Cloning and functional characterization of a novel endo-β-1,4-glucanase gene from a soil-derived metagenomic library

A metagenomic library consisting of 3,024 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B with high-molecular-weight DNA extracted from red soil in South China. A novel cellulase gene with an open reading frame of 1,332 bp, cel5G, encoding an endo-β-1,4-glucanase was cloned using an activity-based screen. The deduced enzyme, Cel5G, belongs to the glycosyl hydrolase family 5 and shares <39% identity with endoglucanases in the GenBank database. cel5G was expressed in E. coli BL21, and the recombinant enzyme Cel5G was purified to homogeneity for enzymatic analysis. Cel5G hydrolyzed a wide range of β-1,4-, β-1,3/β-1,4-, or β-1,3/β-1,6-linked polysaccharides, amorphous cellulose, filter paper, and microcrystalline cellulose. Its highest activity was in 50 mM citrate buffer, pH 4.8, at 50°C. Cel5G is stable over a wide range of pH values (from 2.0 to 10.6) and is thermally stable under 60°C. It is highly tolerant and active in high salt concentrations and is stable in the presence of pepsin and pancreatin. The K _m and V _max values of Cel5G for carboxymethyl cellulose are 19.92 mg/ml and 1,941 μmol min⁻¹ mg⁻¹, respectively. These characteristics indicate that Cel5G has potential for industrial use.     

Screening,identification, and characterization of a novel saccharide-stimulated β-glycosidase from a soil metagenomic library

Applied Microbiology and Biotechnology - MeBglD2, a β-glycosidase that is highly activated in the presence of various monosaccharides and disaccharides, was isolated from a soil metagenomic...     

Cloning and functional characterization of endo-β-1,4-glucanase gene from metagenomic library of vermicompost

In the vermicomposting of paper mill sludge, the activity of earthworms is very dependent on dietetic polysaccharides including cellulose as energy sources. Most of these polymers are degraded by the host microbiota and considered potentially important source for cellulolytic enzymes. In the present study, a metagenomic library was constructed from vermicompost (VC) prepared with paper mill sludge and dairy sludge (fresh sludge, FS) and functionally screened for cellulolytic activities. Eighteen cellulase expressing clones were isolated from about 89,000 fosmid clones libraries. A short fragment library was constructed from the most active positive clone (cMGL504) and one open reading frame (ORF) of 1,092 bp encoding an endo-β-1,4-glucanase was indentified which showed 88% similarity with Cellvibrio mixtus cellulase A gene. The endo-β-1,4-glucanase cmgl504 gene was overexpressed in Escherichia coli. The purified recombinant cmgl504 cellulase displayed activities at a broad range of temperature (25–55°C) and pH (5.5–8.5). The enzyme degraded carboxymethyl cellulose (CMC) with 15.4 U, while having low activity against avicel. No detectable activity was found for xylan and laminarin. The enzyme activity was stimulated by potassium chloride. The deduced protein and three-dimensional structure of metagenome-derived cellulase cmgl504 possessed all features, including general architecture, signature motifs, and N-terminal signal peptide, followed by the catalytic domain of cellulase belonging to glycosyl hydrolase family 5 (GHF5). The cellulases cloned in this work may play important roles in the degradation of celluloses in vermicomposting process and could be exploited for industrial application in future.     

Identification, localization and characterization of the human γ-synuclein gene

We have identified and characterized a new member of the human synuclein gene family, γ-synuclein (SNCG). This gene is composed of five exons, which encode a 127 amino acid protein that is highly homologous to α-synuclein, which is mutated in some Parkinson’s disease families, and to β-synuclein. The γ-synuclein gene is localized to chromosome 10q23 and is principally expressed in the brain, particularly in the substantia nigra. We have determined its genomic sequence, and established conditions for sequence analysis of each of the exons. The γ-synuclein gene, also known as BCSG1, was recently found to be overexpressed in advanced infiltrating carcinoma of the breast. Our survey of the EST database indicated that it might also be overexpressed in an ovarian tumor. Received: 6 February 1998 / Accepted: 8 April 1998     

