Rational surface charge engineering of haloalkane dehalogenase for boosting the enzymatic performance in organic solvent solutions

Biocatalysis in organic solvents(OSs)has numerous important applications,but native enzymes in OSs often exhibit limited catalytic performance.Herein,we proposed a computation-aided surface charge engineering strategy to improve the catalytic performance of haloalkane dehalogenase DhaA in OSs based on the energetic analysis of substrate binding to the DhaA surface.Several variants with enhanced OS resistance were obtained by replacing negative charged residues on the surface with positive charged residue(Arg).Particularly,a four-substitution variant E16R/E93R/E121R/E257R exhibited the best catalytic performance(five-fold improvement in OS resistance and seven-fold half-life increase in 40%(vol)dimethylsulfoxide).As a result,the overall catalytic performance of the variant could be at least 26 times higher than the wild-type DhaA.Fluorescence spectroscopy and molecular dynamics simulation studies revealed that the residue substitution mainly enhanced OS resistance from four aspects:(a)improved the overall structural stability,(b)increased the hydrophobicity of the local microenvironment around the catalytic triad,(c)enriched the hydrophobic substrate around the enzyme molecule,and(d)lowered the contact frequency between OS molecules and the catalytic triad.Our findings validate that computationaided surface charge engineering is an effective and ingenious rational strategy for tailoring enzyme performance in OSs.

Vitrification cryopreservation of ligaments based on zwitterionic betaine

Ligament cryopreservation enables a prolonged shelf life of allogeneic ligament grafts,which is fundamentally important to ligament reconstruction.However,conventional cryopreservation techniques fail to eliminate the damage caused by ice crystal growth and the toxicity of cryopreservation agents(CPAs).Here,we report a novel CPA vitrification formulation primarily composed of betaine for ligament cryopreservation.Comprehensive optimization was conducted on the methods for vitrification and rewarming,as well as the loading and unloading conditions,based on the critical cooling rate(CCR),critical warming rate(CWR),and permeation properties of the CPA.Using biomechanical and histological level tests,we demonstrate the superior performance of our method in ligament cryopreservation.After 30 days of vitrification cryopreservation,parameters such as the Young's modulus,tensile stress,denaturation temperature,and glycosaminoglycans content of the ligament remained essentially unchanged.This work pioneers the application of ice-free cryopreservation for ligament and holds great potential for improving the long-term storage of ligament,providing valuable insights for future cryopreservation technique development.

Bio-Review 2021: Inspiring the future of Biochemical Engineering

Biochemical Engineering(BCE)discipline had largely developed from fermentation technology studies from 1950s through 1960s when fermentation emerged as the core technology able to address multiple industrial needs ranging from health care products such as antibiotics;food products such as single cell proteins,amino acids,organic acids,and vitamins;liquid fuels and chemicals such as ethanol and acetic acid;and industrial enzymes.

Regeneration of the anterior cruciate ligament:Current strategies in tissue engineering

Recent advancements in the field of musculoskeletaltissue engineering have raised an increasing interest in the regeneration of the anterior cruciate ligament(ACL). It is the aim of this article to review the current research efforts and highlight promising tissue engineering strategies. The four main components of tissue engineering also apply in several ACL regeneration research efforts. Scaffolds from biological materials, biodegradable polymers and composite materials are used. The main cell sources are mesenchymal stem cells and ACL fibroblasts. In addition, growth factors and mechanical stimuli are applied. So far, the regenerated ACL constructs have been tested in a few animal studies and the results are encouraging. The different strategies, from in vitro ACL regeneration in bioreactor systems to bio-enhanced repair and regeneration, are under constant development. We expect considerable progress in the near future that will result in a realistic option for ACL surgery soon.

