Directed evolution of beta-galactosidase from Escherichia coli into beta-glucuronidase

In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes. E. coli beta-galactosidase and beta-glucuronidase are widely used, and their biological function, catalytic mechanism, and molecular structures are well characterized. We applied an in vitro directed evolution strategy through DNA shuffling and obtained five mutants named YG6764, YG6768, YG6769, YG6770 and YG6771 after two rounds of DNA shuffling and screening, which exhibited more beta-glucuronidase activity than wild-type beta-galactosidase. These variants had mutations at fourteen nucleic acid sites, resulting in changes in ten amino acids: S193N, T266A, Q267R, V411A, D448G, G466A, L527I, M543I, Q626R and Q951R. We expressed and purified those mutant proteins. Compared to the wild-type protein, five mutant proteins exhibited high beta-glucuronidase activity. The comparison of molecular models of the mutated and wildtype enzymes revealed the relationship between protein function and structural modification.     

A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences

Directed evolution in vitro is a powerful molecular tool for the creation of new biological phenotypes. It is unclear whether it is more efficient to mutate an enzyme randomly or to mutate just the active sites or key sites. In this study, the strategy of a semi-rational design of directed evolution combined with whole sequence and sites was developed. The 1553 bp gene encoding the thermostable beta-galactosidase of Pyrococcus woesei was chemically synthesized and optimized for G+C content and mRNA secondary structures. The synthesized gene product was used as a template or as a wild-type control. On the basis of the first round of DNA shuffling, library construction and screening, one mutant of YH6754 was isolated with higher activity. Eight potential key sites were deduced from the sequence of the shuffled gene, and 16 degenerate oligonucleotides were designed according to those eight amino acids. Two variants of YG6765 and YG8252 were screened in the second part of DNA shuffling, library construction and screening. For comparison, one mutant of YH8757 was screened through the same routine rounds of directed evolution with YH6754 as template. The purified beta-galactosidase from YH8757 exhibited a lower specific activity at 25 degrees C than those purified from mutated YG6755 and YG8252.     

Directed evolution of the surface chemistry of the reporter enzyme beta-glucuronidase.

The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a reporter in gene expression studies is limited due to loss of activity during tissue fixation by glutaraldehyde or formaldehyde. We have directed the evolution of a GUS variant that is significantly more resistant to both glutaraldehyde and formaldehyde than the wild-type enzyme. A variant with eight amino acid changes was isolated after three rounds of mutation, DNA shuffling, and screening. Surprisingly, although glutaraldehyde is known to modify and cross-link free amines, only one lysine residue was mutated. Instead, amino acid changes generally occurred near conserved lysines, implying that the surface chemistry of the enzyme was selected to either accept or avoid glutaraldehyde modifications that would normally have inhibited function. We have shown that the GUS variant can be used to trace cell lineages in Xenopus embryos under standard fixation conditions, allowing double staining when used in conjunction with other reporters.     

Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution

Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting activity and stability properties suitable for applications in laundry and dish washing detergents. However, BgAP suffers from a significant decrease of activity at low temperatures. In order to increase BgAP activity at 15°C, a directed evolution campaign based on the SeSaM random mutagenesis method was performed. An optimized microtiter plate expression system in B. subtilis was established and classical proteolytic detection methods were adapted for high throughput screening. In parallel, the libraries were screened for increased residual proteolytic activity after incubation at 58°C. Three iterative rounds of directed BgAP evolution yielded a set of BgAP variants with increased specific activity (K_cat) at 15°C and increased thermal resistance. Recombination of both sets of amino acid substitutions resulted finally in variant MF1 with a 1.5‐fold increased specific activity (15°C) and over 100 times prolonged half‐life at 60°C (224 min compared to 2 min of the WT BgAP). None of the introduced amino acid substitutions were close to the active site of BgAP. Activity‐altering amino acid substitutions were from non‐charged to non‐charged or from sterically demanding to less demanding. Thermal stability improvements were achieved by substitutions to negatively charged amino acids in loop areas of the BgAP surface which probably fostered ionic and hydrogen bonds interactions. Biotechnol. Bioeng. 2013; 110: 711–720. © 2012 Wiley Periodicals, Inc.     

Identification of a key functional region in harpins from <Emphasis Type="Italic">Xanthomonas</Emphasis> that suppresses protein aggregation and mediates harpin expression in <Emphasis Type="Italic">E. coli</Emphasis>

In the current study, we identified a key functional region in harpins from Xanthomonas that suppressed protein aggregation and mediated its expression in E. coli. Our data suggested that the presence of two common features in harpins [Wei et al. (1992) Science 257:85-88], namely, high glycine content and lack of cysteine residues, were not sufficient for Xanthomonas to elicit hypersensitive response (HR) activity or heat stability. Additionally, bioinformatic analyses revealed that the secondary structure of a conserved N-terminal region consisting of 12 highly hydrophilic amino acids (QGISEKQLDQLL) was α-helical. Following site-directed mutagenesis deletion of this region, the three mutated harpin proteins, in cultures induced at 37°C, failed to elicit a HR in tobacco leaves. However, at 24°C, two mutated harpins retained the ability to elicit HR, albeit with lower expression levels than that noted with the wild-type. SDS-PAGE and Western blot data suggested the HpaG mutant protein was found almost entirely in the inclusion body. These data demonstrated that these conserved amino acid residues played a critical role in protein aggregation and inclusion body formation in harpins from Xanthomonas.     

Cloning and expression of the sucrose phosphorylase gene from <Emphasis Type="Italic">Leuconostoc </Emphasis><Emphasis Type="Italic">mesenteroides</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene encoding sucrose phosphorylase (742sp) in Leuconostoc mesenteroides NRRL B-742 was cloned and expressed in Escherichia coli. The nucleotide sequence of the transformed 742sp comprised an ORF of 1,458 bp giving a protein with calculated molecular mass of 55.3 kDa. 742SPase contains a C-terminal amino acid sequence that is significantly different from those of other Leu. mesenteroides SPases. The purified 742SPase had a specific activity of 1.8 U/mg with a K _m of 3 mM with sucrose as a substrate; optimum activity was at 37°C and pH 6.7. The purified 742SPase transferred the glucosyl moiety of sucrose to cytosine monophosphate (CMP). Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Purification of branched-chain amino acid aminotransferase from <Emphasis Type="Italic">Helicobacter pylori</Emphasis> NCTC 11637

Summary. Branched-chain amino acid aminotransferase was purified by several column chromatographies from Helicobacter pylori NCTC 11637, and the N-terminal amino acid sequence was analyzed. The enzyme gene was sequenced based on a putative branched-chain amino acid aminotransferase gene, ilvE of H. pylori 26695, and the whole amino acid sequence was deduced from the nucleotide sequence. The enzyme existed in a homodimer with a calculated subunit molecular weight (MW) of 37,539 and an isoelectric point (pI) of 6.47. The enzyme showed high affinity to 2-oxoglutarate (K _m = 0.085 mM) and L-isoleucine (K _m = 0.34 mM), and V _max was 27.3 μmol/min/mg. The best substrate was found to be L-isoleucine followed by L-leucine and L-valine. No activity was shown toward the D-enantiomers of these amino acids. The optimal pH and temperature were pH 8.0 and 37 °C, respectively.     

Improvement of the Optimum Temperature of Penicillium expansum Lipase by Site-Directed Mutagenesis

In order to improve the optimum temperature of lipases, the Penicillum expansum lipase (PEL) gene was mutated by site-directed mutagenesis using overlap extension PCR technique. The recombinant plasmid containing mutant E83V pPIC3.5K-lip-E83V was expressed in Pichia pastoris GS115. Comparison experiments of the mutant PEL-E83V-GS and the wild-type PEL-GS showed that the optimum temperature (45°C) of the mutant was 5°C higher than that of the wild type. The thermostability of the mutant was similar to that of the wild type. The enzymatic activity of the mutant was 188 U/ml at 37°C, which was 80% that of the wild type in the same conditions. Hydrophobic interaction may be enhanced in the surface region by the hydrophilic amino acid Glu substituted with the hydrophobic amino acid Val, and may be responsible for the improvement of the optimum temperature. Translated from Microbiology, 2005, 32(1) (in Chinese)     

Enhancement of thermostability of fungal deglycating enzymes by directed evolution

Fructosyl peptide oxidases are valuable for the determination of glycoproteins such as hemoglobin A1c. For practical use in clinical diagnosis, we applied directed evolution to improve the thermostability of these enzymes. After two rounds of random mutagenesis and high-throughput screening, six thermostabilizing amino acid substitutions were identified. Therefore, site-directed and cassette mutageneses were applied to combine these six stabilizing mutations. The simultaneous mutants showed that the stabilizing effect of the amino acid replacement was cumulative. The sextuple mutant enzyme, R94K/G184D/F265L/N272D/H302R/H388Y, had a half-life of thermal inactivation at 50°C that was 79.8-fold longer than that of the parental fructosyl peptide oxidase. The thermostable variants also showed increased tolerance to digestion by a protease. The sextuple mutant enzyme did not lose its activity on incubation with neutral protease, while the wild-type enzyme almost completely lost its activity. Furthermore, three amino acid substitutions were introduced into another fructosyl peptide oxidase with a different substrate specificity. The half-life of inactivation at 50°C was 3.61-fold longer than that of the parent enzyme. These engineered fructosyl peptide oxidases will be useful for industrial application to clinical diagnosis.     

1H, 13C, and 15N NMR assignments of StnII-R29Q, a defective lipid binding mutant of the sea anemone actinoporin Sticholysin II

Several studies have been made to characterize the molecular properties and activity of Sticholysin II (StnII), a 175 amino acid protein secreted by the sea anemone Stichodactyla helianthus. In particular, the biochemical characterization of different mutants of this protein have been shown to be essential for the rational understanding of its activity. Here we report the nearly complete NMR ¹⁵N, ¹³C and ¹H chemical shift assignments, at pH 4.0 and 25°C, of a less hemolytic and defective lipid binding mutant of StnII, the R29Q variant (BMRB no 16362).     

Improvement of low‐temperature caseinolytic activity of a thermophilic subtilase by directed evolution and site‐directed mutagenesis

By directed evolution and subsequent site‐directed mutagenesis, cold‐adapted variants of WF146 protease, a thermophilic subtilase, have been successfully engineered. A four‐amino acid substitution variant RTN29 displayed a sixfold increase in caseinolytic activity in the temperature range of 15–25°C, a down‐shift of optimum temperature by ～15°C, as well as a decrease in thermostability, indicating it follows the general principle of trade‐off between activity and stability. Nevertheless, to some extent RTN29 remained its thermophilic nature, and no loss of activity was observed after heat‐treatment at 60°C for 2 h. Notably, RTN29 exhibited a lower hydrolytic activity toward suc‐AAPF‐pNA, due to an increase in K_m and a decrease in k_cat, in contrast to other artificially cold‐adapted subtilases with increased low‐temperature activity toward small synthetic substrates. All mutations (S100P, G108S, D114G, M137T, T153A, and S246N) identified in the cold‐adapted variants occurred within or near the substrate‐binding region. None of these mutations, however, match the corresponding sites in naturally psychrophilic and other artificially cold‐adapted subtilases, implying there are multiple routes to cold adaptation. Homology modeling and structural analysis demonstrated that these mutations led to an increase in mobility of substrate‐binding region and a modulation of substrate specificity, which seemed to account for the improvement of the enzyme's catalytic activity toward macromolecular substrates at lower temperatures. Our study may provide valuable information needed to develop enzymes coupling high stability and high low‐temperature activity, which are highly desired for industrial use. Biotechnol. Bioeng. 2009; 104: 862–870. © 2009 Wiley Periodicals, Inc.     

Improving the thermostability of <Emphasis Type="Italic">N</Emphasis>-carbamyl-<Emphasis Type="SmallCaps">d</Emphasis>-amino acid amidohydrolase by error-prone PCR

To facilitate the easier production of d-amino acids using N-carbamyl-d-amino acid amidohydrolase (DCase) in an immobilized form, we improved the enzymatic thermostability of highly soluble DCase-M3 of Ralstonia pickettii using directed mutagenesis. Six novel mutation sites were identified in this study, apart from several thermostability-related amino acid sites reported previously. The most thermostable mutant, in which the 12th amino acid had been changed from glutamine to leucine, showed a 7 °C increase in thermostability. Comparative characterization of the parental and mutant DCases showed that although there was a slight reduction in the oxidative stability of the mutants, their kinetic properties and high solubility were not affected. The mutated enzymes are expected to be applied to the development of a fully enzymatic process for the industrial production of d-amino acids.     

Purification and biochemical characterization of a broad substrate specificity thermostable alkaline protease from Aspergillus nidulans

Aspergillus nidulans PW1 produces an extracellular carboxylesterase activity that acts on several lipid esters when cultured in liquid media containing olive oil as a carbon source. The enzyme was purified by gel filtration and ion exchange chromatography. It has an apparent MW and pI of 37 kDa and 4.5, respectively. The enzyme efficiently hydrolyzed all assayed glycerides, but showed preference toward short- and medium-length chain fatty acid esters. Maximum activity was obtained at pH 8.5 at 40°C. The enzyme retained activity after incubation at pHs ranging from 8 to11 for 12 h at 37°C and 6 to 8 for 24 h at 37°C. It retained 80% of its activity after incubation at 30 to 70°C for 30 min and lost 50% of its activity after incubation for 15 min at 80°C. Noticeable activation of the enzyme is observed when Fe²⁺ ion is present at a concentration of 1 mM. Inhibition of the enzyme is observed in the presence of Cu²⁺, Fe³⁺, Hg²⁺, and Zn²⁺ ions. Even though the enzyme showed strong carboxylesterase activity, the deduced N-terminal amino acid sequence of the purified protein corresponded to the protease encoded by prtA gene.     

Enhancement of the activity and pH-performance of chitosanase from Bacilluscereus strains by DNA shuffling

DNA shuffling was carried out with two chitosanase genes belonging to glycoside hydrolase family eight from Bacillus cereus KNUC51 and B. cereus KNUC55. The shuffled products, YM18 and YM20, which showed higher activity than the parents at 40°C, were selected for further studies. The 50 kDa chitosanases were purified using affinity chromatography with glutathione-Sepharose 4B. In general, the specific activity of YM18 is enhanced 250% and that of YM20 is 350% compared to the parents. YM20 exhibits a shift of the optimal pH level from 5.5 to 6.5. DNA sequence analysis revealed that YM18 and YM20 contained 2 amino acid substitutions (I13T and A87V for YM18; K66R and N352S for YM20). We presumed that these amino acid substitutions increase the specific activity and change the property of the two variants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Catalytic mechanism of S-adenosylhomocysteine hydrolase: roles of His 54, Asp130, Glu155, Lys185, and Aspl89

S-adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine (AdoHcy) to form adenosine and homocysteine. The crystal structure of the K185N mutated enzyme, which has weak catalytic activity (0.1%), has been determined at 2.8 A resolution and supports the previously predicted mechanism [Takata, Y., Yamada, T., Huang, Y., Komoto, J., Gomi, T., Ogawa, H., Fujioka, M., & Takusagawa, F. (2002). Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190. J. Biol. Chem. 277, 22670-22676]. The mutated enzyme has an intermediate structure between the open and closed conformation, observed in the substrate-free enzyme and in the inhibitor complexes, respectively. H54, H300, and H352 were mutated to asparagine, respectively, to identify the roles of the histidine residues in catalysis. The kinetic data of H54N, H300N, and H354N mutated enzymes suggest that H54 is the amino acid residue that acts as a general acid/base to cleave the C5'-S(D) bond of AdoHcy. The E155Q mutated enzyme retained a large portion of the catalytic activity (31%), while the E155D mutated enzyme lost most of it (0.3%). The NADH accumulation measurements of the mutated enzymes indicated that the C3'-oxidation and the C4'-proton abstraction are a concerted event and the C5'-S(D) bond cleavage is an independent event. The C4'-proton exchange measurements indicate that the enzyme has an open conformation when AdoHcy is converted to 3'-keto-4', 5'-dehydro-Ado in the active site. With the results of this study and those of the previous studies, a detailed catalytic mechanism of AdoHcyase is described. K185 facilitates the C3'-oxidation, D130 abstracts the C4'-proton, D189, and E155 act as a communicator between the concerted C3'-oxidation and C4'-proton abstraction, and H54 plays as a general acid to cleave the C5'-S(D) bond of AdoHcy.     

Molecular and functional properties of the psychrophilic elongation factor G from the Antarctic Eubacterium <Emphasis Type="Italic">Pseudoalteromonas haloplanktis</Emphasis> TAC 125

