Structural and functional analyses of an L-asparaginase from Geobacillus thermopakistaniensis.

Genome sequence of Geobacillus thermopakistaniensis contains an open reading frame annotated as a type II L-asparaginase (ASNaseGt). Critical structural analysis disclosed that ASNaseGt might be a type I L-asparaginase. In order to determine whether it is a type I or type II L-asparaginase, we have performed the structural-functional characterization of the recombinant protein as well as analyzed the localization of ASNaseGt in G. thermopakistaniensis. ASNaseGt exhibited optimal activity at 52 °C and pH 9.5. There was a > 3-fold increase in activity in the presence of ß-mercaptoethanol. Apparent Vmax and Km values were 2735 U/mg and 0.35 mM, respectively. ASNaseGt displayed high thermostability with >80 % residual activity even after 6 h of incubation at 55 °C. Recombinant ASNaseGt existed in oligomeric form. Addition of ß-mercaptoethanol lowered the degree of oligomerization and displayed that tetrameric form was the most active, with a specific activity of 4300 U/mg. Under physiological conditions, ASNaseGt displayed >50 % of the optimal activity. Localization studies in G. thermopakistaniensis revealed that ASNaseGt is a cytosolic protein. Structural and functional characterization, and localization in G. thermopakistaniensis displayed that ASNaseGt is not a type II but a type I L-asparaginase.

Recombinant production and characterization of a metal ion-independent Lysophospholipase from a hyperthermophilic archaeon Pyrococcus abyssi DSM25543.

Genome sequence of Pyrococcus abyssi DSM25543 contains a coding sequence (PAB_RS01410) for α/ß hydrolase (WP_010867387.1). Structural analysis revealed the presence of a consensus motif GXSXG and a highly conserved catalytic triad in the amino acid sequence of α/ß hydrolase that were characteristic features of lysophospholipases. A putative lysophospholipase from P. abyssi with its potential applications in oil degumming and starch processing was heterologously produced in E. coli Rosetta (DE3) pLysS in soluble form followed by its purification and characterization. The recombinant enzyme was found to be active at temperature of 40-90 °C and pH 5.5-7.0. However, the enzyme exhibited its optimum activity at 65 °C and pH 6.5. None of the metal ions (Mn2+, Mg2+, Ni2+, Cu2+, Fe2+, Co2+, Zn2+ and Ca2+) being tested had stimulatory effect on lysophospholipase activity. Km and Vmax for hydrolysis of 4-nitrophenyl butyrate were calculated to be 1 ± 0.089 mM and 1637 ± 24.434 U/mg, respectively. It is the first report on the soluble production and characterization of recombinant lysophospholipase from P. abyssi which exhibits its lipolytic activity in the absence of divalent metal ions. Broad substrate specificity, activity and stability at elevated temperatures make recombinant lysophospholipase an ideal candidate for potential industrial applications.

Molecular cloning and characterization of Pcal_0039, an ATP-/NAD+-independent DNA ligase from hyperthermophilic archaeon Pyrobaculum calidifontis.

The genome sequence of hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0039, which encodes a putative DNA ligase. Structural analysis disclosed the presence of signature sequences of ATP-dependent DNA ligases. We have heterologously expressed Pcal_0039 gene in Escherichia coli. The recombinant protein, majorly produced in soluble form, was purified and functionally characterized. Recombinant Pcal_0039 displayed nick-joining activity between 40 and 85 °C. Optimal activity was observed at 70 °C and pH 5.5. Nick-joining activity was retained even after heating for 1 h at 90 °C, indicating highly thermostable nature of Pcal_0039. The nick-joining activity, displayed by Pcal_0039, was metal ion dependent and Mg2+ was the most preferred. NaCl and KCl inhibited the nick-joining activity at or above 200 mmol/L. The activity catalyzed by recombinant Pcal_0039 was independent of addition of ATP or NAD+ or any other nucleotide cofactor. A mismatch adjacent to the nick, either at 3'- or 5'-end, abolished the nick-joining activity. These characteristics make Pcal_0039 a potential candidate for applications in DNA diagnostics. To the best of our knowledge, Pcal_0039 is the only DNA ligase, characterized from genus Pyrobaculum, which exhibits optimum nick-joining activity at pH below 6.0 and independent of any nucleotide cofactor.

Structural and functional analyses of Pcal_0917, an α-glucosidase from hyperthermophilic archaeon Pyrobaculum calidifontis.

