DNA-methylases from different strains of Bacillus subtilis and Bacillus natto

DNA-methyltransferase activity has been detected in some of Bacillus subtilis and Bacillus natto strains. Two strains of Bacillus subtilis exhibited DNA-cytosine methyltransferase activity, and the strains of Bacillus natto exhibited DNA-adenine methyltransferase activity. A possible effect of DNA-methyltransferase specificity on transformation efficiency is discussed.     

Directed evolution of a maltogenic alpha-amylase from Bacillus sp. TS-25

Directed evolution coupled with a high-throughput robotic screen was employed to broaden the industrial use of the maltogenic alpha-amylase Novamyl from Bacillus sp. TS-25. Wild-type Novamyl is currently used in the baking industry as an anti-staling agent in breads baked at neutral or near neutral pH. However, the enzyme is rapidly inactivated during the baking process of bread made with low pH recipes and Novamyl thus has very limited beneficial effect for this particular application. In an effort to improve the performance of Novamyl for low pH bread applications such as sourdough and rye, two error-prone PCR libraries were generated, expressed in Bacillus subtilis and screened for variants with improved thermal stability and activity under low pH conditions. Variants exhibiting improved performance were iteratively recombined using DNA shuffling to create two generations of libraries. Relative to wild-type Novamyl, a number of the resulting variants exhibited more than 10 degrees C increase in thermal stability at pH 4.5, one of which demonstrated substantial anti-staling properties in low pH breads.     

Directed evolution for increased chitinase activity

Directed evolution through DNA shuffling and screening was used to enhance the catalytic ability of a fungal, Beauveria bassiana, chitinase, Bbchit1. The Bbchit gene was first linked to various prokaryotic signal sequences and expressed in Escherichia coli. The signal peptide, PelB, from Erwinia carotovora resulted in greatest chitinase secretion into broth. The nucleotide sequence expressing PelB signal peptide was then incorporated into an E. coli vector to express Bbchit1 variants generated by three rounds of DNA shuffling. A Bbchit1 library with 150,000 variants was constructed with a nucleotide point mutation frequency of 0.6% and screened for chitinolytic activity. Two Bbchit1 variants (SHU-1 and SHU-2) were selected that showed increased chitinolytic activity compared to the wild type. Sequence analysis of these variants revealed mutations in amino acid residues that would not normally be considered for rational design of improved chitinase activity. The amino acid substitutions occurred outside of the two putative substrate-binding sites and the catalytic region.     

Directed evolution of transketolase activity on non-phosphorylated substrates

We have used active-site targeted directed evolution by saturation mutagenesis to improve the activity of E. coli transketolase towards non-phosphorylated substrates. Residues were selected for each set based on either structural proximity to substrate, or on phylogenetic variation. Each library was screened towards the reaction between hydroxypyruvate (HPA) and glycolaldehyde (GA) to form L-erythrulose, and the location of improved mutants related to the natural sequence entropy at each residue. A number of mutants from the phylogenetically defined library were found to outperform the wild-type with up to 3-fold specific activity under biocatalytically relevant conditions, though interestingly with substituted residues that differed from those found in nature. Conserved residues which interact with the phosphate group in natural substrates also yielded mutants with almost 5-fold improved specific activity on the non-phosphorylated substrates. These results suggest that phylogenetically variant active-site residues are useful for modulating activity on natural or structurally-homologous substrates, and that conserved residues which no longer interact with modified target substrates are useful sites to apply saturation mutagenesis for improvement of activity.     

Binding activity of natto (a fermented food) and Bacillus natto isolates to mutagenic-carcinogenic heterocyclic amines.

