Cloning, overexpression, and characterization of a novel thermostable penicillin G acylase from Achromobacter xylosoxidans: probing the molecular basis for its high thermostability

The gene encoding a novel penicillin G acylase (PGA), designated pgaW, was cloned from Achromobacter xylosoxidans and overexpressed in Escherichia coli. The pgaW gene contains an open reading frame of 2586 nucleotides. The deduced protein sequence encoded by pgaW has about 50% amino acid identity to several well-characterized PGAs, including those of Providencia rettgeri, Kluyvera cryocrescens, and Escherichia coli. Biochemical studies showed that the optimal temperature for this novel PGA (PGA650) activity is greater than 60 degrees C and its half-life of inactivation at 55 degrees C is four times longer than that of another previously reported thermostable PGA from Alcaligenes faecalis (R. M. D. Verhaert, A. M. Riemens, J. V. R. Laan, J. V. Duin, and W. J. Quax, Appl. Environ. Microbiol. 63:3412-3418, 1997). To our knowledge, this is the most thermostable PGA ever characterized. To explore the molecular basis of the higher thermostability of PGA650, homology structural modeling and amino acid composition analyses were performed. The results suggested that the increased number of buried ion pair networks, lower N and Q contents, excessive arginine residues, and remarkably high content of proline residues in the structure of PGA650 could contribute to its high thermostability. The unique characteristic of higher thermostability of this novel PGA provides some advantages for its potential application in industry.     

Construction, Expression, and Characterization of a Thermostable Xylanase

A hybrid gene, btx, encoding a thermostable xylanase, Btx, was constructed by substituting the 31 N-terminal amino acid residues of the Thermomonospora fusca xylanase A (TfxA) for the corresponding region of 22 amino acid residues of the Bacillus subtilis xylanase A (BsxA). The btx gene was expressed in Escherichia coli BL21. The halo size produced by xylanase Btx on a Remanzol brilliant blue R (RBB) xylan plate at 60°C and pH 6.0 was larger than those of BsxA and TfxA. The molecular weight of Btx was 22 kDa. Temperature and pH optima for Btx were at 50–60°C and 6.0, respectively. Btx showed activity over 80% over a pH range of 5.0–9.0, which was wider than that of BsxA, and was also more acid-resistant than TfxA. Btx exhibited significant thermostability compared with BsxA. The results show the importance of the N-terminal sequence of TfxA in thermostability.     

Cloning, expression and characterization of xylose isomerase, XylA, from Caldanaerobacter subterraneus subsp. yonseiensis

The xylA gene, coding for xylose isomerase, from the extreme thermophile, Caldanaerobacter subterraneus subsp. yonseiensis was cloned, sequenced, and expressed in Escherichia coli. The nucleotide sequence of the xylA gene encoded a polypeptide of 438 residues with a calculated molecular weight of 50,170 Da. The purified XylA showed high sequence homology (92% identity) with that of Thermoanaerobacter thermohydrosulfuricus. The recombinant enzyme expressed in Escherichia coli was purified by heat treatment and gel chromatography. The purified enzyme was thermostable with optimal activity at 95°C. The enzyme required divalent cations including Zn²⁺ for its maximal activity and thermostability.     

A novel thermostable arylesterase from the archaeon Sulfolobus solfataricus P1: purification, characterization, and expression

A novel thermostable arylesterase, a 35-kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 94°C and 7.0, respectively. The enzyme displayed remarkable thermostability: it retained 52% of its activity after 50 h of incubation at 90°C. In addition, the purified enzyme showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity besides showing an arylesterase activity toward aromatic esters: it exhibits not only carboxylesterase activity toward tributyrin and p-nitrophenyl esters containing unsubstituted fatty acids from butyrate (C₄) to palmitate (C₁₆), but also paraoxonase activity toward organophosphates such as p-nitrophenylphosphate, paraoxon, and methylparaoxon. The k_cat/K_m ratios of the enzyme for phenyl acetate and paraoxon, the two most preferable substrates among all tested, were 30.6 and 119.4 s⁻¹·μM⁻¹, respectively. The arylesterase gene consists of 918 bp corresponding to 306 amino acid residues. The deduced amino acid sequence shares 34% identity with that of arylesterase from Acinetobacter sp. strain ADP1. Furthermore, we successfully expressed active recombinant S. solfataricus arylesterase in Escherichia coli. Together, our results show that the enzyme is a serine esterase belonging to the A-esterases and contains a catalytic triad composed of Ser156, Asp251, and His281 in the active site.     