Genome analysis of Pyrobaculum calidifontis revealed the presence of α-glucosidase (Pcal_0917) gene. Structural analysis affirmed the presence of signature sequences of Type II α-glucosidases in Pcal_0917. We have heterologously expressed the gene and produced recombinant Pcal_0917 in Escherichia coli. Biochemical characteristics of the recombinant enzyme resembled to that of Type I α-glucosidases, instead of Type II. Recombinant Pcal_0917 existed in a tetrameric form in solution and displayed highest activity at 95 °C and pH 6.0, independent of any metal ions. A short heat-treatment at 90 °C resulted in a 35 % increase in enzyme activity. A slight structural shift was observed by CD spectrometry at this temperature. Half-life of the enzyme was >7 h at 90 °C. Pcal_0917 exhibited apparent Vmax values of 1190 ± 5 and 3.9 ± 0.1 U/mg against p-nitrophenyl α-D-glucopyranoside and maltose, respectively. To the best of our knowledge, Pcal_0917 displayed the highest ever reported p-nitrophenyl α-D-glucopyranosidase activity among the characterized counterparts. Moreover, Pcal_0917 displayed transglycosylation activity in addition to α-glucosidase activity. Furthermore, in combination with α-amylase, Pcal_0917 was capable of producing glucose syrup from starch with >40 % glucose content. These properties make Pcal_0917 a potential candidate for starch hydrolyzing industry.

Engineering a DNA polymerase from Pyrobaculum calidifontis for improved activity, processivity and extension rate.

Positively charged amino acids in the DNA polymerase domain are important for interaction with DNA. Two potential residues in the palm domain of Pca-Pol, a DNA polymerase from Pyrobaculum calidifontis, were identified and mutated to arginine in order to improve the properties of this enzyme. The mutant proteins were heterologously produced in Escherichia coli. Biochemical characterization revealed that there was no significant difference in pH, metal ion, buffer preferences, 3' - 5' exonuclease activity and error rate of the wild-type and the mutant enzymes. However, the specific activity, processivity and extension rate of the mutant enzymes increased significantly. Specific activity of one of the mutants (G522R-E555R) was nearly 9-fold higher than that of the wild-type enzyme. These properties make G522R-E555R mutant enzyme a potential candidate for commercial applications.

Cost-effective, high-yield production of Pyrobaculum calidifontis DNA polymerase for PCR application.

Polymerase Chain Reaction (PCR) is widely used for cloning, genetic engineering, mutagenesis, detection and diagnosis. A thermostable DNA polymerase is required for PCR. Here we describe low-cost and high-recovery production of Pyrobaculum calidifontis DNA polymerase (Pca-Pol). The gene was cloned in pET-28a and expressed in Escherichia coli BL21CodonPlus. Gene expression conditions were optimized. Eventually, gene expression was induced with 0.1 mM IPTG for 3 hours at 37 °C. Recombinant Pca-Pol produced was purified to homogeneity by immobilized metal-ion affinity chromatography yielding around 9000 U of Pca-Pol per liter of the culture with a recovery of 92%. Stability and PCR amplification efficiency of Pca-Pol was tested under various storage conditions with highest efficiency in 25 mM Tris-Cl buffer (pH 8.5) containing 0.1% Tween 20, 0.2 mg/mL BSA and 20% glycerol. Under this condition, no loss in PCR activity of Pca-Pol was observed, even after one year of storage. Repeated freeze-thaw, however, deteriorated enzyme activity of Pca-Pol. 55% PCR amplification activity retained after 7 prolong freeze-thaw cycles (freezing overnight at -20 °C and thawing for 45 minutes at 28 °C). Purified Pca-Pol possessed 3'-5' exonuclease (proofreading) activity and is expected to have greater fidelity as compared to Taq polymerase which does not have proofreading activity.

Functional analyses of a highly thermostable hexokinase from Pyrobaculum calidifontis.

The gene encoding a repressor open reading frame sugar kinase (ROK) family protein from hyperthermophilic crenarchaeon Pyrobaculum calidifontis, Pcal-HK, was cloned and expressed in Escherichia coli. The recombinant protein was produced in soluble and highly active form. Purified Pcal-HK was highly thermostable and existed in a monomeric form in solution. The enzyme was specific to ATP as phosphoryl donor but showed broad specificity to phosphoryl acceptors. It catalyzed the phosphorylation of a number of hexoses, including glucose, glucosamine, N-acetyl glucosamine, fructose and mannose, at nearly the same rate and similar affinity. The enzyme was metal ion dependent exhibiting highest activity at 90-95 °C and pH 8.5. Mg2+ was most effective metal ion, which could be partially replaced by Mn2+, Ni2+ or Zn2+. Kinetic parameters were determined at 90 °C and the enzyme showed almost similar catalytic efficiency (kcat/Km) towards the above mentioned hexoses. To the best of our knowledge, Pcal-HK is the most active thermostable ROK family hexokinase characterized to date which catalyzes the phosphorylation of various hexoses with nearly similar affinity.