Multiple effects of polydopamine nanoparticles on Cu^(2+)-mediated Alzheimer's β-amyloid aggregation

Deposition of β-amyloid protein(Aβ) is the main hallmark of Alzheimer's disease(AD), and it has been well recognized that Cu^(2+)-mediated Aβ aggregation plays a crucial role in AD pathological processes.Cu^(2+)binding to Aβ can promote the production of reactive oxygen species(ROS) through Fenton-like reactions and produce more toxic Aβ-Cu^(2+)species under Cu^(2+)stimulation. Thus, the development of nanomaterials that can inhibit Cu^(2+)-mediated Aβ aggregation and degrade Aβ-Cu^(2+)complexes is considered an effective strategy for the prevention and treatment of AD. In this study, polydopamine nanoparticles(PDA NPs) were prepared and the results reveal that PDA NPs potently inhibit Cu^(2+)-mediated Aβaggregation and effectively reduce the formation of Aβ-Cu^(2+)complexes. In vitro experiments show that PDA NPs efficiently eliminate ROS generation catalyzed by Cu^(2+)or Aβ-Cu^(2+)complexes, thus rescuing cultured cells by reducing intracellular ROS levels. More importantly, PDA NPs can depolymerize Aβ-Cu^(2+)complexes, and the degradation of Aβ-Cu^(2+)complexes is promoted by near-infrared light irradiation due to their high photothermal conversion ability. In vivo studies reveal that PDA NPs significantly reduce the deposition of Aβ plaques in the presence of Cu^(2+)and extend the lifespan of AD nematodes from 11 to 14 d. Thus, the PDA NPs developed herein are multifunctional against Cu^(2+)-mediated Aβ aggregation for the potential prevention and treatment of AD.

Unraveling structure and performance of protein a ligands at liquid–solid interfaces: A multi-techniques analysis

Oriented ligand immobilization is one of the most effective strategies used in the design and construction of a high-capacity protein A chromatography. In this work, cysteine was introduced as anchoring sites by substituting a specific residue on Helix Ⅰ, Ⅱ, and at C-terminus of antibody binding domain Z from protein A, respectively, to investigate structural evolution and binding behavior of protein A ligands at liquid-solid interfaces. Among the three affinity dextran-coated Fe_(3)O_(4) magnetic nanoparticles(Fe_(3)O_(4)@Dx MNPs), affinity MNPs with the immobilized ligand via N11C on Helix Ⅰ(Fe_(3)O_(4)@Dx-Z_(1) MNPs) had the highest helical content, and MNPs with the immobilized ligand via G29C on Helix Ⅱ(Fe_(3)O_(4)@Dx-Z_(2) MNPs) had the lowest helical content at the same pHs. It was attributed to less electrostatic attraction of ligand to negatively charged surface on Fe_(3)O_(4)@Dx-Z_(1) MNPs because of less positive charged residues on Helix Ⅰ(K6) than Helix Ⅱ(R27/K35). Among the three affinity MNPs, moreover, the highest affinity to immunoglobulin G(IgG) binding was observed on Fe_(3)O_(4)@Dx-Z_(1) MNPs in isothermal titration calorimetry measurement, further validating greater structural integrity of the ligand on Fe_(3)O_(4)@Dx-Z_(1) MNPs. Finally,the study of IgG binding on MNPs and 96-well plates showed that anchoring sites for ligand immobilization had distinct influences on IgG binding and IgG-mediated antigen binding. This work illustrated that anchoring sites of the ligands had a striking significance for the molecular structure of the ligand at liquid-solid interfaces and raised an important implication for the design and optimization of protein A chromatography and protein A-based immunoassay analysis.

Facile purification and immobilization of organophosphorus hydrolase on protein-inorganic hybrid phosphate nanosheets

Oriented immobilization of enzymes helps to maintain their native structure and proper orientation for high-performance engineering to meet extensive biocatalysis demands.However,the supporting materials used for orientated immobilization are usually costly or complicated in preparation,affecting their practical applications.In this work,a facile purification and immobilization method was proposed for enzyme immobilization based on organic-inorganic hybrid calcium phosphate nanocrystal(Ca Ps)induced by Cu^(2+) modified bovine serum albumin(BSA-Cu).Then,the as-prepared hybrid calcium phosphate nanosheet,BSA-Cu@Ca Ps,was utilized for one-pot purification and immobilization of His-tagged organophosphorus hydrolase(OPH)by metal-affinity binding to the incorporated BSA.BSA-Cu@Ca PsOPH exhibited enhanced p H stability and thermal stability compared to the free enzyme.Moreover,BSA-Cu@Ca Ps-OPH could retain more than 75%and 56%of initial activity after reuse 5 and 10 times,respectively.The results demonstrated that this facile strategy was promising for the effective biodegradation of organophosphorus pesticides with the immobilized enzyme.