Improved Thermostability and Acetic Acid Tolerance of Escherichia coli via Directed Evolution of Homoserine o-Succinyltransferase

In Escherichia coli, growth is limited at elevated temperatures mainly because of the instability of a single enzyme, homoserine o-succinyltransferase (MetA), the first enzyme in the methionine biosynthesis pathway. The metA gene from the thermophile Geobacillus kaustophilus cloned into the E. coli chromosome was found to enhance the growth of the host strain at elevated temperature (44°C), thus confirming the limited growth of E. coli due to MetA instability. In order to improve E. coli growth at higher temperatures, we used random mutagenesis to obtain a thermostable MetA_{E. coli} protein. Sequencing of the thermotolerant mutant showed five amino acid substitutions: S61T, E213V, I229T, N267D, and N271K. An E. coli strain with the mutated metA gene chromosomally inserted showed accelerated growth over a temperature range of 34 to 44°C. We used the site-directed metA mutants to identify two amino acid residues responsible for the sensitivity of MetA_{E. coli} to both heat and acids. Replacement of isoleucine 229 with threonine and asparagine 267 with aspartic acid stabilized the protein. The thermostable MetA_{E. coli} enzymes showed less aggregation in vivo at higher temperature, as well as upon acetic acid treatment. The data presented here are the first to show improved E. coli growth at higher temperatures solely due to MetA stabilization and provide new knowledge for designing E. coli strains that grow at higher temperatures, thus reducing the cooling cost of bioprocesses.     

Directed Evolution of Thermus Maltogenic Amylase toward Enhanced Thermal Resistance

The thermostability of maltogenic amylase from Thermus sp. strain IM6501 (ThMA) was improved greatly by random mutagenesis using DNA shuffling. Four rounds of DNA shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme ThMA-DM, which had a total of seven individual mutations. The seven amino acid substitutions in ThMA-DM were identified as R26Q, S169N, I333V, M375T, A398V, Q411L, and P453L. The optimal reaction temperature of the recombinant enzyme was 75°C, which was 15°C higher than that of wild-type ThMA, and the melting temperature, as determined by differential scanning calorimetry, was increased by 10.9°C. The half-life of ThMA-DM was 172 min at 80°C, a temperature at which wild-type ThMA was completely inactivated in less than 1 min. Six mutations that were generated during the evolutionary process did not significantly affect the specific activity of the enzyme, while the M375T mutation decreased activity to 23% of the wild-type level. The molecular interactions of the seven mutant residues that contributed to the increased thermostability of the mutant enzyme with other adjacent residues were examined by comparing the modeled tertiary structure of ThMA-DM with those of wild-type ThMA and related enzymes. The A398V and Q411L substitutions appeared to stabilize the enzyme by enhancing the interdomain hydrophobic interactions. The R26Q and P453L substitutions led potentially to the formation of genuine hydrogen bonds. M375T, which was located near the active site of ThMA, probably caused a conformational or dynamic change that enhanced thermostability but reduced the specific activity of the enzyme.     

AppA C-terminal Plays an Important Role in its Thermostability in Escherichia coli

Due to our previous research, mainly the thermostable mutants Q307D, Y311K, and I427L, we conjectured that Escherichia coli AppA phytase’s C-terminal plays an important role in its thermostability, and AppA begins to collapse from the C-terminal when at a higher temperature. So here we constructed C-lose mutant to prove it. The residual activities of the wild-type AppA phytase and C-lose were 31.42 and 70.49 %, respectively, after being heated at 80 °C for 10 min. The C-terminal deletion mutant C-lose showed 39.07 % thermostability enhancement than the wild-type both without the pH and temperature optimum changed. It proved the C-lose plays a key role in E. coli AppA phytase’s thermostability.     

