Machine learning-assisted substrate binding pocket engineering based on structural information.

Engineering enzyme-substrate binding pockets is the most efficient approach for modifying catalytic activity, but is limited if the substrate binding sites are indistinct. Here, we developed a 3D convolutional neural network for predicting protein-ligand binding sites. The network was integrated by DenseNet, UNet, and self-attention for extracting features and recovering sample size. We attempted to enlarge the dataset by data augmentation, and the model achieved success rates of 48.4%, 35.5%, and 43.6% at a precision of ≥50% and 52%, 47.6%, and 58.1%. The distance of predicted and real center is ≤4 Å, which is based on SC6K, COACH420, and BU48 validation datasets. The substrate binding sites of Klebsiella variicola acid phosphatase (KvAP) and Bacillus anthracis proline 4-hydroxylase (BaP4H) were predicted using DUnet, showing high competitive performance of 53.8% and 56% of the predicted binding sites that critically affected the catalysis of KvAP and BaP4H. Virtual saturation mutagenesis was applied based on the predicted binding sites of KvAP, and the top-ranked 10 single mutations contributed to stronger enzyme-substrate binding varied while the predicted sites were different. The advantage of DUnet for predicting key residues responsible for enzyme activity further promoted the success rate of virtual mutagenesis. This study highlighted the significance of correctly predicting key binding sites for enzyme engineering.

Biosynthesis of a Functional Fragment of Human Collagen II in Pichia pastoris.

Collagen II (COL2) is the major component of cartilage tissue and is widely applied in pharmaceuticals, food, and cosmetics. In this study, COL fragments were extracted from human COL2 for secretory expression in Pichia pastoris. Three variants were successfully secreted by shake flask cultivation with a yield of 73.3-100.7 mg/L. The three COL2 variants were shown to self-assemble into triple-helix at 4 °C and capable of forming higher order assembly of nanofiber and hydrogel. The bioactivities of the COL2 variants were validated, showing that sample 205 exhibited the best performance for inducing fibroblast differentiation and cell migration. Meanwhile, sample 205 and 209 exhibited higher capacity for inducing in vitro blood clotting than commercial mouse COL1. To overexpress sample 205, the expression cassettes were constructed with different promoters and signal peptides, and the fermentation condition was optimized, obtaining a yield of 172 mg/L for sample 205. Fed-batch fermentation was carried out using a 5 L bioreactor, and the secretory protease Pep4 was knocked out to avoid sample degradation, finally obtaining a yield of 3.04 g/L. Here, a bioactive COL2 fragment was successfully identified and can be overexpressed in P. pastoris; the variant may become a potential biomaterial for skin care.

Identification of a human type XVII collagen fragment with high capacity for maintaining skin health.

Collagen XVII (COL17) is a transmembrane protein that mediates skin homeostasis. Due to expression of full length collagen was hard to achieve in microorganisms, arising the needs for selection of collagen fragments with desired functions for microbial biosynthesis. Here, COL17 fragments (27-33 amino acids) were extracted and replicated 16 times for recombinant expression in Escherichia coli. Five variants were soluble expressed, with the highest yield of 223 mg/L. The fusion tag was removed for biochemical and biophysical characterization. Circular dichroism results suggested one variant (sample-1707) with a triple-helix structure at >37 °C. Sample-1707 can assemble into nanofiber (width, 5.6 nm) and form hydrogel at 3 mg/mL. Sample-1707 was shown to induce blood clotting and promote osteoblast differentiation. Furthermore, sample-1707 exhibited high capacity to induce mouse hair follicle stem cells differentiation and osteoblast migration, demonstrating a high capacity to induce skin cell regeneration and promote wound healing. A strong hydrogel was prepared from a chitosan and sample-1707 complex with a swelling rate of >30 % higher than simply using chitosan. Fed-batch fermentation of sample-1707 with a 5-L bioreactor obtained a yield of 600 mg/L. These results support the large-scale production of sample-1707 as a biomaterial for use in the skin care industry.

Mesenchymal stem cell-derived exosome mir-342-3p inhibits metastasis and chemo-resistance of breast cancer through regulating ID4.