a-Synuclein: A fusion chaperone significantly boosting the enzymaticperformance of PET hydrolase

Extensive use of polyethylene terephthalate (PET) has brought about global environmental problems. Arecently reported PET hydrolase (PETase) discovered from Ideonella sakaiensis showed high potentialfor degrading PET at moderate temperatures, but its activity and stability need further improvementfor practical applications. Herein, we proposed to use a-synuclein (aS) as a fusion chaperone and createdsix PETase-aS fusion enzymes with linkers of different types and lengths. All the fusion enzymes exhibited improved enzymatic performance, presenting 1.5 to 2.6-fold higher activity towards bis-2(hydroxyethyl) terephthalate than PETase, as well as significantly increased stabilities. Fluorescencespectroscopy indicated that the chaperone fusion tightened the overall conformation and resulted inthe opening of the substrate binding pocket, which led to the improved thermal stability and catalyticactivity of the fusion enzymes. Remarkably, one of the fusion proteins, PETase-[(GS)(EK)]10-aS, showed3.2 to 5.1 times higher PET degradation capability than PETase. The significantly boosted PET degradationperformance was not only attributed to the enhanced enzymatic activity and stability, but also possiblydue to the binding affinity of the fused aS domain for PET. These findings demonstrated that aS was aneffective fusion chaperone for significantly enhancing the enzymatic performance of PETase.

Conjugation of Candida rugosa lipase with hydrophobic polymer improves esterification activity of vitamin E in nonaqueous solvent

We described a novel polymer-lipase conjugate for high-efficient esterification of vitamin E using vitamin E and succinic anhydride as the substrates in nonaqueous media.In this work,the monomer,N-isopropylacrylamide(NIPAM),was grafted onto Candida rugosa lipase(CRL)to synthesize poly(NIPAM)(pNIPAM)-CRL conjugate by atom transfer radical polymerization via the initiator coupled on the surface of CRL.The result showed that the catalytic efficiencies of pNIPAM-CRL conjugates(19.5-30.3 L·s^(-1)·mmol^(-1))were at least 7 times higher than that of free CRL(2.36 L·s^(-1)·mmol^(-1))in DMSO.It was attributed to a significant increase in Kcat of the conjugates in nonaqueous media.The synthesis catalyzed by pNIPAM-CRL co njugates was influenced by the length and density of the grafted polymer,water content,solvent polarity and molar ratio of the substrates.In the optimal synthesis,the reaction time was shortened at least 7 times,and yields of vitamin E succinate by pNIPAM-g-CRL and free CRL were obtained to be 75.4%and 6.6%at 55℃after the reaction for 1.5 h.The result argued that conjugation with pNIPAM induced conformational change of the lid on CRL based on hydrophobic interaction,thus providing a higher possibility of catalysis-favorable conformation on CRL in nonaqueous media.Moreover,pNIPAM conjugation improved the thermal stability of CRL greatly,and the stability improved further with an increase of chain length of pNIPAM.At the optimal reaction conditions(55℃and 1.5 h),pNIPAM-g-CRL also exhibited good reusability in the enzymatic synthesis of vitamin E succinate and kept~70%of its catalytic activity after ten consecutive cycles.The research demonstrated that pNIPAM-g-CRL was a more competitive biocatalyst in the enzymatic synthesis of vitamin E succinate and exhibited good application potential under harsh industrial conditions.

