Improving the activity and expression level of a phthalate-degrading enzyme by a combination of mutagenesis strategies and strong promoter replacement.

Phthalic acid esters (PAEs) are widely used plasticizers found in consumer products, which enter the environment and pose severe threats to human health. Here, a new PAE-degrading enzyme EstJ6 was modified by combining mutagenesis strategies and a strong promoter replacement to improve its catalytic activity and expression level. Four mutants with enhanced activity were obtained by random mutation, among which EstJ6M1.1 exhibited the highest catalytic activity with an increase in catalytic activity by 2.9-fold toward dibutyl phthalate (DBP) than that of the wild-type (WT) enzyme. With these mutants as a template, a variant EstJ6M2 with 3.1-fold higher catalytic activity and 4.61 times higher catalytic efficiency (Kcat/Km) was identified by staggered extension PCR. Targeting four mutation sites of EstJ6M2, a variant EstJ6M3.1 was gained by site-directed saturation mutagenesis and displayed 4.3-fold higher activity and 5.97 times higher Kcat/Km than WT. The expression level of three mutants EstJ6M1.1, EstJ6M2, and EstJ6M3.1, as well as the WT, increased nearly threefold after a strong promoter replacement. These results provide a proof-theoretical basis and practicable pipeline for applying PAE-degrading enzymes.

Improving Degradation of Polycyclic Aromatic Hydrocarbons by Bacillus atrophaeus Laccase Fused with Vitreoscilla Hemoglobin and a Novel Strong Promoter Replacement.

Laccases catalyze a variety of electron-rich substrates by reducing O2 to H2O, with O2 playing a vital role as the final electron acceptor in the reaction process. In the present study, a laccase gene, lach5, was identified from Bacillus atrophaeus through sequence-based screening. LacH5 was engineered for modification by fusion expression and promoter replacement. Results showed that the purified enzyme LacH5 exhibited strong oxidative activity towards 2,2'-azinobis(3-ehtylbenzothiazolin-6-sulfnic acid) ammonium salt (ABTS) under optimum pH and temperature conditions (pH 5.0, 60 °C) and displayed remarkable thermostability. The activity of the two fusion enzymes was enhanced significantly from 14.2 U/mg (LacH5) to 22.5 U/mg (LacH5-vgb) and 18.6 U/mg (Vgb-lacH5) toward ABTS after LacH5 fusing with Vitreoscilla hemoglobin (VHb). Three of six tested polycyclic aromatic hydrocarbons (PAHs) were significantly oxidized by two fusion laccases as compared with LacH5. More importantly, the expression level of LacH5 and fusion protein LacH5-vgb was augmented by 3.7-fold and 7.0-fold, respectively, by using a novel strong promoter replacement. The results from the current investigation provide new insights and strategies for improving the activity and expression level of bacterial laccases, and these strategies can be extended to other laccases and multicopper oxidases.

Identification and characterization of a novel carboxylesterase EstQ7 from a soil metagenomic library.

A novel lipolytic gene, estq7, was identified from a fosmid metagenomic library. The recombinant enzyme EstQ7 consists of 370 amino acids with an anticipated molecular mass of 42 kDa. Multiple sequence alignments showed that EstQ7 contained a pentapeptide motif GHSMG, and a putative catalytic triad Ser174-Asp306-His344. Interestingly, EstQ7 was found to have very little similarity to the characterized lipolytic enzymes. Phylogenetic analysis revealed that EstQ7 may be a member of a novel family of lipolytic enzymes. Biochemical characterization of the recombinant enzyme revealed that it constitutes a slightly alkalophilic, moderate thermophilic and highly active carboxylesterase against short-chain fatty acid esters with optimum temperature 50 ℃ and pH 8.2. The Km and kcat values toward p-nitrophenyl acetate were determined to be 0.17 mM and 1910s-1, respectively. Moreover, EstQ7 was demonstrated to have acyltransferase activity by GC-MS analysis. Structural modeling of the three-dimensional structure of this new enzyme showed that it exhibits a typical α/ß hydrolase fold, and the catalytic triad residues are spatially close. Molecular docking revealed the interactions between the enzyme and the ligand. The high levels of lipolytic activity of EstQ7, combined with its moderate thermophilic property and acyltransferase activity, render this novel enzyme a promising candidate biocatalyst for food, pharmaceutical and biotechnological applications.

Enhancing the activity and thermal stability of a phthalate-degrading hydrolase by random mutagenesis.