The fermented food, whole meal Natto, viscous polymeric material from Natto, Natto bean, cooked soya bean, and 28 bacterial isolates from Natto were studied for their binding capacity to foodborne mutagenic-carcinogenic heterocyclic amines. The mutagenic heterocyclic amines used were Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole); Trp-P-2 (3-amino-1-methyl-5H-pyrido(4,3-b)indole); Glu-P-1 (2-amino-6-methyldipyrido(1,2-a:3'2'-d)imidazole); PhIP (2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine); IQ (2-amino-3-methylimidazo(4,5-f)quinoline); MeIQ (2-amino-3,4-dimethylimidazo(4,5-f)quinoxaline); MeIQx (2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline); and MeAalphaC (2-amino-3-methyl-9H-pyrido(2,3)indole). The lyophilized Natto and other fractions of Natto exhibited high binding activity towards Trp-P-1, Trp-P-2, PhIP, and MeAalphaC, while Glu-P-1, IQ, and MeIQ were not effectively bound. The binding capacity of bacterial isolates (Bacillus natto) were isolate-mutagen dependent. Heat treated lyophilized cells, cell wall, and cytoplasmic contents of the bacterial isolate with the highest binding capacity were analyzed for their ability to bind different heterocyclic amines. The results indicate the importance of the cell wall in binding to heterocyclic amines, whereas the cytoplasmic contents were less effective. Heat-treated cells were not much different from that of viable cells in their binding. The impact of different factors, such as pH, incubation time, metal ions, different concentrations of sodium chloride and alcohol, various enzymes, and acetylation of mutagens on binding of Trp-P-1 and IQ, were discussed. The significance of the present results is also discussed from the viewpoint that Natto, a fermented food, is able to scavenge dietary mutagenic heterocyclic amines through binding.     

Partial purification of nattokinase from Bacillus subtilis by expanded bed adsorption

Nattokinase was purified from unclarified Bacillus subtilisculture filtrate using an expanded bed with a purification factor of 8.2 and at a yield of 95%. The optimal pHs for adsorption and elution were 6.0 and 7.0, respectively. The expanded bed route shortened the process time by 8–10 h and increased the yield by 50% when compared with the conventional method.     

Directed evolution of the promiscuous esterase activity of carbonic anhydrase II

A promiscuous activity of an existing enzyme can confer an evolutionary advantage by providing an immediate response to a new selection pressure and a starting point for the divergence of a new enzyme. This work seeks to examine how this process might take place. Human carbonic anhydrase II (hCAII) is an enzyme that evolved to catalyze the reversible hydration of CO(2) and performs this task at a remarkable rate (k(cat) approximately 10(6) s(-)(1)). hCAII also exhibits promiscuous activity toward highly activated esters such as 4-nitrophenyl acetate. We describe a much weaker esterase activity of hCAII toward the bulkier and much less activated ester substrate 2-naphthyl acetate (2NA). Directed evolution of hCAII produced a variant with 40-fold higher rates toward 2NA, owing to two mutations: one within the active site (Ala65Val) and one at its mouth (Thr200Ala). Structure-activity studies suggest that these mutations led to adaptation of the active site for bulkier substrates and for the catalysis of nonactivated esters. The mutations did not, however, significantly alter the native activity of hCAII. Our results support the notion that the evolution of a new function can be driven by mutations that increase a promiscuous function (which serves as the starting point for the evolutionary process) but do not harm the native function.     

Directed evolution of the thermostable xylanase from Thermomyces lanuginosus

The thermostability of the endo-beta-1,4-xylanase from Thermomyces lanuginosus (xynA) was improved by directed evolution using error-prone PCR. Transformants expressing the variant xylanases were first selected on 0.4% Remazol Brilliant Blue-xylan and then exposed to 80 degrees C. Whereas the wild type XynA lost 90% activity after 10 min at 80 degrees C, five mutants displayed both higher stabilities and activities than XynA. Four mutants were subjected to further mutagenesis to improve the stability and activity of the xylanase. Subsequent screening revealed three mutants with enhanced thermostability. Mutant 2B7-10 retained 71% of its activity after treatment at 80 degrees C for 60 min and had a half-life of 215 min at 70 degrees C, which is higher than that attained by XynA. Sequence analysis of second generation mutants revealed that mutations were not concentrated in any particular region of the protein and exhibited much variation. The best mutant obtained from this study was variant 2B7-10, which had a single substitution (Y58F) in beta-sheet A of the protein, which is the hydrophilic, solvent-accessible outer surface of the enzyme. Most of the mutants obtained in this study displayed a compromise between stability and activity, the only exception being mutant 2B7-10. This variant showed increased activity and thermostability.     

Directed evolution of biocatalysts

Directed evolution is being used increasingly in academic and industrial laboratories to modify and improve important biocatalysts. Significant advances during this period of review include compartmentalization of genes and the in vitro translation apparatus in emulsions, as well as several impressive demonstrations of catalyst improvement. Shuffling of homologous genes offers a new way to utilize natural diversity in the evolution of novel catalysts.     