深低温保存下高效抗冻多肽的合理设计和机理探讨

The development of effective antifreeze peptides to control ice growth has attracted a significant amount of attention yet still remains a great challenge.Here,we propose a novel design method based on in-depth investigation of repetitive motifs in various ice-binding proteins(IBPs)with evolution analysis.In this way,several peptides with notable antifreeze activity were developed.In particular,a designed antifreeze peptide named AVD exhibits ideal ice recrystallization inhibition(IRI),solubility,and biocompatibility,making it suitable for use as a cryoprotective agent(CPA).A mutation analysis and molecular dynamics(MD)simulations indicated that the Thr6 and Asn8 residues of the AVD peptide are fundamental to its ice-binding capacity,while the Ser18 residue can synergistically enhance their interaction with ice,revealing the antifreeze mechanism of AVD.Furthermore,to evaluate the cryoprotection potential of AVD,the peptide was successfully employed for the cryopreservation of various cells,which demonstrated significant post-freezing cell recovery.This work opens up a new avenue for designing antifreeze materials and provides peptide-based functional modules for synthetic biology.

New Development of Reverse Micelles and Applications in Protein Separation and Refolding

Reverse micelles bring mild and effective microenvironments in organic solvent that contain bitmolecules, which have attracted immense attention for application in the isolation of proteins, protein refolding, and enzymatic reaction. In this review, the application of reverse micelles for protein separation and refolding has been briefly summarized and various reverse micellar systems composed of different surfactants, including ionic, non- ionic, mixed, and affinity-based reverse micelles, have been highlighted. It illustrates especially the potential application of the novel affinity-based reverse micelles consisting of biocompatible surfactant coupled with affinity ligands. Moreover, the importance to develop universal affinity-based reverse micelles for protein separation and refolding in the downstream processing of biotechnology has been pointed out.

Preparation of Conjugated Linoleic Acid and Identification of Its Isomers

Conjugated linoleic acid (CLA) is a kind of fatty acid with physiological activities and potential application prospect. A synthesis method of conjugated linoleic acid and a purification technology were studied. CLA was prepared and purified by urea-complexation and conjugation using safflower oil as raw material. The purity of CLA and total recovery of the product was more than 95% and 48%, respectively. The main isomers produced in alkali-catalyzed conjugation were identified by gas chromatography (GC) linked to mass spectrometry (MS) and Fourier transform infrared spectroscopy (FTIR). The total amount of the two main isomers (9cis, 11trons-and 10trans, 12cis-CLA) determined by GC was more than 90% of the product.

Characterization and analysis of petrochemical wastewater through particle size distribution, biodegradability, and chemical composition

The centralized treatment method is a widely used form of wastewater treatment that tends to be less effective at removing toxic substances. Therefore, a detailed analysis of the composition of wastewater can provide important information for the design of an effective wastewater treatment process. The objective of this paper was to investigate particle size distribution(PSD), biodegradability, and the chemical composition of the petrochemical wastewater discharges. For this purpose, this project selected the petrochemical wastewater and treated wastewater of China National Offshore Oil Corporation Zhongjie Petrochemical Co, Ltd. as the analysis objects.The step-by-step filtration method, along with a molecular weight classification method, was selected to build the chemical oxygen demand(COD) and biochemical oxygen demand(BOD) fingerprints of petrochemical wastewater and treated wastewater. The results showed that the main pollutants were settleable particles in petrochemical wastewater, which contributed to over 54.85% of the total COD. The colloidal particles with particle sizes in the range of 450–1000 nm had the highest COD value in the treated wastewater, which contributed34.17% of the total COD of treated wastewater. The results of the BOD analysis showed that the soluble fractions were the main reason that treated wastewaters did not meet the treatment standards. Tests on the organic compounds in petrochemical wastewater found that there were mainly linear paraffins, branched paraffins, benzene series compounds, and some plasticizers in the influent of the petrochemical wastewater. The most abundant pollutants in treated petrochemical wastewater were the adjacent diisobutyl phthalate and the linear alkanes.Fourier transform infrared(FTIR) transmission spectroscopy analysis showed that the settleable particles of petrochemical wastewater and membrane bioreactor(MBR)-treated wastewater contained multiple types of organic substances. The results also indicated that removing the oil-settleable substances, the colloidal particles(450–1000 nm), and the soluble organics will be necessary for the treatment of petrochemical wastewater.

