Increased thermostability of microbial transglutaminase by combination of several hot spots evolved by random and saturation mutagenesis

The thermostability of microbial transglutaminase (MTG) of Streptomyces mobaraensis was further improved by saturation mutagenesis and DNA-shuffling. High-throughput screening was used to identify clones with increased thermostability at 55°C. Saturation mutagenesis was performed at seven "hot spots", previously evolved by random mutagenesis. Mutations at four positions (2, 23, 269, and 294) led to higher thermostability. The variants with single amino acid exchanges comprising the highest thermostabilities were combined by DNA-shuffling. A library of 1,500 clones was screened and variants showing the highest ratio of activities after incubation for 30 min at 55°C relative to a control at 37°C were selected. 116 mutants of this library showed an increased thermostability and 2 clones per deep well plate were sequenced (35 clones). 13 clones showed only the desired sites without additional point mutations and eight variants were purified and characterized. The most thermostable mutant (triple mutant S23V-Y24N-K294L) exhibited a 12-fold higher half-life at 60°C and a 10-fold higher half-life at 50°C compared to the unmodified recombinant wild-type enzyme. From the characterization of different triple mutants differing only in one amino acid residue, it can be concluded that position 294 is especially important for thermostabilization. The simultaneous exchange of amino acids at sites 23, 24, 269 and 289 resulted in a MTG-variant with nearly twofold higher specific activity and a temperature optimum of 55°C. A triple mutant with amino acid substitutions at sites 2, 289 and 294 exhibits a temperature optimum of 60°C, which is 10°C higher than that of the wild-type enzyme.     

A single-vial analytical and quantitative gas chromatography-mass spectrometry assay for terpene synthases

A quantitative assay for the analysis of sesquiterpene synthases, wherein each reaction mixture is formulated in glass gas chromatography vials, overlaid with organic solvent such as ethyl acetate, and subsequently vortexed to extract hydrocarbon reaction products into the organic phase after a suitable incubation period, was developed. The product-enriched organic phase is then sampled in an automated fashion and injected directly into a gas chromatograph-mass spectrometer without further workup for analysis and quantification of hydrocarbon products. Application of the vial assay to the analysis of amorpha-4,11-diene synthase (ADS), a sesquiterpene synthase, demonstrated the sensitivity of the assay for detection of major and minor reaction products and most notably for the identification of several sesquiterpene products that had escaped previous detection. A steady-state kinetic analysis of tobacco 5-epi-aristolochene synthase (TEAS), another sesquiterpene synthase, validated the quantitative nature of the assay, providing an alternative means to the established method of using radiolabeled substrate, extraction, and scintillation counting. This simplified assay provides a standardized method to facilitate analysis of terpene synthases and diverse mutant enzyme libraries by supplanting the common practice of using larger scale reactions, multiple extractions, and evaporative concentration of the organic phase prior to gas chromatography-mass spectrometry (GC-MS) analysis.     

A novel thermostable xylanase of Paenibacillus macerans IIPSP3 isolated from the termite gut

Xylanase is an enzyme in high demand for various industrial applications, such as those in the biofuel and pulp and paper fields. In this study, xylanase-producing microbes were isolated from the gut of the wood-feeding termite at 50°C. The isolated microbe produced thermostable xylanase that was active over a broad range of temperatures (40-90°C) and pH (3.5-9.5), with optimum activity (4,170 ± 23.5 U mg?1) at 60°C and pH 4.5. The enzyme was purified using a strong cation exchanger and gel filtration chromatography, revealing that the protein has a molecular mass of 205 kDa and calculated pI of 5.38. The half-life of xylanase was 6 h at 60°C and 2 h at 90°C. The isolated thermostable xylanase differed from other xylanases reported to date in terms of size, structure, and mode of action. The novelty of this enzyme lies in its high specific activity and stability at broad ranges of temperature and pH. These properties suggest that this enzyme could be utilized in bioethanol production as well as in the paper and pulp industry.     

Application of a novel thermostable NAD(P)H oxidase from hyperthermophilic archaeon for the regeneration of both NAD⁺ and NADP⁺

A novel thermostable NAD(P)H oxidase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkNOX) catalyzes oxidation of NADH and NADPH with oxygen from atmospheric air as an electron acceptor. Although the optimal temperature of TkNOX is >90°C, it also shows activity at 30°C. This enzyme was used for the regeneration of both NADP(+) and NAD(+) in alcohol dehydrogenase (ADH)-catalyzed enantioselective oxidation of racemic 1-phenylethanol. NADP(+) regeneration at 30°C was performed by TkNOX coupled with (R)-specific ADH from Lactobacillus kefir, resulting in successful acquisition of optically pure (S)-1-phenylethanol. The use of TkNOX with moderately thermostable (S)-specific ADH from Rhodococcus erythropolis enabled us to operate the enantioselective bioconversion accompanying NAD(+) regeneration at high temperatures. Optically pure (R)-1-phenylethanol was successfully obtained by this system after a shorter reaction time at 45-60°C than that at 30°C, demonstrating an advantage of the combination of thermostable enzymes. The ability of TkNOX to oxidize both NADH and NADPH with remarkable thermostability renders this enzyme a versatile tool for regeneration of the oxidized nicotinamide cofactors without the need for extra substrates other than dissolved oxygen from air.     

Highly thermostable xylanase purified from Rhizomucor miehei NRL 3169

A thermostable xylanase was purified and characterized from the thermophilic fungus Rhizomucor miehei (Cooney & Emerson) Schipper. The enzyme was purified to homogeneity by ammonium sulfate precipitation, sephadex G-100 gel filtration and diethylaminoethyl cellulose anion exchange chromatography with a 29.1-fold. The enzyme was highly active within a range of pH from 5.0 to 6.5. The optimum temperature of the purified enzyme was 75°C. The enzyme showed high thermal stability at 70°C and 75°C and the half-life of the xylanase at 90°C was 30 min. Km and Vmax values at 50°C of the purified enzyme were 0.055 mg/ml and 113.5 μmol min?1 mg?1 respectively. The enzyme was activated by Ca2+, Cu2+, K+ and Na+. On the other hand, Ag2+, Hg2+, Ba2+, and Zn2+ inhibited the enzyme. The molecular weight of the xylanase was estimated to be 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The present study is among the first works to examine and describe a secreted highly thermostable endoxylanase from the Rhizomucor miehei fungus. This enzyme displays a number of biochemical properties that make it a potentially strong candidate for industrial and commercial application in pulp bleaching.     

Identification and characterization of UDP-glucose dehydrogenase from the hyperthermophilic archaon, Pyrobaculum islandicum

A gene encoding a UDP-glucose dehydrogenase homologue was identified in the hyperthermophilic archaeon, Pyrobaculum islandicum. This gene was expressed in Escherichia coli, and the product was purified and characterized. The expressed enzyme is the most thermostable UDP-glucose dehydrogenase so far described, with a half-life of 10 min at 90 °C. The enzyme retained its full activity after incubating in a pH range of 5.0-10.0 for 10 min at 80 °C. The temperature dependence of the kinetic parameters for this enzyme was examined at 37-70 °C. A decrease in K(m)s for UDP-glucose and NAD was observed with decreasing temperature. This resulted in the enzyme still retaining high catalytic efficiency (V(max)/K(m)) for the substrate and cofactor, even at 37 °C. These characteristics make the enzyme potentially useful for its application at a much lower temperature such as 37 °C than the optimum growth temperature of 100 °C for P. islandicum.     

Mutations derived from the thermophilic polyhydroxyalkanoate synthase PhaC enhance the thermostability and activity of PhaC from Cupriavidus necator H16

The thermophile Cupriavidus sp. strain S-6 accumulated polyhydroxybutyrate (PHB) from glucose at 50°C. A 9.0-kbp EcoRI fragment cloned from the genomic DNA of Cupriavidus sp. S-6 enabled Escherichia coli XL1-Blue to synthesize PHB at 45°C. Nucleotide sequence analysis showed a pha locus in the clone. The thermophilic polyhydroxyalkanoate (PHA) synthase (PhaC(Csp)) shared 81% identity with mesophilic PhaC of Cupriavidus necator H16. The diversity between these two strains was found dominantly on their N and C termini, while the middle regions were highly homologous (92% identity). We constructed four chimeras of mesophilic and thermophilic phaC genes to explore the mutations related to its thermostability. Among the chimeras, only PhaC(H16β), which was PhaC(H16) bearing 30 point mutations derived from the middle region of PhaC(Csp), accumulated a high content of PHB (65% [dry weight]) at 45°C. The chimera phaC(H16)(β) and two parental PHA synthase genes were overexpressed in E. coli BLR(DE3) cells and purified. At 30°C, the specific activity of the chimera PhaC(H16β) (172 ± 17.8 U/mg) was 3.45-fold higher than that of the parental enzyme PhaC(H16) (50 ± 5.2 U/mg). At 45°C, the half-life of the chimera PhaC(H16β) (11.2 h) was 127-fold longer than that of PhaC(H16) (5.3 min). Furthermore, the chimera PhaC(H16β) accumulated 1.55-fold (59% [dry weight]) more PHA content than the parental enzyme PhaC(H16) (38% [dry weight]) at 37°C. This study reveals a limited number of point mutations which enhance not only thermostability but also PhaC(H16) activity. The highly thermostable and active PHA synthase will provide advantages for its promising applications to in vitro PHA synthesis and recombinant E. coli PHA fermentation.     