The molecular and functional properties of the elongation factor (EF) G from the psychrophilic Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph) were studied. PhEF-G catalyzed protein synthesis in vitro that was inhibited by fusidic acid, an antibiotic specifically acting on EF-G. The EF interacted with GDP only in the presence of P. haloplanktis ribosome and fusidic acid with an affinity similar to that displayed by Escherichia coli EF-G. The psychrophilic translocase elicited a ribosome-dependent GTPase that was competitively inhibited by GDP, the slowly hydrolyzable GTP analog GppNHp, and the protein synthesis inhibitor ppGDP. The temperature dependence of the activity of PhEF-G reached its maximum at least 26°C beyond the growth temperature of P. haloplanktis (4–20°C). The heat inactivation profile of the ribosome-dependent GTPase of PhEF-G gave a temperature for half inactivation (46°C), significantly lower than that for half denaturation measured by either UV- (57°C) or fluorescence-melting (62°C). This finding was attributed to a different effect of the temperature on the catalytic domain with respect to that elicited on the other domains constituting the EF, thus confirming the differential molecular flexibility present in psychrophilic enzymes. A molecular model, based on the 3D coordinates of a thermophilic EF-G, showed differences only in connecting loops. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Engineering of NADPH-dependent aldo-keto reductase from <Emphasis Type="Italic">Penicillium citrinum</Emphasis> by directed evolution to improve thermostability and enantioselectivity

Penicillium citrinum β-keto ester reductase (KER) can catalyze the reduction of methyl 4-bromo-3-oxobutyrate (BAM) to methyl (S)-4-bromo-3-hydroxybutyrate with high optical purity. To improve the thermostability of KER, protein engineering was performed using error-prone polymerase chain reaction-based random mutagenesis. Variants with the highest levels of thermostability contained the single amino acid substitutions L54Q, K245R, and N271D. The engineered L54Q variant of KER retained 62% of its initial activity after heat treatment at 30°C for 6 h, whereas wild-type KER showed only 15% activity. The L54Q substitution also conferred improved enantioselectivity by KER. An Escherichia coli cell biocatalyst that overproduced the L54Q mutant of KER and glucose dehydrogenase as a cofactor regeneration enzyme showed the highest level of BAM reduction in a water/butyl acetate two-phase system.     

Isolation and characterization of a <Emphasis Type="Italic">Mastigocladus</Emphasis> species capable of growth,N<Subscript>2</Subscript>-fixation and N-assimilation at elevated temperature

A Mastigocladus species was isolated from the hot spring of Jakrem (Meghalaya) India. Uptake and utilization of nitrate, nitrite, ammonium and amino acids (glutamine, asparagine, arginine, alanine) were studied in this cyanobacterium grown at different temperatures (25°C, 45°C). There was 2–3 fold increase in the heterocyst formation and nitrogenase activity in N-free medium at higher temperature (45°C). Growth and uptake and assimilation of various nitrogen sources were also 2–3 fold higher at 45°C indicating that it is a thermophile. The extent of induction and repression of nitrate uptake by NO₃ ⁻ and NH₄ ⁺, respectively, differed from that of nitrite. It appeared that Mastigocladus had two independent nitrate/nitrite transport systems. Nitrate reductase and nitrite reductase activitiy was not NO₃ ⁻-inducible and ammonium or amino acids caused only partial repression. Presence of various amino acids in the media partially repressed glutamine synthetase activity. Ammonium (methylammonium) and amino acid uptake showed a biphasic pattern, was energy-dependent and the induction of uptake required de novo protein synthesis. Ammonium transport was substrate (NH₄ ⁺)-repressible, while the amino acid uptake was substrate inducible. When grown at 25°C, the cyanobacterium formed maximum akinetes that remained viable upto 5 years under dry conditions.     

Some required events in conidial germination of Neurospora crassa.

A temperature-sensitive mutant of Neurospora crassa, with reduced levels of protein synthesis at 37°C, was used to identify some essential events in conidial germination. Conidia of mutant strain psi-1 were incubated for 2 hr at 37°C and then shifted to 20°C. Germination was inhibited at 37°C, but commenced after 1.5 hr at 20°C. Increases in aspartate transcarbamylase activity, cell wall synthesis, and nuclear number preceded germination. However, increases in glutamate dehydrogenase activity, amino acid uptake, and DNA synthesis were inhibited prior to germination. Although all of these events were correlated with germination in control cultures of the mutant at 20°C and of its parent strain at 20 and 37°C, some events were apparently not essential for germination. The requirement for aspartate transcarbamylase activity was demonstrated independently by the failure of strain pyr-3d (lacking the activity) to germinate in the absence of uridine. The dispensability of glutamate dehydrogenase activity and DNA synthesis for the germination of some conidia was verified by the germination of strain am-1 (lacking glutamate dehydrogenase activity) in the absence of glutamate and by the germination of the parent strain in the presence of hydroxyurea (an inhibitor of DNA synthesis). These findings identify some landmarks in germination which may be useful in further studies of the regulation of a developmental program. They also provide preliminary evidence that the resting conidia may contain nuclei arrested at different stages of their division cycle.