Zwitterionic polymer-coated porous poly(vinyl acetate–divinyl benzene)microsphere: A new support for enhanced performance of immobilized lipase

Enzyme immobilization has attracted great attention for improving the performance of enzymes in industrial applications.This work was designed to create a new support for Candida rugosa lipase(CRL)immobilization.A porous poly(vinyl acetate–divinyl benzene)microsphere coated by a zwitterionic polymer,poly(maleic anhydride-alt-1-octadecene)and N,N-dimethylethylenediamine derivative,was developed for CRL immobilization via hydrophobic binding.The catalytic activity,reaction kinetics,stabilities and reusability of the immobilized CRL were investigated.It demonstrated the success of the zwitterionic polymer coating and subsequent CRL immobilization on the porous microsphere.The immobilized lipase(p2-MS-CRL)reached27.6 mg·g^-1 dry carrier and displayed a specific activity 1.5 times higher than free CRL.The increase of Vmax and decrease of Kmwere also observed,indicating the improvement of catalytic activity and enzyme-substrate affinity of the immobilized lipase.Besides,p2-MS-CRL exhibited significantly enhanced thermal stability and pH tolerance.The improved performance was considered due to the interfacial activation regulated by the hydrophobic interaction and stabilization effect arisen by the zwitterionic polymer coating.This study has thus proved the advantages of the zwitterionic polymer-coated porous carrier for lipase immobilization and its potential for further development in various enzyme immobilizations.

Fabrication and characterization of epoxylated zwitterionic copolymergrafted silica nanoparticle as a new support for lipase immobilization

Our previous work proved that the thermal stability of Candida rugosa lipase(CRL)immobilized on zwitterionic polymer(poly(carboxybetaine methacrylate))grafted silica nanoparticle(SNP)was much higher than that on poly(glycidyl methecrylate)(pGMA)grafted SNP,while the latter showed significantly increased activity.Inspired by the research,we have herein proposed to synthesize copolymers of zwitterionic sulfobetaine methacrylate(SBMA)and GMA for CRL immobilization.The copolymers were grafted onto SNP surface at three GMA/SBMA(G/S)molar ratios(G100/S0,G50/S50,G10/S90),followed by the covalent coupling of CRL to the surface copolymers.The immobilized CRLs on the corresponding supports were denoted as p(G100-S0)-CRL,p(G50-S50)-CRL and p(G10-S90)-CRL.The enzyme loading increased with the increase of GMA content in the copolymer,while the activity varied with the grafted copolymer composition.Kinetic study proved the improvement of enzyme-substrate affinity after immobilization.In comparison to p(G100-S0)-CRL,p(G50-S50)-CRL and p(G10-S90)-CRL presented remarkably enhanced thermal stability and pH tolerance,and p(G10-S90)-CRL showed the highest stability.These results suggest that the copolymer design is promising for development as a versatile platform for enzyme immobilization.

Enhanced Riboflavin Production by Expressing Heterologous Riboflavin Operon from B.cereus ATCC14579 in Bacillus subtilis

Fragment containing the whole riboflavin(rib)operons of B.cereus ATCC14579 was detected from GenBank and annotated.The rib operon of ATCC14579 was cloned with Pn,its native promoter,or with P43,the vegetative growth promoter,into the plasmid.Expression analysis showed that heterologous rib operon was operative in B.subtilis.Integrative plasmid with P43-rib fragment was integrated into the chromosome of B.subtilis RH33,yielding transformant B.subtilis PY.With optimized medium components,4.3 g·L -1 of riboflavin was achieved in batch culture of B.subtilis PY,which was 27%enhancement compared to the host strain.Real-time reverse transcription polymerase chain reaction(RT-PCR)analysis indicated that the transcriptional level of ribA maintained 2.8-fold higher with the expression of herterologous rib operon.Furthermore,the stability of B.subtilis PY was increased form 45%to 87%.The high transcriptional level of rib gene and higher stability of B.subtilis PY could explain the increased riboflavin production.

