Ultrasensitive detection and identification of BRAF V600 mutations in fresh frozen,FFPE, and plasma samples of melanoma patients by E-ice-COLD-PCR

A number of molecular diagnostic methods have been developed for the detection and identification of mutations in tumor samples, which are important for the choice of treatment in the context of personalized medicine. For the treatment of metastatic melanoma, Vemurafenib is recommended for patients with BRAF V600 activating mutations. However, the different assays developed to date for the detection of these mutations lack sensitivity or specificity or do not allow a sequencing-based identification or validation of the mutation. Recently, enhanced improved and complete enrichment co-amplification at lower denaturation temperature-polymerase chain reaction (E-ice-COLD-PCR) has been developed as a sensitive method for the detection and identification of mutations in KRAS codons 12/13. Here, we present the first E-ice-COLD-PCR assay for the detection and identification of BRAF codon 600 mutations, which has a large dynamic range, as 25 pg to 25 ng can be used as DNA input without any reduction in mutation enrichment efficiency, and which can detect down to 0.01 % of mutated alleles in a wild-type background. The assay has been validated on fresh frozen, formalin-fixed paraffin-embedded (FFPE), and plasma samples of melanoma patients and has allowed the detection and identification of BRAF mutations present in samples appearing as wild type using standard pyrosequencing, endpoint genotyping, or Sanger sequencing. Thus, the BRAF V600 E-ice-COLD-PCR assay is currently one of the most powerful molecular diagnostic tools for the ultrasensitive detection and identification of BRAF codon 600 mutations.     

Development of a novel class of B-Raf(V600E)-selective inhibitors through virtual screening and hierarchical hit optimization

Oncogenic mutations in critical nodes of cellular signaling pathways have been associated with tumorigenesis and progression. The B-Raf protein kinase, a key hub in the canonical MAPK signaling cascade, is mutated in a broad range of human cancers and especially in malignant melanoma. The most prevalent B-Raf(V600E) mutant exhibits elevated kinase activity and results in constitutive activation of the MAPK pathway, thus making it a promising drug target for cancer therapy. Herein, we describe the development of novel B-Raf(V600E) selective inhibitors via multi-step virtual screening and hierarchical hit optimization. Nine hit compounds with low micromolar IC(50) values were identified as B-Raf(V600E) inhibitors through virtual screening. Subsequent scaffold-based analogue searching and medicinal chemistry efforts significantly improved both the inhibitor potency and oncogene selectivity. In particular, compounds and possess nanomolar IC(50) values with selectivity for B-Raf(V600E)in vitro and exclusive cytotoxicity against B-Raf(V600E) harboring cancer cells.     

Quantitative analysis of wild-type and V600E mutant BRAF proteins in colorectal carcinoma using immunoenrichment and targeted mass spectrometry

The BRAF V600E mutation is one of the most common mutations implicated in the development of several types of cancer including colorectal cancer (CRC), where it is associated with aggressive disease phenotypes and poor outcomes. The status of the BRAF V600E mutation is frequently determined by direct DNA sequencing. However, no previous study has sought to quantify the BRAF V600E protein in cancer specimens. Here, we evaluated immunoenrichment coupled with two MS-based quantitative techniques, namely multiple reaction monitoring (MRM) and single ion monitoring conjugated accurate inclusion mass screening (SIM-AIMS), to detect and precisely quantify wild-type (WT) and V600E mutant BRAF proteins in DNA sequence-confirmed CRC tissue specimens. WT and V600E BRAF proteins were immunoprecipitated from a CRC cell line (HT-29), and their representative peptides (⁵⁹²IGDFGLATVK⁶⁰¹ and ⁵⁹²IGDFGLATEK⁶⁰¹, respectively) were confirmed by LC-MS/MS analysis and then quantified by MRM or SIM-AIMS with spiked stable isotope-labeled peptide standards. Both assays worked well for measuring WT BRAF from different amounts of HT-29 cell lysates, but the MRM assay was more sensitive than SIM-AIMS assay for quantifying lower levels of V600E BRAF. In protein extracts (2 mg) from 11 CRC tissue specimens, the MRM assay could measure WT BRAF in all 11 cases (0.32–1.66 ng) and the V600E BRAF in two cases (0.1–0.13 ng; mutant-to-WT ratio, 0.16–0.17). The SIM-AIMS assay could also detect WT and V600E BRAF in CRC specimens, but the measured levels of both targets were lower than those determined by MRM assay. Collectively, this study provides an effective method to precisely quantify WT and V600E BRAF proteins in complex biological samples using immunoenrichment-coupled targeted MS. Since the V600E BRAF protein has emerged as an important therapeutic target for cancer, the developed assay should facilitate future BRAF-related basic and clinical studies.     

Design,synthesis, docking and mechanistic studies of new thiazolyl/thiazolidinylpyrimidine-2,4-dione antiproliferative agents

In this article, we display on the synthesis and biological evaluation of a new series of thiazolylpyrimidine 3a-l and thiazolidinylpyrimidine derivatives 5a-e. The structures of the new compounds were confirmed by using different spectral techniques including NMR, IR, mass spectroscopy in addition to elemental analyses. The cell viability of the new compounds was assessed against normal human mammary gland epithelial (MCF-10A) cell line. Data revealed that none of the compounds examined exhibited cytotoxic effects, and the cell viability for the compounds examined at 50 µM was greater than 87%. The antiproliferative activity of 3a-l and 5a-e was evaluated against four human cancer cell lines where the compounds showed promising activity. The most potent derivatives were compounds 3a, 3c, 3f, 3i, and 5b with GI₅₀ values ranging from 0.90 µM to 1.70 µM against the four cancer cell lines in comparison to doxorubicin (GI₅₀ = 1.10 µM). Compounds 3a, 3c and 3i showed potent antiproliferative activity with dual inhibitory action against EGFR and BRAF^V600E. Compounds 3a, 3c, and 3i demonstrated promising AutoDock scores towards EGFR and BRAF^V600E with values of ? 9.1 and ? 8.6, ?9.0 and ? 8.5, and ? 8.4 and ? 8.0 kcal/mol, respectively. The physicochemical and pharmacokinetic characteristics of 3a, 3c, and 3i were anticipated, demonstrating their oral bioavailability.     

Embedded passivated-electrode insulator-based dielectrophoresis (EπDEP)

Here, we introduce a new technique called embedded passivated-electrode insulator-based dielectrophoresis (EπDEP) for preconcentration, separation, or enrichment of bioparticles, including living cells. This new method combines traditional electrode-based DEP and insulator-based DEP with the objective of enhancing the electric field strength and capture efficiency within the microfluidic channel while alleviating direct contact between the electrode and the fluid. The EπDEP chip contains embedded electrodes within the microfluidic channel covered by a thin passivation layer of only 4 μm. The channel was designed with two nonaligned vertical columns of insulated microposts (200 μm diameter, 50 μm spacing) located between the electrodes (600 μm wide, 600 μm horizontal spacing) to generate nonuniform electric field lines to concentrate cells while maintaining steady flow in the channel. The performance of the chip was demonstrated using Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacterial pathogens in aqueous media. Trapping efficiencies of 100 % were obtained for both pathogens at an applied AC voltage of 50 V peak-to-peak and flow rates as high as 10 μl/min.     

Expression and characterization of mutant forms of penicillin acylase from Alcaligenes faecalis

The sequencing of six plasmids carrying a gene of penicillin acylase from Alcaligenes faecalis VKM B1518 (AfPA) revealed the presence of random mutations in the gene; they occurred during a polymerase chain reaction. Six mutant AfPAs and a wild-type enzyme were expressed in E. coli cells. The activity assay of mutant AfPAs in E. coli cells indicated that several amino acid substitutions affect the expression level of the AfPA gene and the rate of cell growth. Four mutant AfPAs were purified; their catalytic properties and thermal stability were studied. It is shown that the amino acid substitutions under study do not affect the catalytic efficiency value. Within the experimental error, the βQ133R and βK184E (the AfPA M2 mutant) substitutions had no effect on the thermal stability of the enzyme; in the case of mutants AfPA M4 (βY90H), M5 (αD132G, βR97C), and M6 (αV5E, αN183S, and βE439G), the inactivation rate constant increased 2.4, 2.75, and 8.3 times, respectively, as compared to that of the wild-type enzyme.     

Site-Directed Mutagenesis Improves the Thermostability and Catalytic Efficiency of Aspergillus niger N25 Phytase Mutated by I44E and T252R

Aspergillus niger phytase (PhyA) has been used as a feed supplement to improve the bioavailability of phytate phosphorus to swine and poultry. However, it is unable to maintain its stability due to high temperature during the feed pelleting process. In this study, we performed site-directed mutagenesis in the Aspergillus niger N25 phyA ^m gene at residue 44I and 252 T, and they were replaced by glutamic acid and arginine. Single-site mutants I44E-PhyA and T252R-PhyA, as well as double-site mutant I44E/T252R-PhyA, were constructed to improve the thermostability of PhyA through hydrogen bondings and ionic interactions. The three mutant enzymes all showed more than 20 % improvement in thermostability compared to the wild-type enzyme after being heated at 80 °C for 10 min. Their melting temperatures (T _m) were increased by 1, 1, and 1.2 °C, respectively. The k _m values of I44E-PhyA, T252R-PhyA, and I44E/T252R-PhyA for sodium phytate were 78, 44, and 79 % lower (P <0.05) than that of the wild-type enzyme. Overall catalytic efficiency (k _cat/k _m) of I44E-PhyA, T252R-PhyA, and I44E/T252R-PhyA was improved by 310, 155, and 84 % (P <0.05) than that of the wild type, respectively. The catalytic efficiency did not seem to be negatively affected by the improvement in thermostability.     

High-throughput virtual screening and preclinical analysis identifies CB-1, a novel potent dual B-Raf/c-Raf inhibitor,effective against wild and mutant variants of B-Raf expression in colorectal carcinoma

Paradoxical Raf activation via Raf dimerization is a major drawback of wild/mutant B-Raf inhibitors. Herein, we report that CB-1 a novel, potent B-Raf/c-Raf dual inhibitor, effective against colon cancer cells, irrespective of their genetic status. High-throughput virtual screening of the ChemBridge library against wild B-Raf (B-Raf^WT), mutant B-Raf (B-Raf^V600E), and c-Raf was performed using an automated protocol with the AutoDock-VINA. Caco-2 and HT-29 cells were used. Of the 23,365 compounds screened computationally, CB-1 showed the highest binding energy towards B-Raf^WT with a ΔG_binding score of ? 13.0 kcal/mol. The compound was also predicted to be effective against B-Raf^V600E and c-Raf molecules with ΔG_binding energies of ? 10.6 and ? 10.1 kcal/mol, respectively. The compound inhibited B-Raf^WT, B-Raf^V600E and c-Raf kinases with IC₅₀ values of 27.13, 51.70, and 40.23 nM, respectively. The GI₅₀ value of CB-1 was 247.9 nM in B-Raf^WT-expressing Caco-2 cells and 352.4 nM in B-RafV600E-expressing HT-29 cells. Dose-dependent increases in total apoptosis and G₁ cell cycle phase arrest was observed in CB-1-treated colon cancer cells. The compound decreased B-Raf expression in both wild and mutant colon cancer cells. CB-1, a novel, potent dual B-Raf/c-Raf inhibitor was effective against colon cancer cells bearing wild-type and mutant variants of B-Raf expression.

     

Direct electrochemistry of double stranded DNA on ionic liquid modified carbon paste electrode

The direct electrochemistry of herring sperm double-stranded DNA (dsDNA) on an ionic liquid N-butylpyridinium hexafluorophosphate modified carbon paste electrode was investigated. The cyclic voltammogram showed two irreversible oxidation peaks at 0.868 V and 1.188 V (vs. SCE), which corresponded to the oxidation of guanine and adenine residues, respectively. Compared to the common carbon paste electrode the electrochemical response was greatly improved. The electrochemical behavior of dsDNA on the modified electrode was carefully investigated with the electrochemical parameters were calculated. Under optimal conditions the dsDNA can be directly determined in the concentration range from 50 to 600 μg mL^?1 with a detection limit of 17 μg mL^?1 (3σ).     

Self-Cloning Significantly Enhances the Production of Catalase in Bacillus subtilis WSHDZ-01

The katA gene that encodes catalase (CAT) in Bacillus subtilis WSHDZ-01 was overexpressed in B. subtilis WB600 and B. subtilis WSHDZ-01. The CAT yield in both transformed strains was significantly improved compared to that in the wild-type WSHDZ-01 in shake flask culture. When cultured in a 3-L stirred tank reactor (STR), the recombinant CAT activity in B. subtilis WSHDZ-01 could be improved by 419 %, reaching up to 39,117 U/mL and was 8,149.4 U/mg dry cell weight, which is the highest activity reported in Bacillus sp. However, the recombinant CAT in B. subtilis WB600 cultured in a 3-L STR was not significantly improved by any of the common means for process optimization, and the highest CAT activity was 3,673.5 U/mg dry cell weight. The results suggest that self-cloning of the complete expression cassette in the original strain is a reasonable strategy to improve the yield of wild-type enzymes.     

Methiothepin Increases Chemotherapy Efficacy against Resistant Melanoma Cells

We previously reported that methiothepin, a small molecule known as a nonselective serotonin 5-HT receptor antagonist, inhibited the doxorubicin efflux activity of the Hedgehog receptor Ptch1 and enhanced the cytotoxic, pro-apoptotic, anti-proliferative, and anti-clonogenic effects of doxorubicin on adrenocortical carcinoma cells. Here, we show that methiothepin also inhibits doxorubicin efflux and increases doxorubicin cytotoxicity in melanoma cells which endogenously overexpress Ptch1. Melanoma patients having the BRAF^V600E mutation are treated with vemurafenib, an inhibitor of BRAF^V600E, often in combination with trametinib, an inhibitor of MEK. Almost all patients ultimately acquire resistance to the treatment leading to disease progression. Here, we report that methiothepin overcomes the resistance of BRAF^V600E melanoma cells by enhancing the cytotoxicity of vemurafenib and trametinib on these cells leading to melanoma cells death. We observe that the addition of methiothepin to vemurafenib prevents migration of resistant melanoma cells more efficiently than vemurafenib alone. Our results provide an additional proof that Ptch1 drug efflux inhibition increases the effectiveness of anti-cancer treatments and overcomes resistance of melanoma cells expressing Ptch1.     

Quantitative HPLC-MS analysis of nucleotide sugars in plant cells following off-line SPE sample preparation

An analytical workflow was developed for the absolute quantification of uridine diphosphate (UDP)-sugars in plant material in order to compare their metabolism both in wild-type Arabidopsis thaliana and mutated plants (ugd2,3) possessing genetic alterations within the UDP-glucose dehydrogenase genes involved in UDP-sugar metabolism. UDP-sugars were extracted from fresh plant material by chloroform-methanol-water extraction and further purified by solid-phase extraction with a porous graphitic carbon adsorbent with extraction efficiencies between 80?±?5 % and 90?±?5 %. Quantitative determination of the UDP-sugars was accomplished through HPLC separation with a porous graphitic carbon column (Hypercarb^TM) which was interfaced to electrospray ionization Orbitrap mass spectrometry. The problem of instable retention times due to redox processes on the stationary phase were circumvented by grounding of the column effluent and incorporation of a column regeneration procedure using acetonitrile-water containing 0.10 % trifluoroacetic acid. The method was calibrated using external calibration and UDP as internal standard. Calibration functions were approximated by first- or second-order regression analysis for concentrations spanning three orders of magnitude. Upon injecting sample volumes of 2.65 μL, the limits of detection for the UDP-sugars were in the 70 nmol L^?1 range. Six different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-arabinose, UDP-xylose, UDP-glucuronic acid, and UDP-galacturonic acid were found in concentrations of 0.4 to 38 μg/g plant material. Data evaluation by analysis of variance (ANOVA) revealed statistically significant differences in UDP-sugar concentrations between wild-type and mutant plants, which were found to conclusively mirror the impaired metabolic pathways in the mutant plants.

Effect of Extracellular Tyrosinase on the Expression Level of P450, Fpr,and Fdx and Ortho-hydroxylation of Daidzein in <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis>

We have reported that the expression of CYP105D7 in Streptomyces avermitilis produces 112.5 mg L^?1 of 7,3′,4′-trihydroxyisoflavone (3'ODI) in 15 h of the reaction time, when 7,4′-dihydroxyisoflavone (daidzein) is used as a substrate. Although production is significant, rapid degradation of 3'ODI after 15 h was observed in a whole-cell biotransformation system, suggesting the further modification of 3'ODI by endogenous enzymes. In this present study, the effect of deletion of extracellular tyrosinase (melC2) in S. avermitilis for 3'ODI production as well as the expressions of CYP105D7, ferredoxin (Fdx), and ferredoxin reductase (Fpr) were investigated. The result revealed that daidzein hydroxylation activity in the ?melC2 mutant decreased by 40% compared with wild-type S. avermitilis. Further, melC2 deletion significantly affects the messenger RNA (mRNA) expression profile of CYP105D7 and its electron transfer counterparts. Real-time PCR analysis of 9 Fdx, 6 Fpr, and CYP105D7 revealed a significant decrease in mRNA expression level compared to wild-type S. avermitilis. The result clearly shows that the decrease in daidzein hydroxylation activity is due to the lower expression level of CYP105D7 and its electron transfer counterpart in the ?melC2 mutant. Furthermore, melC2 deletion prevents the degradation of 3'ODI.     

A Practical System for High-Throughput Screening of Mutants of <Emphasis Type="Italic">Bacillus fastidiosus</Emphasis> Uricase

For high-throughput screening (HTS) of Bacillus fastidiosus uricase mutants, a practical system was proposed. By error-prone PCR with final 1.5 mM MnCl₂, two focused libraries of mutants for A1-V158 and V150-D212 were generated separately. After induced expression of individual clones in 48-well microplates, Escherichia coli cells (BL21) were lyzed by 1.0 M Tris-HCl at pH 9.0 in 96-well microplates at 25 °C for 7.5 ~ 10.5 h; uricase reaction was continuously monitored with 0.15 mM uric acid in 96-well plates by absorbance at 298 nm to estimate V _m/K _m by kinetic analysis of reaction curve for comparison. V _m/K _m was resistant to initial uric acid levels with an upper limit 3-fold over that of initial rates. By receiver-operator-characteristic analysis of the recognition of the one of higher activity in uricase pair whose specific activity ratio was 1.8 or 3.3, the area-under-the-curve was comparable to that with cell lysates prepared by sonication treatment. A cutoff for the maximum Youden index was thus developed to recognize positive mutants of 1-fold higher activity. Indeed, mutant L171I/Y182F/Y187F/A193S of higher activity but lower thermostability at pH 7.4 and mutant V144A of higher activity and consistent thermostability were discovered. Therefore, the proposed system was practical for HTS of uricase mutants.     

ARMS TaqMan real-time PCR for genotyping factor V Leiden mutation in Han Chinese

Factor V Leiden (FV_Leiden) is a missense mutation of 1691 position (G1691A) in exon 10 of FV gene, and being a genetic risk for venous thrombosis. Currently, there are several PCR-based methods for detecting FV_Leiden mutation; however, these methods have disadvantages such as time-consuming, cumbersome steps and potentially hazardous gels. The aims of present study were to develop a simple, time-saving, accurate, and gel-free method, called amplification refractory mutation system (ARMS) TaqMan real-time PCR, for detecting FV_Leiden mutation. We severally designed two specific reverse primers for mutant and wild-type through intentional introduction of mismatched nucleotide at the penultimate 3′ position. Although target amplicon amplification efficiency is reduced, but another corresponding amplicon is almost completely inhibited. Then, specific TaqMan-probe was designed to detect target amplicon. Established method was used to detect 500 unselected samples in Han Chinese, the results showed 499 cases of wild-type and one heterozygote. Afterward, 50 randomly picked wild-type cases and one heterozygote were reexamined by bidirectional DNA sequencing, which is considered as “Gold standard method.” Exhilaratingly, the results detected by the two methods were completely consistent. At last, allelic frequency of FV_Leiden was calculated the in Han Chinese. Given the above results, A FV_Leiden heterozygote has been found in 500 random samples in Han Chinese, and the allelic frequency was 0.1%. In conclusion, the ARMS TaqMan real-time PCR is an ideal detecting system for genotyping FV_Leiden mutation in clinical application, and FV_Leiden mutation exists in Han Chinese despite extremely low prevalence.     

Volumetric Properties of 1,3-Dioxolane + Toluene + o- or p-Xylene Ternary Mixtures at 25.00 °C and Atmospheric Pressure

Excess molar volumes, $ V_{123}^{\text{E}} $ V 123 E , of 1, 3-dioxolane (1) + toluene (2) + o- or p-xylene (3) ternary mixtures have been determined dilatometrically over the entire composition range at 298.15 K. For thermodynamic consistency the experimental values were fitted to Redlich–Kister Equation. The $ V_{123}^{\text{E}} $ V 123 E values of 1, 3-dioxolane (1) + toluene (2) + o- or p-xylene (3) ternary mixtures have been found to be negative over the whole composition range. It has been observed that $ V_{123}^{\text{E}} $ V 123 E values calculated by graph theory are of the same sign and magnitude with respect to their experimental values.     

Effect of mutations to amino acid A301 and F361 in thermostability and catalytic activity of the β-galactosidase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> VTCC-DVN-12-01

Background

Beta-galactosidase (EC 3.2.1.23), a commercially important enzyme, catalyses the hydrolysis of β-1,3- and β-1,4-galactosyl bonds of polymer or oligosaccharidesas well as transglycosylation of β-galactopyranosides. Due to catalytic properties; β-galactosidase might be useful in the milk industry to hydrolyze lactose and produce prebiotic GOS. The purpose of this study is to characterize β-galactosidase mutants from B. subtilis.

Results

Using error prone rolling circle amplification (epRCA) to characterize some random mutants of the β-galactosidase (LacA) from B. subtilisVTCC-DVN-12-01, amino acid A301 and F361 has been demonstrated significantly effect on hydrolysis activity of LacA. Mutants A301V and F361Y had markedly reduced hydrolysis activity to 23.69 and 43.22 %, respectively. Mutants the site-saturation of A301 reduced catalysis efficiency of LacA to 20–50 %, while the substitution of F361 by difference amino acids (except tyrosine) lost all of enzymatic activity, indicating that A301 and F361 are important for the catalytic function. Interestingly, the mutant F361Y exhibited enhanced significantly thermostability of enzyme at 45–50 °C. At 45 °C, LacA-361Y retained over 93 % of its original activity for 48 h of incubation, whereas LacA-WT and LacA-301Vwere lost completely after 12 and 24 h of incubation, respectively. The half-life times of LacA-361Y and LacA-301 V were about 26.8 and 2.4 times higher, respectively, in comparison to the half-life time of LacA-WT. At temperature optimum 50 °C, LacA-361Y shows more stable than LacA-WT and LacA-301 V, retaining 79.88 % of its original activities after 2 h of incubation, while the LacA-WT and LacA-301 V lost all essential activities. The half-life time of LacA-361Y was higher 12.7 and 9.39 times than that of LacA-WT and LacA-301 V, respectively. LacA-WT and mutant enzymes were stability at pH 5–9, retained over 90 % activity for 72 h of incubation at 30 °C. However, LacA-WT showed a little bit more stability than LacA-301 V and LacA-361Y at pH 4.