Engineering methionine γ-lyase from Citrobacter freundii for anticancer activity

Methionine deprivation of cancer cells, which are deficient in methionine biosynthesis, has been envisioned as a therapeutic strategy to reduce cancer cell viability. Methionine γ-lyase (MGL), an enzyme that degrades methionine, has been exploited to selectively remove the amino acid from cancer cell environment. In order to increase MGL catalytic activity, we performed sequence and structure conservation analysis of MGLs from various microorganisms. Whereas most of the residues in the active site and at the dimer interface were found to be conserved, residues located in the C-terminal flexible loop, forming a wall of the active site entry channel, were found to be variable. Therefore, we carried out site-saturation mutagenesis at four independent positions of the C-terminal flexible loop, P357, V358, P360 and A366 of MGL from Citrobacter freundii, generating libraries that were screened for activity. Among the active variants, V358Y exhibits a 1.9-fold increase in the catalytic rate and a 3-fold increase in K_M, resulting in a catalytic efficiency similar to wild type MGL. V358Y cytotoxic activity was assessed towards a panel of cancer and nonmalignant cell lines and found to exhibit IC₅₀ lower than the wild type. The comparison of the 3D-structure of V358Y MGL with other MGL available structures indicates that the C-terminal loop is either in an open or closed conformation that does not depend on the amino acid at position 358. Nevertheless, mutations at this position allosterically affects catalysis.     

Molecular cloning and characterization of a novel γ-CGTase from alkalophilic Bacillus sp.

We found a novel cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. G-825-6. The enzyme was expressed in the culture broth by recombinant Bacillus subtilis KN2 and was purified and characterized. The enzyme named CGTase825-6 showed 95% amino acid sequence identity with a known enzyme β-/γ-CGTase from Bacillus firmus/lentus 290-3. However, the product specificity of CGTase825-6 differed from that of β-/γ-CGTase. CGTase825-6 produced γ-cyclodextrin (CD) as the main product, but degradation of γ-CD was observed with prolonged reaction. The product specificity of the enzyme was positioned between γ-CGTase produced by Bacillus clarkii 7364 and B. firmus/lentus 290-3 β-/γ-CGTase. It showed that the difference of product specificity was dependent on only 28 amino acid residues in 671 residues in CGTase825-6. We compared the amino acid sequence of CGTase825-6 and those of other CGTases and constructed a protein structure model of CGTase825-6. The comparison suggested that the diminished loop (Val138-Asp142) should provide subsite -8 for γ-CD production and that Asp142 might have an important role in product specificity. CGTase825-6 should be a useful tool to produce γ-CD and to study the differences of producing mechanisms between γ-CD and β-CD.     

Identification and characterization of a novel antibacterial peptide,avian β-defensin 2 from ducks

In this study, a novel avian β-defensin (AvBD) was isolated from duck pancreas. The complete nucleotide sequence of the gene contained an 195 bp open reading frame encoding 64 amino acids. Homology, characterization and comparison of the gene with AvBD from other avian species confirmed that it was duck AvBD2. The mRNA expression of the gene was analyzed in 17 tissues from 21-day-old ducks. AvBD2 was highly expressed in the trachea, crop, heart, bone marrow, and pancreas; moderately expressed in the muscular stomach, small intestine, kidney, spleen, thymus, and bursa of Fabricius; and weakly expressed in skin. We produced and purified recombinant AvBD2 by expressing the gene in Escherichia coli. As expected, the recombinant peptide exhibited strong bactericidal properties against Bacillus cereus, Staphylococcus aureus, and Pasteurella multocida, and weak bactericidal properties against E. coli and Salmonella choleraesuis. In addition, the recombinant protein retained antimicrobial activity against S. aureus under different temperatures (range, −20°C to 100°C) and pH values (range, 3 to 12)     

A continuous spectrophotometric assay and nonlinear kinetic analysis of methionine γ-lyase catalysis

In this article, we present a new, easy-to-implement assay for methionine γ-lyase (MGL)-catalyzed γ-elimination reactions of l-methionine and its analogues that produce α-ketobutyrate (α-KB) as product. The assay employs ultraviolet–visible (UV–Vis) spectrophotometry to continuously monitor the rate of formation of α-KB by its absorbance at 315 nm. We also employ a nonlinear data analysis method that obviates the need for an “initial slope” determination, which can introduce errors when the progress curves are nonlinear. The spectrophotometric assay is validated through product analysis by ¹H NMR (nuclear magnetic resonance), which showed that under the conditions of study l-methionine (l-met) and l-methionine sulfone (l-met sulfone) substrates were converted to α-KB product with greater than 99% yield. Using this assay method, we determined for the first time the Michaelis–Menten parameters for a recombinant form of MGL from Porphyromonas gingivalis, obtaining respective k_cat and K_m values of 328 ± 8 min⁻¹ and 1.2 ± 0.1 mM for l-met γ-elimination and 2048 ± 59 min⁻¹ and 38 ± 2 mM for l-met sulfone γ-elimination reactions. We envisage that this assay method will be useful for determining the activity of MGL γ-elimination reactions that produce α-KB as the end product.     

Identification of a halophilic α-amylase gene from Escherichia coli JM109 and characterization of the recombinant enzyme

A halophilic α-amylase (EAMY) gene from Escherichia coli JM109 was overexpressed in E. coli XL10-Gold and the recombinant protein was purified and characterized. The activity of the EAMY depended on the presence of both Na⁺ and Cl^?, and had maximum activity in 2 M NaCl at 55 °C and pH 7.0. When 2 % (w/v) soluble starch was used as substrate, the specific activity was about 1,090 U mg^?1 protein. This is the first report on identifying a halophilic α-amylase with high specific activity from non-halophilic bacteria.     

Isolation and characterization of a novel <Emphasis Type="Italic">α</Emphasis>-amylase from a metagenomic library of Western Ghats of Kerala,India

In the present study, metagenomic library of Western Ghats soil sample was constructed in a fosmid vector (pCC1FOS) and screened for biocatalytic properties. The clones showed amylolytic activity on Luria-Bertani starch agar plates and one of them was studied in detail. The enzyme exhibited stability at elevated temperature with 60°C being the optimal temperature. The enzyme retained more than 30% activity after 60 min incubation at 80°C. It also showed more than 70% activity retention in 1.5 M NaCl solution. The pH optimum of the enzyme was at pH = 5.0. The enzyme possesses good activity in the presence of chelating and strong reducing agents with activity enhancements or retention being observed at 5 mM β-mercaptoethanol, dithiothreitol and N-bromosuccinimide. However, almost complete loss of activity was observed with 5 mM EDTA, while activity enhancement was observed upon incubation with Ca²⁺ suggesting it to be a Ca²⁺-dependent α-amylase, which was further confirmed by a thin-layer chromatography (TLC). The TLC run revealed that digestion pattern was similar to commercial α-amylase. The 16S rRNA gene sequence (GenBank accession number HQ680979) BLAST showed 95% similarities with Exiguobacterium sp. AFB-11 and AFB 18, with query sequence coverage of 99%.     

L-methionine γ-lyase from Citrobacter freundii: Cloning of the gene and kinetic parameters of the enzyme

It is shown for the first time for the Enterobacteriaceae family that a gene encoding L-methionine gamma-lyase (MGL) is present in the genome of Citrobacter freundii. Homogeneous enzyme has been purified from C. freundii cells and its N-terminal sequence has been determined. The hybrid plasmid pUCmgl obtained from the C. freundii genomic library contains an EcoRI insert of about 3000 bp, which ensures the appearance of MGL activity when expressed in Escherichia coli TG1 cells. The nucleotide sequence of the EcoRI fragment contains two open reading frames. The first frame (the megL gene) encodes a protein of 398 amino acid residues that has sequence homology with MGLs from different sources. The second frame encodes a protein with sequence homology with proteins belonging to the family of permeases. To overexpress the megL gene it was cloned into pET-15b vector. Recombinant enzyme has been purified and its kinetic parameters have been determined. It is demonstrated that a presence of a hybrid plasmid pUCmgl, containing the megL gene in the E. coli K12 cells, leads to a decrease in efficiency of EcoKI-restriction. It seems likely that decomposition of L-methionine under the action of MGL leads to a decrease in the intracellular content of S-adenosylmethionine. Expression of the megL gene in the C. freundii genome occurs only upon induction by a significant amount of L-methionine.     