Cloning of a gene encoding β-glucosidase from Chaetomium thermophilum CT2 and its expression in Pichia pastoris

A new thermostable β-glucosidase gene (bgl) from Chaetomium thermophilum CT2 was cloned, sequenced and expressed. The full-length DNA of bgl was 3,101 bp and included three introns. The full-length cDNA contained an open reading frame of 2,604-bp nucleotides, encoding 867 amino acids with a potential secretion signal. The C. thermophilum CT2 β-glucosidase gene was functionally expressed in Pichia pastoris. The purified recombinant β-glucosidase was a 119-kDa glycoprotein with an optimum catalytic activity at pH 5.0 and 60°C. The enzyme was stable at 50°C, and retained 67.7% activity after being kept at 60°C for 1 h; the half-time of the enzyme at 65°C was approximately 55 min, and even retained 29.7% activity after incubation at 70°C for 10 min.     

耐热荧光素酶的表达及热稳定焦磷酸测序体系的建立

焦磷酸测序是一种基于生物发光反应的实时DNA序列测定技术,其反应体系中的荧光素酶决定测序反应的灵敏度。传统焦测序反应使用的是野生型北美荧光素酶Photinus pyralis luciferase(PpL),该酶热稳定性较差,因此由该酶配制的焦磷酸测序反应体系对热不稳定,影响焦磷酸测序的灵敏度。为了建立热稳定的焦磷酸测序反应体系,全合成了耐热突变日本萤火虫荧光素酶的编码序列,并将其插入到表达载体pET28a(+)中构建重组质粒,将重组质粒转化到大肠杆菌中,筛选得到的阳性重组菌成功表达了N端带有6×His(组氨酸)标签的耐热突变日本萤火虫荧光素酶。利用镍亲和层析法,纯化得到了分子量约为60 kDa且比活为4.29×1010RLU/mg的重组蛋白。对重组耐热日本萤火虫荧光素酶的热稳定性研究结果表明,该酶在50℃时仍具有活性,在40℃下孵育25 min后活性保留了90%以上。与基于野生型北美荧光素酶的焦磷酸测序体系相比,由耐热日本萤火虫荧光素酶配制的测序反应体系对热稳定性有了极大的提高,该测序反应液在37℃放置1 h后仍能够得到正确的测序结果。研究结果为建立稳定可靠的现场及时快速焦磷酸测序反应体系奠定了基础。     

Isolation and characterization of N-acylhomoserine lactonase from the thermophilic bacterium, Geobacillus caldoxylosilyticus YS-8

Geobacillus caldoxylosilyticus YS-8, which was isolated from volcanic soil in Indonesia, was found to degrade various N-acylhomoserine lactones (AHLs) with different lengths and acyl side-chain substitutions over a wide temperature range of 30-70 °C. The purified AHL-degrading enzyme showed a single band of 32 kDa, and its N-terminal amino acid sequence was determined to be ANVIKARPKLYVMDN, tentatively suggesting that the AHL-degrading enzyme was AHL lactonase. The AHL-degrading activity of the purified enzyme was maximized at pH 7.5 and 50 °C, and it retained about 50% of its activity even after a heat treatment at 60 °C for 3 h, exhibiting properties consistent with a thermostable enzyme. The mass spectrometric analysis demonstrated that the AHL-degrading enzyme catalyzed lactone ring opening of N-3-oxohexanoyl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone by hydrolyzing the lactones and working as an AHL lactonase.     

Screening for thermostable mutant of kanamycin nucleotidyltransferase by the use of a transformation system for a thermophile, Bacillus stearothermophilus

A structural gene of kanamycin nucleotidyltransferase cloned into a single-stranded bacteriophage M13 was subjected to mutagenesis with hydroxylamine. Having recloned the mutagenized gene of the enzyme in a vector plasmid pTB922, the recombinant plasmid was used to transform Bacillus stearothermophilus with a purpose of screening for the more thermostable enzyme than the wild type. Out of greater than 8 X 10(3) transformants, 12 clones that were suspected to harbor the mutant gene encoding the more thermostable enzyme were isolated by shifting from a permissive (55 degrees C) to a nonpermissive (61 degrees C) temperature that inactivates the wild-type enzyme. DNA sequence analysis of the mutant genes revealed two types of mutation of single base substitution and hence a single amino acid replacement. The first type was the replacement of an aspartate by a tyrosine at position 80 of the wild-type enzyme, while the second was that of a threonine by a lysine at position 130. Purified enzymes from the two mutant genes were confirmed to be substantially more thermostable than the wild type in vitro. The method of screening for a thermostable kanamycin nucleotidyltransferase presented here could be applied to any other enzyme, if a transformation system of a thermophile were available. Indeed, thermostable mutants with a subtle amino acid change would be of value for better understanding of forces and interactions that contribute to the stability of a protein.     

Thermostabilization of porcine kidney D-amino acid oxidase by a single amino acid substitution

D-amino acid oxidase (DAO) is of considerable practical importance, such as bioconversion and enzymatic assay. In this study, we succeeded in obtaining a thermostable mutant DAO from porcine kidney by a single amino acid substitution. This mutant enzyme, F42C, was stable at 55 degrees C, while the wild-type enzyme was stable only up to 45 degrees C. The Km values of F42C for D-amino acids was about half of those of the wild-type enzyme. This mutant DAO with improved stability and affinity for its substrates is advantageous for the determination of D-amino acids.     

Biosynthetic potential of sesquiterpene synthases: alternative products of tobacco 5-epi-aristolochene synthase

Nicotiana tabacum (tobacco) 5-epi-aristolochene synthase (TEAS) serves as an useful model for understanding the enzyme mechanisms of sesquiterpene biosynthesis. Despite extensive bio-chemical and structural characterization of TEAS, a more detailed analysis of the reaction product spectrum is lacking. This study reports the discovery and quantification of several alternative sesquiterpene products generated by recombinant TEAS in the single-vial GC-MS assay. The combined use of chiral and non-polar stationary phases for gas chromatography separations proved critical for resolving the numerous sesquiterpene products of TEAS for mass spectral analysis and identification. Co-injection studies with available authentic standards from both synthetic and natural sources further corroborated the assignment of several compounds, resulting in an annotated reaction mechanism accounting for their biosynthesis. Moreover, a previously undocumented farnesyl trans-cis isomerization pathway was observed.     

Differences in the catalytic mechanisms of mesophilic and thermophilic indole-3-glycerol phosphate synthase enzymes at their adaptive temperatures

Thermophilic enzymes tend to be less catalytically-active at lower temperatures relative to their mesophilic counterparts, despite having very similar crystal structures. An often cited hypothesis for this general observation is that thermostable enzymes have evolved a more rigid tertiary structure in order to cope with their more extreme, natural environment, but they are also less flexible at lower temperatures, leading to their lower catalytic activity under mesophilic conditions. An alternative hypothesis, however, is that complementary thermophilic-mesophilic enzyme pairs simply operate through different evolutionary-optimized catalytic mechanisms. In this communication, we present evidence that while the steps of the catalytic mechanisms for mesophilic and thermophilic indole-3-glycerol phosphate synthase (IGPS) enzymes are fundamentally similar, the identity of the rate-determining step changes as a function of temperature. Our findings indicate that while product release is rate-determining at 25°C for thermophilic IGPS, near its adaptive temperature (75°C), a proton transfer event, involving a general acid, becomes rate-determining. The rate-determining steps for thermophilic and mesophilic IGPS enzymes are also different at their respective, adaptive temperatures with the mesophilic IGPS-catalyzed reaction being rate-limited before irreversible CO2 release, and the thermophilic IGPS-catalyzed reaction being rate limited afterwards.     