First Thermostable Endo-β-1,4-Glucanase from Newly Isolated Xanthomonas sp. EC102

A novel gene encoding thermostable endoglucanase was identified in Xanthomonas sp. EC102 from soil. The gene had 1,458 base pairs of open reading frame, which encode a 52-kDa protein of 486 amino acid residues. Sequence of the amino acid residues was similar with the endoglucanase from Xanthomonas campestris pv. campestris ATCC33913 (GenBank Accession No. NP_638867.1) (94 % identity). The endoglucanase was overexpressed in Escherichia coli BL21 and purified. Temperature for the highest enzymatic activity was 70 °C and pH optima was pH 5.5. The specific activity of the endoglucanase toward carboxymethylcellulose (CMC) was approximately 2 μmol min^?1 mg^?1, V _max for CMC was 1.44 μmol mg^?1 min^?1, and K _m values was 25.6 mg mL^?1. The EC102 endoglucanase was stable at temperatures up to 60 °C, and it was activated by 0.1 mM of Mn²⁺ and Co²⁺. This is the first report about thermostable endoglucanase from Xanthomonas sp.     

Expression of Soluble Active Interferon αA in <Emphasis Type="Italic">Escherichia coli</Emphasis> Periplasm by Fusion with Thermostable Lichenase Using the Domain Insertion Approach

A recombinant DNA in which the interferon αA (IFN–αA) gene sequence is integrated into a loop region of the gene coding thermostable lichenase was constructed. This approach of insertion fusion with thermostable lichenase is advantageous in terms of increasing the solubility, stability, and production of the fusion partner in soluble form in general and in the periplasm of bacterial cells in particular. Thus, the insertion of IFN–αA into the loop (53 a.a.) of thermostable lichenase from Clostridium thermocellum resulted in effective expression of the soluble form of the recombinant protein in the periplasm of Escherichia coli without any compromise in biological activity of IFN–αA, while the thermostable lichenase retained its ability for functional folding without dramatic loss of its basic activity and thermostability.     

Residue Tyr224 is critical for the thermostability of Geobacillus sp. RD-2 lipase

A thermophilic lipase (lipGRD) from Geobacillus sp. RD-2, isolated from a hot spring in Yunnan, China, was cloned and over-expressed in Escherichia coli. The function of the conserved residue, Tyr224, near the presumed temperature switch site was analyzed by site-directed saturation mutagenesis. The activity of the wild type lipGRD was optimal at 55??C and pH?7.5, but that from mutant Y224C was optimally active at 35??C, whereas Y224P lipase was optimally active at 65??C. Furthermore, the latter lipase retained 60% of its activity after incubation at 65??C for 5?h. The conserved residue Tyr224, which is close to the lid helix, is the key amino acid residue determining the thermostability of the thermostable lipase.     

Adopting Selected Hydrogen Bonding and Ionic Interactions from Aspergillus fumigatus Phytase Structure Improves the Thermostability of Aspergillus niger PhyA Phytase

Although it has been widely used as a feed supplement to reduce manure phosphorus pollution of swine and poultry, Aspergillus niger PhyA phytase is unable to withstand heat inactivation during feed pelleting. Crystal structure comparisons with its close homolog, the thermostable Aspergillus fumigatus phytase (Afp), suggest associations of thermostability with several key residues (E35, S42, R168, and R248) that form a hydrogen bond network in the E35-to-S42 region and ionic interactions between R168 and D161 and between R248 and D244. In this study, loss-of-function mutations (E35A, R168A, and R248A) were introduced singularly or in combination into seven mutants of Afp. All seven mutants displayed decreases in thermostability, with the highest loss (25% [P < 0.05]) in the triple mutant (E35A R168A R248A). Subsequently, a set of corresponding substitutions were introduced into nine mutants of PhyA to strengthen the hydrogen bonding and ionic interactions. While four mutants showed improved thermostability, the best response came from the quadruple mutant (A58E P65S Q191R T271R), which retained 20% greater (P < 0.05) activity after being heated at 80°C for 10 min and had a 7°C higher melting temperature than that of wild-type PhyA. This study demonstrates the functional importance of the hydrogen bond network and ionic interaction in supporting the high thermostability of Afp and the feasibility of adopting these structural units to improve the thermostability of a homologous PhyA phytase.     