Structural characterization and promoter activity analysis of the γ-kafirin gene from sorghum

A genomic clone encoding the γ-kafirin gene from sorghum was isolated and sequenced. A 2938 bp sequenced fragment includes an intronless open reading frame of 636 nucleotides encoding a putative polypeptide of 212 amino acids. Comparison of the deduced amino acid sequence of γ-kafirin with the published sequences of γ-prolamins of maize, and Coix revealed highly conserved domains. The N-terminal region of these proteins contains the conserved hexapeptide PPPVHL, which is repeated eight times in γ-zein, four times in γ-kafirin and three times in γ-coixin. The number of PPPVHL repeats accounts predominantly for the differences in the molecular weights of γ-prolamins. Several putative regulatory sequences common to the γ-kafirin and γ-zein genes were identified in both the 5′ and the 3′ flanking regions. Putative GCN4-like regulatory sequences were found at positions ?192 and ?476 in the 5′ flanking region of γ-kafirin. In the 3′ noncoding region, three putative polyadenylation signals, two AATAAT and one AATGAA, were found at positions + 658, + 716, and + 785, respectively. In order to investigate the role of the putative GCN4-like motifs and other possible cis-acting element(s) of the γ-kafirin promoter, a series of deleted and chimeric promoter constructs were introduced into maize, Coix and sorghum tissues by particle bombardment. Histochemical analysis of β-glucuronidase (GUS) activity in different tissues indicated that the element(s) responsible for tissue specificity is probably located in the 285-bp proximal region of the promoter, while the remaining promoter sequence seems to carry the element(s) responsible for the quantitative response.     

Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120

Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO₃ ^? dehydration and the subsequent fixation of CO₂. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein–protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k _cat of 2.0 × 10⁴ s^?1 and k _cat/K _m of 4.1 × 10⁶ M^?1 s^?1 at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25–35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO₂ analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes.     

Identification of a phosphofructokinase-encoding gene from Streptococcus thermophilus CNRZ1205--a novel link between carbon metabolism and gene regulation?

In order to isolate genes encoding so-called Two-Component Regulatory Systems from the lactic acid bacterium Streptococcus thermophilus, a cloning strategy was employed based on suppression of the alkaline phosphatase-negative phenotype displayed by the Escherichia coli strain ANCC22. Several suppressing clones were obtained which were shown to produce alkaline phosphatase activity. Sequence analysis of four of these clones revealed the presence of overlapping DNA inserts representing two ORFs, designated pfkT and pykT, whose deduced protein products exhibit significant similarity to phosphofructokinases and pyruvate kinases, respectively, from a variety of bacteria. A plasmid bearing pfkT was shown to complement a phosphofructokinase-negative mutant of E. coli, showing that this gene indeed specifies phosphofructokinase activity. It was shown that suppression of the alkaline phosphatase-negative phenotype of E. coli ANCC22 due to the presence of pfkT is caused by modulation of the intracellular level of acetyl phosphate.     

Identification and characterization of a novel alkaline α-amylase Amy703 belonging to a new clade from Bacillus pseudofirmus

Alkaline α-amylases are of great interest in desizing processes and detergent industries. Here, an alkaline α-amylase gene amy703 from an alkaliphilic Bacillus pseudofirmus strain was cloned and sequenced. Its encoding product, Amy703, might represent a new clade of α-amylase family, because it shared only 35 % highest identity with all amylases characterized up to date and was not clustered into any subfamilies with amylase activity in glycoside hydrolase family 13. Heterologous expression and characterization of Amy703 showed that it is a metalloenzyme with maximal activity at 40 °C and pH 9.0. Its activity was significantly enhanced by 2- and 2.48-fold at the presence of 10 mM Ca²⁺ and Mg²⁺, respectively, while Hg²⁺ was a strong inhibitor of Amy703. Amy703 has a higher affinity (K _m  = 3.92 mg/ml) for soluble starch compared to many other alkaline amylases. The computer modeling of its structure indicated that Amy703 contains typical amylase domains and a loop region appearing to bind the substrates. Site-directed mutagenesis suggested that a conserved residue Glu550 was essential for the activity of Amy703, and proposed it working together with other two residues to constitute a catalytic triad (Asp521, Glu550, and Asp615).