Adsorption of Heavy Metal Ions by Adsorbent from Waste Mycelium Chitin

The adsorption properties of chitin adsorbent from mycelium of fermentation industries for the removal of heavy metal ions were studied. The result shows that the chitin adsorbent has high adsorption capacity for many heavy metal ions and Ni2+ in citric acid. The influence of pH was significant:When pH is higher than 4.0, the high adsorption capacity is obtained,otherwise H+ ion inhibits the adsorption of heavy metal ions. The comparison of the chitin adsorbent with some other commercial adsorbents was made, in which that the adsorption behaviorchitin adsorbent is close to that of commercial cation exchange adsorbents, and its cost is much lower than those commercial adsorbents.

Protein retention in dextran-grafted cation exchange chromatography:The influence of pHs,counterions and polymer structure

Polymer-grafted ion exchange adsorbents were of great interest for the development of high-performance protein chromatography in biopharmaceutical and related fields.In this work,protein retention was systematically investigated in ion exchange chromatography packed respectively with dextran-grafted cation exchange adsorbents containing sulphopropyl(SP)ligand,SP Sepharose XL and Capto S,and non-grafted cation exchange adsorbent,SP Sepharose FF,using five proteins.With an increase of buffer p Hs,retention factors of proteins decreased among all the adsorbents,demonstrating the dominant role of electrostatic interaction for protein binding on cation exchange adsorbents.The evidences further revealed that the scattered positive charges on the surface of protein molecules,rather than net charge of protein molecule,determined protein retention on cation exchange adsorbent.Likely,counterions including NH4^+,K^+,Na^+and Mg^2+exhibited distinct influence on protein retention.It was well ascribed to solvent-mediated indirect ion-macromolecule interactions and direct ion-macromolecule interactions.Compared with SP Sepharose FF,polymer structure in dextran-grafted cation exchange adsorbents ultimately brought about different ligand distributions and smaller pore sizes,thereby regulating protein retention in cation exchange chromatography.By comparing the retention of myoglobin andβ-lactoglobulin B in SP Sepharose XL and Capto S,we reasonably speculated that the enhancement of nonelectrostatic interaction caused by reducing the space arm length was a major reason for an increasing retention factor of myoglobin in Capto S.The results in this research help us understand adsorption mechanism of protein in polymer-grafted adsorbents and give scientific guidance for the development of chromatographic materials.

Lysozyme adsorption to cation exchanger derivatized by sequential modification of poly(ethylenimine)-Sepharose with succinic anhydride and ethanolamine: Effect of p H and ionic strength

In our previous work, a series of polyethylenimine(PEI)-derived cation exchangers were synthesized using PEIgrafted resin FF-PEI-L740(ionic capacity, 740 mmol·L^-1) as the basic resin to study lysozyme adsorption and chromatographic behavior. It was found that the resin with an ionic capacity of 630 mmol·L^-1(FF-PEI-CR630)possessed high adsorption performance towards lysozyme at 0–100 mmol·L^-1 Na Cl. Therefore, in this work,FF-PEI-CR630 was selected to study the influences of pH and ionic strength(IS) on protein adsorption and chromatographic behavior towards lysozyme. The increase of lysozyme adsorption capacity in the pH range of 6 to 10 was observed. However, the uptake rate decreased in the pH range of 6 to 8 and then remained essentially unchanged from pH 8 to pH 10. Increasing IS led to decreased protein adsorption capacity and increased uptake rate in different pH ranges. Besides, FF-PEI-CR630 maintained dynamic binding capacity as high as over150 mg·ml^-1 at pH 8–10 without NaCl. The research has thus provided insight into the selection of proper pH and IS conditions for protein purification by using FF-PEI-CR630.

Design and regulation of the surface and interfacial behavior of protein molecules

Surface and interfacial behavior of protein molecules are crucial for the protein function involved in many biochemical processes and biomedical products such as enzyme design,bio-separation,drug design and delivery.This article is devoted to an overview of design and regulation of the surface and interfacial behavior of protein molecules.The improvement of enzyme surface such as the directed evolution and the rational design of enzymes is introduced at first,followed by the rational design of protein interface for the protein assembly.Thereafter,the design of micro-environment and ligands are described as two examples for the design guided by protein surface.Then the design of protein surface and interface with the help of artificial intelligence will be discussed.