Directed evolution of alpha-aspartyl dipeptidase from Salmonella typhimurium.

Model-free approaches (error-prone PCR to introduce random mutations, DNA shuffling to combine positive mutations, and screening of the resultant mutant libraries) have been used to enhance the catalytic activity and thermostability of alpha-aspartyl dipeptidase from Salmonella typhimurium, which is uniquely able to hydrolyze Asp-X dipeptides (where X is any amino acid) and one tripeptide (Asp-Gly-Gly). Under double selective pressures of activity and thermostability, through two rounds of error-prone PCR and three sequential generations of DNA shuffling, coupled with screening, a mutant pepEM3074 with approximately 47-fold increased enzyme activity compared with its wild-type parent was obtained. Moreover, the stability of pepEM3074 is increased significantly. Three amino acid substitutions (Asn89His, Gln153Glu, and Leu205Arg), two of them are near the active site and substrate binding pocket, were identified by sequencing the genes encoding this evolved enzyme. The mechanism of the enhancement of activity and stability was analyzed in this paper.     

纳豆杆菌对白假丝酵母菌的拮抗作用

目的探讨纳豆杆菌对白假丝酵母菌的拮抗作用。方法将纳豆杆菌和白假丝酵母菌混合培养24 h后,应用沙保弱平板培养基分离白假丝酵母菌,计数菌落,计算纳豆杆菌对白假丝酵母菌的拮抗率。结果纳豆杆菌对白假丝酵母菌的拮抗作用明显,拮抗率高达91.91%;纳豆杆菌肉汤培养物的除菌滤液对白假丝酵母菌也有明显的拮抗作用,拮抗率为79.05%。结论纳豆杆菌对白假丝酵母菌具有明显拮抗作用,是白假丝酵母菌的理想拮抗菌株。     

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.     

Purification and properties of a fibrinolytic enzyme from Bacillus subtilis

A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis. The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2 Copper ions significantly increased enzyme activity, while Zn++ and Mn++ caused marked inhibition.     

Directed evolution and characterization of atrazine chlorohydrolase variants with enhanced activity

Atrazine chlorohydrolase (AtzA, EC 3.8.1.8) has attracted widespread interests as it catalyzes conversion of toxic atrazine to nontoxic hydroxyatrazine and can be used in the biodegradation of atrazine. To facilitate this application, a Haematococcus pluvialis-based method was applied to screen AtzA variants from a random mutagenesis library. Eight variants with enhanced enzyme activity were obtained. They showed 2.7- to 5.0-fold increase in specific activity compared with the wild type. Sequencing revealed that the two most active variants contained single substitution at Val12 and Leu395, respectively, while several improved variants contained substitutions at the four sites of Met315, His399, Asn429, and Val466 simultaneously, indicating that these residues contribute to the enzyme activity of AtzA. Kinetic analysis showed that five variants decreased the K _m value 0.6- to 0.9-fold, whereas all the variants increased the catalytic efficiency (k _cat/K _m value) 2.5- to 4.1-fold compared to the wild type. The modeled three-dimensional structure showed that AtzA is comprised of a typical (β/α)₈ domain of the amidohydrolase superfamily and a dual β-sheet domain. An iron ion and five ligand-binding residues are located in the β-barrel core of the (β/α)₈ domain. Some substituted residues are involved in hydrogen bond formation in the (β/α)₈-neighboring β-sheet.     

Computational design of glutamate dehydrogenase in Bacillus subtilis natto

Bacillus subtilis natto is widely used in industry to produce natto, a traditional and popular Japanese soybean food. However, during its secondary fermentation, high amounts of ammonia are released to give a negative influence on the flavor of natto. Glutamate dehydrogenase (GDH) is a key enzyme for the ammonia produced and released, because it catalyzes the oxidative deamination of glutamate to alpha-ketoglutarate using NAD⁺ or NADP⁺ as co-factor during carbon and nitrogen metabolism processes. To solve this problem, we employed multiple computational methods model and re-design GDH from Bacillus subtilis natto. Firstly, a structure model of GDH with cofactor NADP⁺ was constructed by threading and ab initio modeling. Then the substrate glutamate were flexibly docked into the structure model to form the substrate-binding mode. According to the structural analysis of the substrate-binding mode, Lys80, Lys116, Arg196, Thr200, and Ser351 in the active site were found could form a significant hydrogen bonding network with the substrate, which was thought to play a crucial role in the substrate recognition and position. Thus, these residues were then mutated into other amino acids, and the substrate binding affinities for each mutant were calculated. Finally, three single mutants (K80A, K116Q, and S351A) were found to have significant decrease in the substrate binding affinities, which was further supported by our biochemical experiments.     

Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-l-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of k_cat/K_m of 72-fold toward 3-oxo-N-dodecanoyl-l-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-l-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.     

Directed evolution of the alpha-L-fucosidase from Thermotoga maritima into an alpha-L-transfucosidase

The alpha-L-fucosidase from Thermotoga maritima (Tm alpha fuc) was converted into alpha-L-transfucosidase variants by directed evolution. The wild-type enzyme catalyzes oligosaccharide synthesis by transfer of a fucosyl residue from a pNP-fucoside donor to pNP-fucoside (self-condensation) with alpha-(1-->3) regioselectivity or pNP-galactoside (transglycosylation) with alpha-(1-->2) regioselectivity at low yields (7%). The wild-type enzyme was submitted to one cycle of mutagenesis, followed by rational recombination of the selected mutations, which allowed identification of variants with improved transferase activity. The transferase and hydrolytic kinetics of all the mutants were assessed by NMR methods and capillary electrophoresis. It was shown that the best mutant exhibited a dramatic 32-fold increase in the transferase/hydrolytic kinetic ratio, while keeping 60% of the overall wild-type enzyme activity. Accordingly, the maximum yield of a specific transglycosylation product [pNP-Gal-alpha-(1-->2)-Fuc] reached more than 60% compared to 7% with WT enzyme at equimolar and low concentrations of donor and acceptor (10 mM). Such an improvement was obtained with only three mutations (T264A, Y267F, L322P), which were all located in the second amino acid shell of the fucosidase active site. Molecular modeling suggested that some of these mutations (T264A, Y267F) cause a reorientation of the amino acids that are in direct contact with the substrates, resulting in a better docking energy. Such mutants with high transglycosidase activity may constitute novel enzymatic tools for the synthesis of fucooligosaccharides.     

Directed molecular evolution of antibodies

Antibodies play a key role in the immune system, are characterized by a homogeneous overall structure and by their ability to interact with an almost unlimited number of compounds. Encoded by a fixed number of genes, they acquire their specificity and affinity of recognition after a succession of genetic recombination and molecular mutation processes. Since the pioneer works of Kohler and Milstein in 1975 describing the possibility of producing monoclonal antibodies with pre-determined specificity, the use of antibodies in the fields of research, diagnosis and therapy has never stopped increasing. Thus, about twenty monoclonal antibodies have yet been authorized to be used in human immunotherapy. However, a majority of these molecules have been engineered to bring them into line with their clinical use: chimerization, humanization, recombinant expression of single or fused fragments. Furthermore, the recent development of in vitro molecular evolution approaches now make it possible to engineer the affinity, the specificity as well as the stability of monoclonal antibodies. The potential of in vitro molecular evolution of antibodies will be illustrated through the example of the specificity improvement of an anti-progesterone antibody.     

Directed evolution of enzyme stability

Modern enzyme development relies to an increasing extent on strategies based on diversity generation followed by screening for variants with optimised properties. In principle, these directed evolution strategies might be used for optimising any enzyme property, which can be screened for in an economically feasible way, even if the molecular basis of that property is not known. Stability is an interesting property of enzymes because (1) it is of great industrial importance, (2) it is relatively easy to screen for, and (3) the molecular basis of stability relates closely to contemporary issues in protein science such as the protein folding problem and protein folding diseases. Thus, engineering enzyme stability is of both commercial and scientific interest. Here, we review how directed evolution has contributed to the development of stable enzymes and to new insight into the principles of protein stability. Several recent examples are described. These examples show that directed evolution is an effective strategy to obtain stable enzymes, especially when used in combination with rational or semi-rational engineering strategies. With respect to the principles of protein stability, some important lessons to learn from recent efforts in directed evolution are (1) that there are many structural ways to stabilize a protein, which are not always easy to rationalize, (2) that proteins may very well be stabilized by optimizing their surfaces, and (3) that high thermal stability may be obtained without forfeiture of catalytic performance at low temperatures.