BACKGROUND: The mesenchymal stem cell-derived exosome (MSCs-exo) carrying microRNAs have been proved to regulate tumor biological activities. Clarifying molecular mechanism and identifying predictive microRNAs will be of great value in anti-tumor therapy improvement. OBJECTIVE: We aimed to investigate the regulatory role of microRNA-342-3p (miR-342-3p) in MSCs-exo on breast cancer. METHODS: Breast cancer tissues and cell lines were used to evaluate miR-342-3p expression in patients with or without lymph node/distal organ metastasis. The impact of MSCs-exo expression on tumor cell chemo-resistance and invasion/migration was measured. Dual-luciferase reporter gene assay was applied to identify binding site. Inhibitor of differentiation 4 (ID4) siRNA and miR-342-3p inhibitor transfection was conducted to further explore the miR-342-3p/ID4 axis on chemo-resistance and metastasis of breast cancer cells. RESULTS: Breast cancer cells revealed significantly lower level of miR-342-3p in patients with metastatic diseases. miR-342-3p suppressed invasive and chemo-resistant behavior of breast cancer tumor cells. Binding site between miR-342-3p and ID4 was proved. ID4 could reverse the influence of miR-342-3p on chemo-resistance. The tumor inhibition effect of IDA siRNA in vivo was also identified. CONCLUSIONS: This study demonstrated that miR-342-3p acted as potential tumor suppressor by inhibiting metastasis and chemo-resistance of breast cancer cells through targeting ID4. This study might provide potential therapy targets for the treatment of breast cancer.

HCB dechlorination combined with heavy metals immobilization in MSWI fly ash by using n-Al/CaO dispersion mixture.

Municipal solid waste incineration fly ash (MSWI-FA) has been strictly controlled as hazardous waste globally because it contains various heavy metals and dioxins. This study prepared a nanometallic Al/CaO (n-Al/CaO) dispersion mixture via ball-milling as a reductive stabilization reagent for the simultaneous immobilization of heavy metals and detoxification of POPs like substance in MSWI-FA. Under optimal conditions, Cu, Zn, Cd, Cr, Ni, and Pb had been significantly immobilized (over 99.9 %) and the leaching concentration of Zn, Cd, Cr, Ni, and Pb were below the detectable limit. Simultaneously, 82.43 % of HCB can be destructed into alkanes and amorphous carbon. The porous structure of the fly ash and alkaline surface of n-Al/CaO promoted the adsorption and cracking of HCB. The highly active n-Al/CaO interacted with water as the hydrogen donor to promote the reductive dechlorination process. Hydrocalumite was a new mineral formed from the adsorption and complexation of heavy metal. Therefore, n-Al/CaO can strengthen the control of heavy metals in the S/S treatment of MSWI-FA, effectively detoxify chlorinated organics, and reduce environmental health risks.

Novel method for comprehensive utilization of MSWI fly ash through co-reduction with red mud to prepare crude alloy and cleaned slag.

Municipal solid waste incineration fly ash (MSWI-FA) is classified as hazardous waste that requires an effective processing method. This study proposed an innovative technique process, co-reduction of MSWI-FA and red mud followed by magnetic separation, to prepare crude alloy and cleaned slag. In this process, MSWI-FA acted not only as a reductant to reduce metal minerals in MSWI-FA and red mud to form an alloy, but also as a calcium additive to enhance the reduction of metal minerals and alter the melting point of the CaO-SiO2-Al2O3 system. Under optimal conditions, 85.52% Fe, 80.10% Cu, 92.96% Ni, and 66.74% Cr can be recovered in the form of a Fe-Cu-Ni-Cr alloy. The Fe-Cu-Ni-Cr alloy containing 96.47% Fe, 0.81% Cu, 0.65% Ni, and 0.42% Cr can be used for weathering steel production. Other heavy metals, including Cd, Pb, and Zn, were removed via volatilization. The toxicity characteristic leaching procedure test indicated that the leaching toxicity of the cleaned slag was substantially below the standard limits. The characteristics of the cleaned slag were similar to those of ground granulated blast furnace slag, suggesting its potential application in cement production.

A novel insight into the influence of thermal pretreatment temperature on the anaerobic digestion performance of floatable oil-recovered food waste: Intrinsic transformation of materials and microbial response.

The intrinsic reason determining digestion performance of 100-160 °C preheated food waste after recovering floatable oil (FO-recovered FW) was investigated using two-dimensional correlated infrared spectroscopy, three-dimensional fluorescence spectroscopy and high-throughput 16S rRNA amplicon sequencing. The results indicated that thermal temperature significantly affected CH4 production of FO-recovered FW due to different structural alteration degree of starch, protein, cellulose and lipid components. Fragmentation of starch mainly occurred at 100 °C. The hydrolytic and acidogenic rate of starch was promoted and accordingly induced rapid growth of carbohydrate-fermenting bacteria, which resulted in severe acidification. Protein hydrolysis and cellulose H-bonds cleavage occurring at 120-160 °C accelerated the accessible sites interacting with microbial hydrolytic enzymes, and growth of Cloacimonetes and Syntrophomonas enhanced CH4 production. Non-degradable humic acid-like organics remarkably formed at 160 °C caused a carbon loss and digestion inhibiting/deteriorating. Pretreatment at 120 °C was feasible for promoted methane production based on energy assessment.

Characterization of naturally aged cement-solidified MSWI fly ash.

Solidification/stabilization (S/S) is the most common treatment for municipal solid waste incineration fly ash (MSWI-FA), and is widely applied in developed countries but has a history barely longer than 10 years in China. However, our understanding of the physicochemical characteristics of the solidified FA body after long-term natural aging is comparatively poor. Focusing on cement-solidified FA that was naturally aged for 6 years (hereafter referred to as FA-6), the physicochemical characteristics including elemental composition, mineral composition, microstructure, thermogravimetry, distribution of heavy metals in mineral phases, and leaching characteristics of inorganic salts (Na, K, Ca), anions (Cl and SO4) and heavy metals (Cd, Cr, Cu, Pb, Zn) were investigated in this study. By combining pH-dependent leaching results with the geochemical model LeachXS, the chemical forms of heavy metals in the FA solid phase was determined. The main conclusion was as follows: (1) soluble salts of FA-6 decreased by more than 92% compared with fresh FA. (2) In FA-6, the proportions of Pb, Cd and Zn in the non-mineral phase were 100%, 100% and 58%, respectively, which may cause potential environmental risk of heavy metal release. The leaching concentration of Pb was 4007.37 µg/L according to compliance batch test of HJ300, which was far higher than the landfill requirement of 250 µg/L. (3) The controlling phase for Pb in FA-6 was Pb5(PO4)3Cl (pH 2-12) and Pb2(OH)3Cl (pH > 12). (4) Carbonates, hydrous Fe oxides (HFO) and dissolved organic carbon (DOC) in FA-6 also affected the phase-controlled leaching of heavy metals. The carbonate fraction partly controlled the leaching of Cd, Cu and Zn. For example, smithsonite (ZnCO3) controlled the release of Zn (pH 2-13). Adsorption to solid humic acid (SHA) controlled the Cr leaching at pH < 7 and the Cu leaching except pH > 12.

Dechlorination of Hexachlorobenzene in Contaminated Soils Using a Nanometallic Al/CaO Dispersion Mixture: Optimization through Response Surface Methodology.

Hexachlorobenzene (HCB) contamination of soils remains a significant environmental challenge all over the world. Reductive stabilization is a developing technology that can decompose the HCB with a dechlorination process. A nanometallic Al/CaO (n-Al/CaO) dispersion mixture was developed utilizing ball-milling technology in this study. The dechlorination efficiency of HCB in contaminated soils by the n-Al/CaO grinding treatment was evaluated. Response surface methodology (RSM) was employed to investigate the effects of three variables (soil moisture content, n-Al/CaO dosage and grinding time) and the interactions between these variables under the Box-Behnken Design (BBD). A high regression coefficient value (R² = 0.9807) and low p value (<0.0001) of the quadratic model indicated that the model was accurate in predicting the experimental results. The optimal soil moisture content, n-Al/CaO dosage, and grinding time were found to be 7% (m/m), 17.7% (m/m), and 24 h, respectively, in the experimental ranges and levels. Under optimal conditions, the dechlorination efficiency was 80%. The intermediate product analysis indicated that dechlorination was the process by stepwise loss of chloride atoms. The main pathway observed within 24 h was HCB → pentachlorobenzene (PeCB) → 1,2,3,4-tetrachlorobenzene (TeCB) and 1,2,4,5-TeCB. The results indicated that the moderate soil moisture content was crucial for the hydrodechlorination of HCB. A probable mechanism was proposed wherein water acted like a hydrogen donor and promoted the hydrodechlorination process. The potential application of n-Al/CaO is an environmentally-friendly and cost-effective option for decontamination of HCB-contaminated soils.

Enhancing syntrophic associations among Clostridium butyricum, Syntrophomonas and two types of methanogen by zero valent iron in an anaerobic assay with a high organic loading.

The impacts of ZVI on microbial community diversity in an anaerobic assay with high organic loading were investigated. The relative abundance of bacteria, archaea, and the functional methyl coenzyme-M reductase (mcrA) gene were investigated using high-throughput sequencing, and variations in their quantity were determined by qPCR. The results showed that ZVI significantly increased both the relative abundance and quantity of Methanobacteriales and Methanosarcinales during hydrogenotrophic and acetoclastic methanogenesis. The relative abundance of syntrophic Methanobacteriales at the hydrolysis and acidogenesis stages resulted in H2 partial pressure decrease through an interspecies hydrogen transfer (IHT) network, which further induced butyric conversion to acetic by Syntrophomonas. The primary microbial metabolism then converted to acetoclastic methanogensis in the assay with ZVI addition. The short duration of this process and high relative abundance of Syntrophomonas, Clostridium butyricum and Methanosarcinales potentially indicated the existence of a novelty syntrophic mechanism for extracellular electron transfer, which promoted CH4 generation.

Effect of Fe0 addition on volatile fatty acids evolution on anaerobic digestion at high organic loading rates.

Excessive acidification frequently occurs in the anaerobic digestion of the organic fraction of municipal solid waste (OFMSW) at high organic loading rates (OLR), due to the accumulation of non-acetic volatile fatty acids (VFA). In this study, the performance of Fe0 in enhancing various VFA production and metabolism was investigated. The butyric acid concentration in a high OLR reactor with Fe0 addition decreased from 7200 to 0mg/L after a short lag phase, and the total VFA (TVFA) concentration also decreased substantially. The corresponding dominant acidogenesis type also changed from butyric type to propionic type fermentation. Furthermore, the CH4 yield of the reactor with added Fe0 was approximately 595ml CH4/g VSadded, which was an increase of 41.7% compared with the biochemical methane potential (BMP) test results in controls without added ZVI. A microbial diversity analysis, using high throughput sequencing, showed that Methanofollis and Methanosarcina were dominant in terms of the archaeal structures of the Fe0 reactor at the butyric converting stage; however, Methanosaeta was predominant in the reactor during the control BMP test. These results suggested that Fe0 can convert non-acetic VFA to acetic VFA and improve the CH4 yield by enhancing the activity of methanogens.

Inhibiting excessive acidification using zero-valent iron in anaerobic digestion of food waste at high organic load rates.

Excessive acidification occurs frequently in food waste (FW) anaerobic digestion (AD) due to the high carbon-to-nitrogen ratio of FW. In this study, zero-valent iron (ZVI) was applied to prevent the excessive acidification. All of the control groups, without ZVI addition (pH∼5.3), produced little methane (CH4) and had high volatile fatty acids/bicarbonate alkalinity (VFA/ALK). By contrast, at OLR of 42.32gVS/Lreactor, the pH of effluent from the reactors with 0.4g/gVSFWadded of ZVI increased to 7.8-8.2, VFA/ALK decreased to <0.1, and the final CH4 yield was ∼380mL/gVSFWadded, suggesting inhibition of excessive acidification. After adding powdered or scrap metal ZVI to the acidogenic reactors, the fractional content of butyric acid changed from 30-40% to 0%, while, that of acetic acid increased. These results indicate that adding ZVI to FW digestion at high OLRs could eliminate excessive acidification by promoting butyric acid conversion and enhancing methanogen activity.