Role of C-terminal domain in a manganese-catalase from Geobacillus thermopakistaniensis.

Catalases catalyze the decomposition of hydrogen peroxide into water and oxygen. We have characterized two manganese-catalases from Geobacillus thermopakistaniensis, CatGt and Cat-IIGt, which exhibited significant variation in their sequence, structure and properties. There was only 23% sequence identity between the two. The striking structural difference was the presence of an extended C-terminal domain in CatGt. Molecular modelling and docking studies revealed that deletion of the C-terminal domain removes non-specific binding, which results in increased substrate affinity. To verify experimentally, a C-terminal truncated version of CatGt, named as CatGt-ΔC, was produced in Escherichia coli and effects of deletion were analyzed. There was no significant difference in optimal pH, optimal temperature and substrate specificity of CatGt and CatGt-ΔC. However, Km value was reduced from 259 to 157 mM and CatGt-ΔC exhibited ∼1.5-fold higher catalytic efficiency as compared to CatGt. Furthermore, removal of the C-terminal domain converted the tetrameric nature to monomeric, and reduced the thermostability of the truncated protein. These results demonstrate that C-terminal domain of CatGt might have little role in maintaining enzyme function but provides additional structural stability to the protein, which is a desired property for industrial applications.

Temperature dependent autocleavage and applications of recombinant L-asparaginase from Thermococcus kodakarensis for acrylamide mitigation.

This manuscript describes enhancement of soluble production, auto-cleavage analysis and assessment of acrylamide mitigation potential of Tk2246, a plant-type L-asparaginase from Thermococcus kodakarensis. The gene encoding Tk2246 was cloned and expressed in Escherichia coli. Recombinant Tk2246 was produced mainly in insoluble form. Various strategies were utilized to enhance the soluble production, which significantly increased the soluble yield. Interestingly, recombinant Tk2246 was produced even without addition of the inducer, though relatively in a lower amount. To our surprise, Tk2246 was produced in partially cleaved form when the inducer was not added in the culture. When applied for acrylamide mitigation, Tk2246 reduced the acrylamide formation more than 80% in French fries, chapati and yeast-leavened bread. In addition to acrylamide mitigation, Tk2246 exhibited antistaling activity without loss of sensory properties of the food. High activity, thermostability and efficient acrylamide reduction capability make Tk2246 a potential candidate for industrial applications.

Molecular cloning, expression in Escherichia coli and structural-functional analysis of a pyruvate kinase from Pyrobaculum calidifontis.

This manuscript describes recombinant production, characterization and structural analysis of wild-type and mutant Pcal_0029, a pyruvate kinase from Pyrobaculum calidifontis. Recombinant Pcal_0029 was produced in soluble and highly active form in Escherichia coli. Purified protein exhibited divalent metal-dependent activity which increased with the increase in temperature till 85 °C. Recombinant Pcal_0029 was highly thermostable with no significant loss in activity even after an incubation of 120 min at 100 °C. The enzyme exhibited apparent S0.5 and Vmax values of 0.44 ± 0.05 mM and 840 ± 39 units, respectively, towards phosphoenolpyruvate. These values towards adenosine-5'-diphosphate were 0.5 ± 0.07 mM and 870 ± 26 units, respectively. In silico structural analysis and comparison with the characterized enzymes revealed the presence of eight conserved regions. Two substitutions, K130E and S155G, resulted in a 10-fold decrease in activity. Secondary structure analysis indicated similar structures for the wild-type and the mutant enzymes. Bioinformatics analysis revealed disruption of interatomic interactions and hydrogen bonds, leading to a decreased flexibility and solvent accessibility, which may have led to decrease in activity. To the best of our knowledge, Pcal_0029 is the most thermostable pyruvate kinase reported so far. Moreover, this is the first study on the role of non-catalytic residues in a pyruvate kinase.

Investigating the role of carbohydrate-binding module 34 in cyclomaltodextrinase from Geobacillus thermopakistaniensis: structural and functional analyses.

Carbohydrate-binding modules (CBMs) are noncatalytic regions found in several enzymes of glycoside hydrolase family 13 and are proposed to orient substrates to the catalytic site. In this study, a substantial information on the conserved aromatic residues in CBM34 regions of characterized bacterial cyclolmaltodextrinases (CDases) has been presented. Molecular modeling of CDase from Geobacillus thermopakistaniensis (CDase Gt ) revealed a change in the active site geometry due to CBM34 truncation. The binding energies of full-length (CDase Gt ) and CBM34 truncated (CDase Gt -ΔN) models showed opposite trends. The least preferred substrate molecule by the full-length model was the most preferred by the CBM34 truncated one. These exciting in silico findings were experimentally verified by recombinant production and characterization of the full-length and the CBM34 truncated proteins. Both the enzymes showed similar optimum pH and temperature. However, substrate specificity was in the reverse order. These experimental verifications matched the homology modeling and docking predictions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03089-9.

Looking into a highly thermostable and efficient recombinant manganese-catalase from Geobacillusthermopakistaniensis.

Catalases, heme or non-heme, are catalysts that decompose hydrogen peroxide. Among them, non-heme or manganese-catalases have been studied from limited organisms. We report here heterologous production of a manganese-catalase, Cat-IIGt, previously annotated as a hypothetical protein, from a thermophilic bacterium Geobacillus thermopakistaniensis. Recombinant Cat-IIGt, produced as inactive inclusion bodies in Escherichia coli, was solubilized and refolded into a soluble and highly active form. Sequence homology, absorption spectra, resistance to sodium azide inhibition and activation by Mn2+ indicated that it was a manganese-catalase. Metal analysis revealed the presence of ∼2 Mn2+ and ∼2 Ca2+ per subunit of Cat-IIGt. Recombinant Cat-IIGt exhibited highest activity at pH 10.0 and 70°C. The enzyme was highly active with a specific activity of 40,529 µmol min-1 mg-1. The apparent Km and kcat values were 75 mM and 1.5 × 104 s-1 subunit-1, respectively. Recombinant Cat-IIGt was highly thermostable with a half-life of 30 min at 100°C. The structural attributes of Cat-IIGt, including the metal and substrate binding residues, were predicted by homology modeling and molecular docking studies. High activity and thermostability and alkaline nature make Cat-IIGt a potential candidate for textile and paper processing industries.

A novel RdRp-based colorimetric RT-LAMP assay for rapid and sensitive detection of SARS-CoV-2 in clinical and sewage samples from Pakistan.

Novel corona virus SARS-CoV-2, causing coronavirus disease 2019 (COVID-19), has become a global health challenge particularly for developing countries like Pakistan where overcrowded cities, inadequate sanitation, little health awareness and poor socioeconomic conditions exist. The SARS-CoV-2 has been known to spread primarily through direct contact and respiratory droplets. However, detection of SARS-CoV-2 in stool and sewage have raised the possibility of fecal-oral mode of transmission. Currently, quantitative reverse-transcriptase PCR (qRT-PCR) is the only method being used for SARS-CoV-2 detection, which requires expensive instrumentation, dedicated laboratory setup, highly skilled staff, and several hours to report results. Considering the high transmissibility and rapid spread, a robust, sensitive, specific and cheaper assay for rapid SARS-CoV-2 detection is highly needed. Herein, we report a novel colorimetric RT-LAMP assay for naked-eye detection of SARS-COV-2 in clinical as well as sewage samples. Our SARS-CoV-2 RdRp-based LAMP assay could successfully detect the virus RNA in 26/28 (93%) of RT-PCR positive COVID-19 clinical samples with 100% specificity (n = 7) within 20 min. We also tested the effect of various additives on the performance of LAMP assay and found that addition of 1 mg/ml bovine serum albumin (BSA) could increase the sensitivity of assay up to 101 copies of target sequence. Moreover, we also successfully applied this assay to detect SARS-CoV-2 in sewage waters collected from those areas of Lahore, a city of Punjab province of Pakistan, declared as virus hotspots by local government. Our optimized LAMP assay could provide a sensitive first tier strategy for SARS-CoV-2 screening and can potentially help diagnostic laboratories in better handling of high sample turnout during pandemic situation. By providing rapid naked-eye SARS-CoV-2 detection in sewage samples, this assay may support pandemic readiness and emergency response to any possible virus outbreaks in future.

Structural and functional analyses of a novel manganese-catalase from Bacillus subtilis R5.

Catalases catalyze the decomposition of hydrogen peroxide into water and oxygen. Limited reports are available on characterization of manganese-catalases. We describe here molecular cloning and expression in Escherichia coli of a putative manganese-catalase gene from mesophilic bacterium, Bacillus subtilis R5. The gene product, CatBsu, produced as a soluble protein, was purified to apparent homogeneity and biochemically characterized. The absorption spectra and nonsignificant inhibition by sodium azide indicated that it is a manganese-catalase. The protein was in homohexameric form in solution, with a subunit molecular weight of 30 kDa, containing ~2 Mn2+ and ~1 Ca2+ per subunit. CatBsu showed highest activity at pH 8.0 and 55 °C. It was found to be highly active with a specific activity of 25,290 µmol min-1 mg-1 and apparent Km and kcat values of 98 mM and 1.27 × 104 s-1 subunit-1, respectively. Although from a mesophilic source, it exhibited a half-life of 2 h at 80 °C. Furthermore, the active site and metal binding residues in CatBsu were predicted by homology modelling and molecular docking. To the best of our knowledge, this is the first characterization of a manganese-catalase from genus Bacillus.

ADP-dependent glucose/glucosamine kinase from Thermococcus kodakarensis: cloning and characterization.

The genome sequence of Thermococcus kodakarensis contains an open reading frame, TK1110, annotated as ADP-dependent glucokinase. The encoding gene was expressed in Escherichia coli and the gene product, TK-GLK, was produced in soluble and active form. The recombinant enzyme was extremely thermostable. Thermostability was increased significantly in the presence of ammonium sulfate. ADP was the preferred co-factor for TK-GLK, which could be replaced with CDP but with a 60% activity. TK-GLK was a metal ion-dependent enzyme which exhibited glucokinase, glucosamine kinase and glucose 6-phosphatase activities. It catalyzed the phosphorylation of both glucose and glucosamine with nearly the same rate and affinity. The apparent Km values for glucose and glucosamine were 0.48 ± 0.03 and 0.47 ± 0.09 mM, respectively. The catalytic efficiency (kcat/Km) values against these two substrates were 6.2 × 105 ± 0.25 and 5.8 × 105 ± 0.75 M-1 s-1. The apparent Km value for dephosphorylation of glucose 6-phosphate was ~14-fold higher than that of glucose phosphorylation. Similarly, catalytic efficiency (kcat/Km) for phosphatase reaction was ~19-fold lower than that for the kinase reaction. To the best of our knowledge, this is the first report that describes the reversible nature of a euryarchaeal ADP-dependent glucokinase.

Effect of Y50H and S187G substitutions on thermostability and exonuclease activity of TK1646 from Thermococcus kodakarensis.

TK1646 is a highly thermostable single strand specific 3'-5' exonuclease. Exonucleases play important role in maintaining the genome integrity at elevated temperatures. Therefore, it is important to examine the factors contributing to thermostability of these exonucleases. In this study we report on production, purification and characterization of S187G and Y50H mutants of TK1646, focusing on the factors leading to thermostability of TK1646. Characterization of the recombinant proteins indicated that these substitutions did not drastically affect the catalysis of single stranded DNA. However, both of these substitutions reduced the thermostability of the recombinant proteins. Half-lives of Y50H and S187G mutants were 95 and 155 min, respectively, at 100 °C in comparison to 180 min of the wild type. Bioinformatics analysis indicated an increase in solvent accessibility of the mutated residues and disruption of hydrogens bonds. Molecular modelling and superimposition of the 3D structures of the mutants and the wild type demonstrated that one of the active site residues, Glu145, was shifted away from the metal ion in both the mutants which may be responsible for the decrease in catalytic activity. Compact secondary structure, hydrophobicity and hydrogen bonding might be the major factors contributing to the thermostability of TK1646.

Isocitrate dehydrogenase 1 gene variants analysis of glioma patients from Pakistan.

Various somatic isocitrate dehydrogenase 1 (IDH1) gene variants have been reported to drive lower-grade gliomas and secondary glioblastomas. In the current study, we explored the IDH1 variants in the glioma biopsy samples of patients from Pakistan. We explored the incidence of isocitrate dehydrogenase 1 gene variants by hotspot sequencing in 80 formalin-fixed paraffin-embedded tissues of different types of glioma biopsy samples. Structural modeling of the identified variants in isocitrate dehydrogenase 1 protein was done to see their possible consequences. The frequently described p.Arg132 variants were not found in any of the glioma types. However, in our study, we identified nonsynonymous variants at the residues p.R109 and p.G136 in astrocytomas and p.R100 in oligodendroglioma. These variants are affecting a part of the conserved domain in isocitrate dehydrogenase 1. Both of p.R100 and p.R109 variants are rare and described before, whereas the p.G136 variant identified in this study has never been described previously. Structural modeling showed that variants of these residues would directly affect the substrate binding and hence the enzyme activity.

Pcal_0842, a highly thermostable glycosidase from Pyrobaculum calidifontis displays both α-1,4- and ß-1,4-glycosidic cleavage activities.

The gene encoding Pcal_0842, annotated as a cellulase (accession no. ABO08268), was cloned and expressed in Escherichia coli. The gene product was produced in insoluble form in E. coli in high amounts even without addition of the inducer isopropyl ß-D-1-thiogalactopyranoside. The recombinant protein was solubilized in 8 M urea and refolded by gradual removal of urea. The refolded protein exhibited both α-1,4- and ß-1,4-glycosidic cleavage activities. The enzyme activity increased with the increase in temperature till 120 °C. Apart from very high optimal temperature, recombinant Pcal_0842 was extremely thermostable. There was no significant loss in activity even after heating for 100 h at 100 °C. The half-lives of Pcal_0842 were 6 and 2.5 h at 110 and 120 °C, respectively. To the best of our knowledge, Pcal_0842 is the most thermostable glycosidase characterized to date and this is the first report on cloning and characterization of an enzyme from archaea that displays both α-1,4- and ß-1,4-glycosidic cleavage activities.

Mutation in pvcABCD operon of Pseudomonas aeruginosa modulates MexEF-OprN efflux system and hence resistance to chloramphenicol and ciprofloxacin.

Pseudomonas aeruginosa harbors pvcABCD operon that is responsible for the synthesis of paerucumarin. Here we report the involvement of pvcABCD operon in chloramphenicol and ciprofloxacin resistance. P. aeruginosa mutant defective in pvcB (PW4832) was more sensitive to chloramphenicol and ciprofloxacin in comparison with its parent strain (MPAO1). A mutation in pvcA gene in MPAO1 (PW4830) did not alter the sensitivity to either antibiotic. As chloramphenicol and ciprofloxacin are substrates of MexEF-OprN efflux pump, so we decided to investigate the modulation of MexEF-OprN and its transcriptional regulator MexT in PW4832, PW4830 and MPAO1 strains. We isolated and sequenced mexT gene from MPAO1, PW4830 and PW4832. The nucleotide sequence of mexT gene in all three strains was identical. Expression levels of mexEF-oprN, mexT and mexS genes were checked via quantitative real-time RT-PCR. All these genes showed significant repression in mRNA levels in PW4832 as compared to MPAO1. These results indicate that chloramphenicol and ciprofloxacin sensitivity in PW4832 is due to transcriptional repression of mexT and mexEF-oprN genes. Exogenous addition of paerucumarin resumed the expression of mexT and mexEF-oprN genes as well as resistance against chloramphenicol and ciprofloxacin in PW4832 strain. This is a novel finding linking pvcB gene of P. aeruginosa with chloramphenicol and ciprofloxacin resistance and MexEF-OprN pump modulation which needs to be further explored.

Gene cloning, expression enhancement in Escherichia coli and biochemical characterization of a highly thermostable amylomaltase from Pyrobaculum calidifontis.

Pcal_0768 gene encoding an amylomaltase, a 4-α-glucanatransferase belonging to family 77 of glycosyl hydrolases, from Pyrobaculum calidifontis was cloned and expressed in Escherichia coli. The recombinant protein was produced in E. coli in soluble and active form. However, the expression level was not very high. Analysis of the mRNA of initial seven codons at the 5'-end of the gene revealed the presence of a hair pin like secondary structure. This secondary structure was removed by site directed mutagenesis, without altering the amino acids, which resulted in enhanced expression of the cloned gene. Recombinant Pcal_0768 exhibited optimal amylomaltase activity at 80 °C and pH 6.9. Under these conditions, the specific activity was 690 U/ mg. Recombinant Pcal_0768 was highly thermostable with a half-life of 6 h at 100 °C. It exhibited the highest kcat value among the characterized glucanotransferases. No metal ions were required for activity or stability of the enzyme. Recombinant Pcal_0768 was successfully employed in the synthesis of modified starch for producing thermoreversible gel. To the best of our knowledge, till now this is the most thermostable enzyme among the characterized amylomaltases. High thermostability and starch modification potential make it a novel and distinct amylomaltase.