Our previous work has reported that EstJ6 was a phthalate-degrading hydrolase. In the study, a random mutant library was constructed by two rounds of error-prone PCR, three mutants (ET1.1, ET2.1, and ET2.2) with enhanced hydrolytic activity against dibutyl phthalate (DBP) were obtained. The best mutant ET2.2, accumulated three amino acid substitutions (Thr91Met, Ala67Val, and Val249Ile) and exhibited 2.8-fold increase enzyme activity and 2.3-fold higher expression level. Meanwhile, compared with EstJ6, ET2.2 showed over 50% improvement in thermostability (at 50 °C for 1 h) and 1.2-fold increase in 50% methanol tolerance. Kinetic parameters analysis revealed that the Km value for ET2.2 decreased by 60% and the kcat/Km value increased by 166%. The molecular docking indicated that the shortening of hydrogen bond between Ser146-OH and DBP-CO, which may led to an increase in enzyme activity and catalytic efficiency, the enhancement of hydrophobicity of hydrophobic pocket was related to the improvement of organic solvents tolerance, and three hydrophobic amino acid substitutions Thr91Met, Ala67Val, and Val249Ile facilitated to improve the thermal stability and organic solvents tolerance. These results confirmed that random mutagenesis was an effective tool for improving enzyme properties and lay a foundation for practical applications of phthalate-degrading hydrolase in biotechnology and industrial fields.

A Method for Broccoli Seedling Recognition in Natural Environment Based on Binocular Stereo Vision and Gaussian Mixture Model.

Illumination in the natural environment is uncontrollable, and the field background is complex and changeable which all leads to the poor quality of broccoli seedling images. The colors of weeds and broccoli seedlings are close, especially under weedy conditions. The factors above have a large influence on the stability, velocity and accuracy of broccoli seedling recognition based on traditional 2D image processing technologies. The broccoli seedlings are higher than the soil background and weeds in height due to the growth advantage of transplanted crops. A method of broccoli seedling recognition in natural environments based on Binocular Stereo Vision and a Gaussian Mixture Model is proposed in this paper. Firstly, binocular images of broccoli seedlings were obtained by an integrated, portable and low-cost binocular camera. Then left and right images were rectified, and a disparity map of the rectified images was obtained by the Semi-Global Matching (SGM) algorithm. The original 3D dense point cloud was reconstructed using the disparity map and left camera internal parameters. To reduce the operation time, a non-uniform grid sample method was used for the sparse point cloud. After that, the Gaussian Mixture Model (GMM) cluster was exploited and the broccoli seedling points were recognized from the sparse point cloud. An outlier filtering algorithm based on k-nearest neighbors (KNN) was applied to remove the discrete points along with the recognized broccoli seedling points. Finally, an ideal point cloud of broccoli seedlings can be obtained, and the broccoli seedlings recognized. The experimental results show that the Semi-Global Matching (SGM) algorithm can meet the matching requirements of broccoli images in the natural environment, and the average operation time of SGM is 138 ms. The SGM algorithm is superior to the Sum of Absolute Differences (SAD) algorithm and Sum of Squared Differences (SSD) algorithms. The recognition results of Gaussian Mixture Model (GMM) outperforms K-means and Fuzzy c-means with the average running time of 51 ms. To process a pair of images with the resolution of 640×480, the total running time of the proposed method is 578 ms, and the correct recognition rate is 97.98% of 247 pairs of images. The average value of sensitivity is 85.91%. The average percentage of the theoretical envelope box volume to the measured envelope box volume is 95.66%. The method can provide a low-cost, real-time and high-accuracy solution for crop recognition in natural environment.

Cytotoxic trichothecene macrolides from the endophyte fungus Myrothecium roridum.

A new cytotoxic roridin-type trichothecene macrolide named epiroridin acid (1) and two known compounds epiroridin E (2) and mytoxin B (3) were isolated from the liquid culture of Myrothecium roridum A553, which was isolated from the medicinal plant Pogostemon cablin. The structure of the new macrolide (1) was elucidated by extensive spectroscopic measurements (UV, IR, MS, and 1D and 2D NMR) analyses. All isolated compounds (1-3) were evaluated for their cytotoxic activities against SF-268, MCF-7, NCI-H460, and HepG-2 tumor cell lines. The new compound (1) exhibited well cytotoxicity against the four selected tumor cell lines.

[Protective effect of total glycosides of Ranunculus japonicus on myocardial ischemic-reperfusion injury in isolated rat hearts].

OBJECTIVES: To study the protective effect of total glycosides of Ranunculus japonicus (TGRJ) on myocardial ischemic-reperfusion injury in isolated rat hearts. METHODS: The SD rats were randomly divided into normal control group, ischemia-reperfusion group, and TGRJ in 0.05, 0.10 and 0.20 mg/mL groups. The ischemia-reperfusion injury model was built using Langendroff isolated rats hearts perfusion system. The indexes of heart function such as heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular systolic pressure maximum rate of rise (+ dp/dt(max)), left ventricular diastolic pressure maximum rate of descent (-dp/dt(max)) and coronary flow (CF) before ischemia and later at 1, 5, 10, 20, 30 and 40 min after reperfusion were recorded. The activity of Lactate dehydrogenase (LDH) and creatine kinase (CK) at 20 and 40 min after reperfusion were determined. The myocardial tissues were stained with Triphenyltetrazolium chloride (TTC) and the percentage of myocardial infarction area was calculated. RESULTS: HR, LVDP, +dp/dt(max) and CF of the I/R group were significantly decreased after reperfusion, LDH and CK levels were increased and the area of myocardial infraction was 58.78%. TGRJ had improving effect on all above indexes. CONCLUSION: TGRJ has protective effet on the myo- cardial ischemic-reperfusion injury.

[Effect of TGRJ on blood press, NO and Ang II in renal hypertensive rats].

OBJECTIVE: To study the effect of total glycosides of Ranunculus japoniucus (TGRJ) on blood pressure, nitric oxide (NO) calcium and angiotensin II (Ang II) in renal hypertensive rats (RHR). METHODS: 1) RHR were established by two kidney one clip (2K1C) and drugs were given by intragastric administration for 5 week, the blood pressure were measured at the end of 5 week,detected the concentration of NO in the serum and Ang II in the blood plasma,heart and kidney tissue. 2) Used a new generations of Ca2+ fluorescent probe (Fluo-3/AM) to mark calcium in vascular smooth muscle cells. Observed the fluorescence imaging by inverted fluorescence microscope and measured the fluorescence intensity of calcium by fluorescence spectrophotometer in vascular smooth muscle cells. RESULTS: 1) The high, medium doses of TGRJ could decrease blood pressure of RHR (P<0.05), TGRJ could significantly increase the concentration of NO in the serum (P<0.01), but it showed no significant effect on the concentration of Ang II (P>0.05); 2) The Ang II could significantly promote the calcium ions in extracellular inner flow. The different doses of TGRJ could reduce the calcium ions in cells which were mediated by Ang II. CONCLUSION: TGRJ could decrease blood pressure in RHR. the mechanism might be related to increasing the rats' NO level and reducing the calcium ions level in cells which are increased by Ang II.

Rapid analysis of the main components of the total glycosides of Ranunculus japonicus by UPLC/Q-TOF-MS.

A rapid method for the analysis of the main components of the total glycosides of Ranunculus japonicus (TGOR) was developed using ultra-performance liquid chromatography with quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS). The separation analysis was performed on a Waters Acquity UPLC system and the accurate mass of molecules and their fragment ions were determined by Q-TOF MS. Twenty compounds, including lactone glycosides, flavonoid glycosides and flavonoid aglycones, were identified and tentatively deduced on the basis of their elemental compositions, MS/MS data and relevant literature. The results demonstrated that lactone glycosides and flavonoids were the main constituents of TGOR. Furthermore, an effective and rapid pattern was established allowing for the comprehensive and systematic characterization of the complex samples.

[Detecting the information of cucumber in greenhouse for picking based on NIR image].

For the cucumber harvesting robot, the identification of target information is one of important tasks in the automation of fruit-picking. In order to implement spatial fruit localization and quality discrimination in greenhouse, this paper presented a machine vision algorithm for the recognition and detection of cucumber fruits based on near-infrared spectral imaging. By comparing the spectral reflectance of cucumber plant (fruit, leaf and stem) from visible to infrared region (325-1 075 nm) measured by ASD FieldSpec Pro VNIR spectrometer, a monospectral near-infrared image at the 850 nm sensitive wavelength was captured to cope with the similar-color segmentation problem in complex environment. Then, a method of fruit extraction was developed on the basis of the following steps. Firstly, from the gray level histogram it was observed that the pixels of fruit distributed on the right are lesser than that of background, so "P parameter threshold method" was used to image segmentation. Subsequently, divided local image was partitioned into several sub-blocks by the application of adaptive template mining, which was feasible for processing the fruit with long-column feature. Finally, noises including parts of stem and leaf were eliminated using estimation condition of barycentre position and area size, proved by relative experiment In addition, the region for robotic grasping was established by gray variation between fruit-handle and fruit pedicel, as the quality feature was extracted with morphological characteristics of the centre-line length and the fruit flexure degree. A detecting experiment was carried out on 30 images with cucumber fruits and 10 images with no fruits, which were taken in a changing greenhouse environment. The results indicate that the accuracy rate of the recognition was 83.3% and 100%, while the success rate of effectively acquiring the grasping region was 83.3%, which can meet the demand of robotic fruit-harvesting.