Genomic organization of plant terpene synthases and molecular evolutionary implications

Terpenoids are the largest, most diverse class of plant natural products and they play numerous functional roles in primary metabolism and in ecological interactions. The first committed step in the formation of the various terpenoid classes is the transformation of the prenyl diphosphate precursors, geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate, to the parent structures of each type catalyzed by the respective monoterpene (C(10)), sesquiterpene (C(15)), and diterpene synthases (C(20)). Over 30 cDNAs encoding plant terpenoid synthases involved in primary and secondary metabolism have been cloned and characterized. Here we describe the isolation and analysis of six genomic clones encoding terpene synthases of conifers, [(-)-pinene (C(10)), (-)-limonene (C(10)), (E)-alpha-bisabolene (C(15)), delta-selinene (C(15)), and abietadiene synthase (C(20)) from Abies grandis and taxadiene synthase (C(20)) from Taxus brevifolia], all of which are involved in natural products biosynthesis. Genome organization (intron number, size, placement and phase, and exon size) of these gymnosperm terpene synthases was compared to eight previously characterized angiosperm terpene synthase genes and to six putative terpene synthase genomic sequences from Arabidopsis thaliana. Three distinct classes of terpene synthase genes were discerned, from which assumed patterns of sequential intron loss and the loss of an unusual internal sequence element suggest that the ancestral terpenoid synthase gene resembled a contemporary conifer diterpene synthase gene in containing at least 12 introns and 13 exons of conserved size. A model presented for the evolutionary history of plant terpene synthases suggests that this superfamily of genes responsible for natural products biosynthesis derived from terpene synthase genes involved in primary metabolism by duplication and divergence in structural and functional specialization. This novel molecular evolutionary approach focused on genes of secondary metabolism may have broad implications for the origins of natural products and for plant phylogenetics in general.     

Thermostabilization of bacterial fructosyl-amino acid oxidase by directed evolution

We succeeded in isolating several thermostable mutant fructosyl-amino acid oxidase (FAOX; EC 1.5.3) without reduction of productivity by directed evolution that combined an in vivo mutagenesis and membrane assay screening system. Five amino acid substitutions (T60A, A188G, M244L, N257S, and L261M) occurred in the most thermostable mutant obtained by a fourth round of directed evolution. This altered enzyme, FAOX-TE, was stable at 45 degrees C, whereas the wild-type enzyme was not stable above 37 degrees C. The K(m) values of FAOX-TE for D-fructosyl-L-valine and D-fructosyl-glycine were 1.50 and 0.58 mM, respectively, in contrast with corresponding values of 1.61 and 0.74 mM for the wild-type enzyme. This altered FAOX-TE will be useful in the diagnosis of diabetes.     

Directed evolution to improve the thermostability of prolyl endopeptidase

Prolyl endopeptidase is the only endopeptidase that specifically cleaves peptides at proline residues. Although this unique specificity is advantageous for application in protein chemistry, the stability of the enzyme is lower than those of commonly used peptidases such as subtilisin and trypsin. Therefore, we attempted to apply a directed evolution system to improve the thermostability of the enzyme. First, an efficient expression system for the enzyme in Escherichia coli was established using the prolyl endopeptidase gene from Flavobacterium meningosepticum. Then, a method for screening thermostable variants was developed by combining heat treatment with active staining on membrane filters. Random mutagenesis by error-prone PCR and screening was repeated three times, and as a result the thermostability of the enzyme was increased step by step as the amino acid substitutions accumulated. The most thermostable mutant obtained after the third cycle, PEP-407, showed a half-life of 42 min at 60 degrees C, which was 60 times longer than that of the wild-type enzyme. The thermostable mutant was also more stable with a high concentration of glycerol, which is a necessary condition for in vitro amidation.     

Microbial production of levansucrase for synthesis of fructooligosaccharides and levan

A newly isolated thermophilic bacterial strain from Tunisian thermal source was identified as Bacillus sp. and was selected for its ability to produce extracellular levansucrase. Following the optimization of carbon source, nitrogen source, temperature and initial pH of the growth medium in submerged liquid cultures. In fact, sucrose was found to be a good inducer of levansucrase enzymes. The optimal temperature and pH of the levansucrase were 50°C and 6.5, respectively and its activity increased four folds in the presence of 50mM Fe(2+). This enzyme exhibited a remarkable stability and retained 100% of its original activity at 50°C for more than 1h at pH 6.5. The half-life of the enzyme was 1h at 90°C. Crude enzyme of Bacillus sp. rich in levansucrase was established for the synthesis of fructooligosaccharides and levan. Bacillus sp. could therefore be considered as a satisfactory and promising producer of thermostable levansucrases. Contrary to other levansucrases, the one presented in the current study was able to produce high levels of levan with high molecular weight at 50°C and having an important effect as a hypoglycemic agent which was demonstrated in our previous publications (Dahech et al., 2011 [25]) and as a hypo-cholesterolemic agent which will be investigated in further research.