Molecular cloning,nucleotide sequence and expression of the structural gene for a thermostable alkaline protease from Bacillus sp. no. AH-101

Summary Alkaliphilic Bacillus sp. no. AH-101 produces an extremely thermostable alkaline serine protease that has a high optimum pH (pH 12–13) and shows keratinolytic activity. The gene encoding this protease was cloned in Escherichia coli and expressed in B. subtilis. The cloned protease was identical to the AH-101 protease in its optimum pH and thermostability at high alkaline pH. An open reading frame of 1083 bases, identified as the protease gene, was preceded by a putative Shine-Dalgarno sequence (AAAGGAGG) with a spacing of 11 bases. The deduced amino acid sequence revealed a pre-pro-peptide of 93 residues followed by the mature protease comprising 268 residues. AH-101 protease showed slightly higher homology to alkaline proteases from alkaliphilic bacilli (61.2% and 65.3%) than to those from neutrophilic bacilli (54.9–56.7%). Also AH-101 protease and other proteases from alkaliphilic bacilli shared common amino acid changes and a four amino acid deletion when compared to the proteases from neutrophilic bacilli. AH-101 protease, however, was distinct among the proteases from alkaliphilic bacilli in showing the lowest homology to the others.Correspondence to: H. Takami     

Cell surface of the fish pathogenic bacterium Aeromonas salmonicida: II. Purification and characterization of a major cell envelope protein related to autoagglutination,adhesion and virulence

The purification of the major protein of the membrane fraction of an autoagglutinating strain of Aeromonas salmonicida is described. This protein, designated as additional cell envelope protein, is water-insoluble, has a molecular weight of about 54 000 and its amino terminal sequence is H₂N-Asp-Val-Leu-Leu. Neither sulphur-containing amino acids nor sugar residues were detected. Its amino acid composition, which shows that the additional cell envelope protein is hydrophobic in nature, is remarkably similar to those of various proteins known to be present in additional surface layers of other bacteria, to the adhesive K88 fimbriae of enteropathogenic Escherichia coli and to a pore protein of the outer membrane of E. coli K12.     

Improving the Thermostability and Activity of a Thermophilic Subtilase by Incorporating Structural Elements of Its Psychrophilic Counterpart

The incorporation of the structural elements of thermostable enzymes into their less stable counterparts is generally used to improve enzyme thermostability. However, the process of engineering enzymes with both high thermostability and high activity remains an important challenge. Here, we report that the thermostability and activity of a thermophilic subtilase were simultaneously improved by incorporating structural elements of a psychrophilic subtilase. There were 64 variable regions/residues (VRs) in the alignment of the thermophilic WF146 protease, mesophilic sphericase, and psychrophilic S41. The WF146 protease was subjected to systematic mutagenesis, in which each of its VRs was replaced with those from S41 and sphericase. After successive rounds of combination and screening, we constructed the variant PBL5X with eight amino acid residues from S41. The half-life of PBL5X at 85°C (57.1 min) was approximately 9-fold longer than that of the wild-type (WT) WF146 protease (6.3 min). The substitutions also led to an increase in the apparent thermal denaturation midpoint temperature (T_m) of the enzyme by 5.5°C, as determined by differential scanning calorimetry. Compared to the WT, PBL5X exhibited high caseinolytic activity (25 to 95°C) and high values of K_m and k_cat (25 to 80°C). Our study may provide a rational basis for developing highly stable and active enzymes, which are highly desired in industrial applications.     

Residues affecting hydrolysis of soy isoflavone glycosides, stability and catalytic properties of Thermotoga maritima β-glucosidase

The thermostable β-glucosidase A (TmBglA) from Thermotoga maritima is a promising biocatalyst for production of isoflavone aglycones. Use of enzymes with high specificity for soy isoflavone conjugates is however essential for efficient hydrolysis. The effect of the amino acids located in the aglycone binding pocket with non-conserved residues between specificity groups in family 1 glycoside hydrolase (GH1) was studied using wild-type TmBglA and 3 exchange mutants (M1-TmBglA, M2-TmBglA, M1M2-TmBglA). Three mutants were expressed in Escherichia coli, purified and characterized. They had shifts in both optimum temperature and thermal stability, and their narrowing pH-activity curve caused by removing the ionized side chain in mutation. All mutants demonstrated the decreased catalytic efficiency more effectively revealed with natural glycoside, salicin, than with artificial substrate, p-nitrophenyl-β-D-glucopyranoside, suggesting that these amino acids are the key residues to determine aglycone specificity. A lower hydrolysis of genistin and daidzin for M2-TmBglA than M1-TmBglA indicated that L400, A407 and E408 being preferable to V170, A171, V173, G174 and H180 residues of Tm-BglA could be essential for soy isoflavone glycoside binding and catalysis.     

