. The deduced amino acid sequence encoded a protein with a molecular weight of 30,278 and showed no significant homology to amino acid sequences currently deposited at the relevant data bases. A presumed NAD(P)H-binding site was identified in the amino-terminal region of the azoreductase. The enzyme was heterologously expressed in Escherichia coli and the azoreductase activities of resting cells and cell extracts were compared. The results suggested that whole cells of the recombinant E. coli strains were unable to take up sulfonated azo dyes and therefore did not show in vivo azoreductase activity. The turnover of several industrially relevant azo dyes by cell extracts from the recombinant E. coli strain was demonstrated.     

Purification and characterization of a Bacillus circulans No. 4.1 chitinase expressed in Escherichia coli

Summary A DNA fragment encoding for 598 amino acids of chitinase protein from Bacillus circulans No. 4.1 was subcloned into pQE-30 expression vector and transformed into Escherichia coli M15 (pREP4). The molecular weight of the expressed protein was approximately 66 kDa. Enzymatic activity of the recombinant protein was assayed after purification using affinity chromatography on a nickel chelating resin. The enzyme hydrolyzed N-acetylchitooligosaccharides mainly to N-acetylchitobiose, and was active toward chitin, carboxymethyl-chitin, colloidal chitin, glycol chitin and 4-methylumbelliferyl-β-d-N, N′-diacetylchitobiose. The pH and temperature optima of the chitinase enzyme were 7.0 and 45 °C, respectively. This enzyme was stable in the pH range of 5.0–9.0 and at temperatures up to 50 °C. In addition, when cleaved by a proteolytic enzyme, the 20-kDa product could retain high chitinolytic activity.     

Cloning of Catechol 2,3-Dioxygenase Gene and Construction of a Stable Genetically Engineered Strain for Degrading Crude Oil

Pseudomonas putida strain BNF1 was isolated to degrade aromatic hydrocarbons efficiently and use phenol as a main carbon and energy source to support its growth. Catechol 2,3-dioxygenase was found to be the responsible key enzyme for the biodegradation of aromatic hydrocarbons. Catechol 2,3-dioxygenase gene was cloned from plasmid DNA of P. putida strain BNF1. The nucleotide base sequence of a 924 bp segment encoding the catechol 2,3-dioxygenase (C23O) was determined. This segment showed an open reading frame, which encoded a polypeptide of 307 amino acids. C23O gene was inserted into NotI-cut transposon vector pUT/mini-Tn5 (Km^r) to get a novel transposon vector pUT/mini-Tn5-C23O. With the helper plasmid PRK2013, the transposon vector pUT/mini-Tn5-C23O was introduced into one alkanes degrading strain Acinetobacter sp. BS3 by triparental conjugation, and then the C23O gene was integrated into the chromosome of Acinetobacter sp. BS3. And the recombinant BS3-C23O, which could express catechol 2,3-dioxygenase protein, was obtained. The recombinant BS3-C23O was able to degrade various aromatic hydrocarbons and n-alkanes. Broad substrate specificity, high enzyme activity, and the favorable stability suggest that the BS3-C23O was a potential candidate used for the biodegradation of crude oil.