Conclusions

Our findings demonstrated that the amino acids A301V and F361 play important role in hydrolysis activity of β -galactosidase from B. subtilis. Specially, amino acid F361 had noteworthy effect on both catalytic and thermostability of LacA enzyme, suggesting that F361 is responsible for functional requirement of the GH42 family.

     

The detection of T-Nos,a genetic element present in GMOs,by cross-priming isothermal amplification with real-time fluorescence

An isothermal cross-priming amplification (CPA) assay for Agrobacterium tumefaciens nopaline synthase terminator (T-Nos) was established and investigated in this work. A set of six specific primers, recognizing eight distinct regions on the T-Nos sequence, was designed. The CPA assay was performed at a constant temperature, 63 °C, and detected by real-time fluorescence. The results indicated that real-time fluorescent CPA had high specificity, and the limit of detection was 1.06?×?10³ copies of rice genomic DNA, which could be detected in 40 min. Comparison of real-time fluorescent CPA and conventional polymerase chain reaction (PCR) was also performed. Results revealed that real-time fluorescent CPA had a comparable sensitivity to conventional real-time PCR and had taken a shorter time. In addition, different contents of genetically modified (GM)-contaminated rice seed powder samples were detected for practical application. The result showed real-time fluorescent CPA could detect 0.5 % GM-contaminated samples at least, and the whole reaction could be finished in 35 min. Real-time fluorescent CPA is sensitive enough to monitor labeling systems and provides an attractive method for the detection of GMO.

Enhancing the Thermostability of a Cold-Active Lipase from Penicillium cyclopium by In Silico Design of a Disulfide Bridge

Cysteine mutants of a cold-active lipase (PcLipI) from Penicillium cyclopium were designed by the software Disulfide by Design Ver. 1.20 in an effort to improve enzyme thermostability by addition of a disulfide bridge. Those mutants predicted by molecular dynamics simulation to have better thermostability than the wild type were first expressed in Escherichia coli BL21(DE3) and then, for further investigation, in Pichia pastoris GS115. By replacing Val²⁴⁸ and Thr²⁵¹ with cysteines to create a disulfide bridge, the recombinant lipases reE-PcLip^V248C-T251C (expressed in E. coli) and reP-PcLip^V248C-T251C (expressed in P. pastoris) were obtained. Both had enhanced thermostability with half-lives at 35 °C about 4.5- and 12.8-fold longer than that of the parent PcLipI expressed in E. coli and P. pastoris, respectively. The temperature optima of reE-PcLip^V248C-T251C and reP-PcLip^V248C-T251C were 35 and 30 °C, which were each 5 °C higher than those of the parent PcLipI expressed in E. coli and P. pastoris. The K _ms of reE-PcLip^V248C-T251C and reP-PcLip^V248C-T251C toward tributyrin were 53.2 and 39.5 mM, while their V _maxs were 1,460 and 3,800 U/mg, respectively. PcLip^V248C-T251C had better thermostability and catalytic efficiency than the other mutants and the parent PcLipI.     

Improving the Activity of Cytochrome P450 BM-3 Catalyzing Indole Hydroxylation by Directed Evolution

Cytochrome P450 BM-3 (A74G/F87V/L188Q) could catalyze indole to produce indigo. To further improve this capability, random mutagenesis was performed on the heme domain of P450 BM-3 (A74G/F87V/L188Q) with error-prone PCR. A single mutant V445A was selected out from the error-prone library and exhibited the highest specific activity toward indole among the mutants obtained. The kinetic parameters of V445A were also highly improved. Compared with the parent enzyme, the turnover rate (k _cat) of V445A was increased by 7.5 times, while its K _m value decreased by 9.2 %. Consequently, the catalytic efficiency (k _cat/K _m) of V445A was raised to 8.2 times than that of the parent enzyme. Moreover, alanine was confirmed as the best amino acid substitution by saturated mutagenesis in Val445 position. Three-dimensional structure analysis was also used to rationalize the effect on the enzyme properties of the mutation. This study showed that random mutagenesis was efficient to identify mutants with potential values in industry and increased our insight into P450 BM-3.