A mutant phospholipase D with enhanced thermostability from Streptomyces sp.

To investigate the contribution of amino acid residues to the thermostability of phospholipase D (PLD), a chimeric form of two Streptomyces PLDs (thermolabile K1PLD and thermostable TH-2PLD) was constructed. K/T/KPLD, in which residues 329–441 of K1PLD were recombined with the homologous region of TH-2PLD, showed a thermostability midway between those of K1PLD and TH-2PLD. By comparing the primary structures of Streptomyces PLDs, the seven candidates of thermostability-related amino acid residues of K1PLD were identified. The K1E346DPLD mutant, in which Glu346 of K1PLD was substituted with Asp by site-directed mutagenesis, exhibited enhanced thermostability, which was almost the same as that of TH-2PLD.     

A thermostable leucine dehydrogenase from Bacillus coagulansNL01: Expression,purification and characterization

L-Tert-leucine is the most representative unnatural amino acid and its production is valuable in industry. At present, l-tert-leucine is mainly produced by bioconversion, in which leucine dehydrogenase (LeuDH) plays a major role. In this study, a highly thermo- and pH-stable LeuDH from Bacillus coagulans NL01 (Bc-LeuDH) was reported and successfully expressed in Escherichia coli BL21(DE3). The enzyme was purified and its enzymatic properties were characterized. The specific activity of Bc-LeuDH at optimum condition (pH 8.0 and 50 °C) is 1337.97 U/mg, and the Km and kcat for sodium α-ketoisocaproate was 1.369 mM and 0.125 _S^-1, respectively. Furthermore, Bc-LeuDH possessed excellent thermostability that held nearly 80 % activity after 72 h incubation at temperature 50 °C and 55 °C. Notably, the half-life of Bc-LeuDH activity also was long for near 12 h at a high temperature of 70 °C, which was the longest in previous reports. Based on the sequence and structure analysis, the hydrophobic amino acid residues and hydrophobic patches are considered to have an important contribution to the thermostability of Bc-LeuDH. Furthermore, the Bc-LeuDH demonstrated better pH stability over a wide pH ranging from 7.0-10.0. These results indicate the potential industrial application of Bc-LeuDH in the future.     

In silico characterization of thermostable lipases

Thermostable lipases are of high priority for industrial applications as they are endowed with the capability of carrying out diversified reactions at elevated temperatures. Extremophiles are their potential source. Sequence and structure annotation of thermostable lipases can elucidate evolution of lipases from their mesophilic counterparts with enhanced thermostability hence better industrial potential. Sequence analysis highlighted the conserved residues in bacterial and fungal thermostable lipases. Higher frequency of AXXXA motif and poly Ala residues in lid domain of thermostable Bacillus lipases were distinguishing characteristics. Comparison of amino acid composition among thermostable and mesostable lipases brought into light the role of neutral, charged and aromatic amino acid residues in enhancement of thermostability. Structural annotation of thermostable lipases with that of mesostable lipases revealed some striking features which are increment of gamma turns in thermostable lipases; being first time reported in our paper, longer beta strands, lesser beta-branched residues in helices, increase in charged-neutral hydrogen bonding pair, hydrophobic-hydrophobic contact and differences in the N-cap and C-cap residues of the α helices. Conclusively, it can be stated that subtle changes in the arrangement of amino acid residues in the tertiary structure of lipases contributes to enhanced thermostability.     

Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans—A Nucleic Acid Binding Protein with Broad Substrate Specificity

BackgroundSSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis.ResultsThis paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein). This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity). The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7±1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100°C and melting temperature (T_m) is 100.2°C as shown by differential scanning calorimetry (DSC) analysis.ConclusionNeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids.