Enhanced biodegradation activity toward polyethylene by fusion protein of anchor peptide and Streptomyces sp. strain K30 latex clearing protein.

Polyethylene is the most commonly used plastic product, and its biodegradation is a worldwide problem. Latex clearing protein derived from Streptomyces sp. strain K30 (LcpK30) has been reported to be able to break the carbon-carbon double bond inside oxidized polyethylene and is an effective biodegradation enzyme for polyethylene. However, the binding of the substrate to the enzyme was difficult due to the hydrophobic nature of polyethylene. Therefore, to further improve the efficiency of LcpK30, the effect of different anchor peptides on the binding capacity of LcpK30 to the substrate was screened in this study. The results of fluorescence confocal microscopy showed that the anchoring peptide LCI had the most significant improvement in effect and was finally selected for further application in a UV-irradiated PE degradation system. The degradation results showed that LCI was able to improve the degradation efficiency of LcpK30 by approximately 1.15 times in the presence of equimolar amounts of protein compared with wild-type. This study further improves the application of LcpK30 in the field of polyethylene degradation by modification.

Enhancing the heterologous expression of latex clearing protein from Streptomyces sp. strain K30 in Escherichia coli through fermentation condition optimization and molecular modification.

Latex clearing protein from Streptomyces sp. strain K30 (LcpK30) is a natural oxidoreductase that can catalyse the cleavage of rubber through dioxygenation. It has significant potential applications in polymer degradation. However, its limited expression in engineered strains restricts its utility. This study aimed to enhance the soluble expression and enzyme activity of LcpK30 in E. coli BL21 (DE3) by optimizing fermentation conditions and making molecular modifications. The enzyme activity reached 5.05 U·mL-1 by optimizing the induction conditions, adding cofactors, and using chemical chaperones, which was 237.1 % of the initial case. Further enhancements in soluble expression were achieved through site mutations guided by the PROSS server, resulting in 8 out of 13 mutants with increased protein expression, a high positive mutation rate of 61.5 %. Subsequently, combined mutants were created by merging single mutants with enhanced protein expression and enzyme activity. The top three double mutants, G91D/S149A, G91D/A210H, and G91D/H296P, displayed expression levels at 173.3 %, 173.3 %, and 153.3 % of the wild-type LcpK30, respectively. These mutants also exhibited enhanced fermentation enzyme activity, reaching 149.5 %, 250.0 %, and 420.2 % compared to the wild-type, along with improved specific activities. This study provides insights for the efficient production of LcpK30 and a practical foundation for its application.

Inverse design of an ultra-compact and large-bandwidth bent subwavelength grating wavelength demultiplexer.

Inverse design has attracted significant attention as a method to improve device performance and compactness. In this research, we employed a combination of forward design and the inverse algorithm using particle swarm optimization (PSO) to design a bent ultra-compact 1310/1550 nm broadband wavelength demultiplexer assisted by a subwavelength grating (SWG). Through the phase matching at 1550 nm and the phase mismatch at 1310 nm, we rapidly designed the width parameters of SWG in the forward direction. Then the PSO algorithm was used to optimize the SWG parameters in a certain range to achieve the best performance. Additionally, we introduced a new bent dimension significantly reducing the device length while maintaining low insertion loss (IL) and high extinction ratios (ERs). It has been verified that the length of the device is only 7.8 µm, and it provides a high ER of 24 dB at 1310 nm and 27 dB at 1550 nm. The transmitted spectrum shows that the IL values at both wavelengths are below 0.1 dB. Meanwhile, the 1 dB bandwidth exceeds 150 nm, effectively covering the entire O-band and C-band. This approach has been proven successful in enhancing performance and significantly reducing the device footprint.

All-optical spiking neural network and optical spike-time-dependent plasticity based on the self-pulsing effect within a micro-ring resonator.

In this paper, we proposed an all-optical version of photonic spiking neurons and spike-time-dependent plasticity (STDP) based on the nonlinear optical effects within a micro-ring resonator. In this system, the self-pulsing effect was exploited to implement threshold control, and the equivalent pulse energy required for spiking, calculated by multiplying the input pulse power amplitude with its duration, was about 14.1 pJ. The positive performance of the neurons in the excitability and cascadability tests validated the feasibility of this scheme. Furthermore, two simulations were performed to demonstrate that such an all-optical spiking neural network incorporated with STDP could run stably on a stochastic topology. The essence of such an all-optical spiking neural network is a nonlinear spiking dynamical system that combines the advantages of photonics and spiking neural networks (SNNs), promising access to the high speed and lower consumption inherent to optical systems.

Tunable and switchable multi-wavelength pulsed fiber laser based on a nonlinear optical loop mirror and an improved Sagnac filter.

We propose and experimentally demonstrate a tunable and switchable multi-wavelength erbium-doped fiber ring pulsed laser based on a nonlinear optical loop mirror (NOLM) and an improved Sagnac filter. To achieve multi-wavelength pulsed laser output, we adopt a NOLM as a quasi-saturable absorber and an improved Sagnac loop as a wavelength selected filter. The constructed laser has a maximum output wavelength number of five with a pulse repetition frequency of 40.45 kHz and pulse duration of 108 ns. The laser can output single-wavelength and dual-wavelength pulsed lasers within a certain wavelength tuning range and a five-wavelength pulsed laser with a constant wavelength interval of 3 nm by adjusting the polarization controller. Dual-wavelength, three-wavelength, and four-wavelength pulsed lasers with various wavelength intervals are also obtained. The output performance of the constructed laser is tested with a maximum average output power of 127.45 µW and minimum pulse duration of 52 ns, and the stability of the laser output is also tested with a maximum power fluctuation of 0.62 dB and minimum wavelength drift of 0.51 nm with pump power of 350 mW.

Tunable and switchable multi-wavelength actively Q-switched random fiber laser based on an electro-optic modulator and Sagnac loop filter.

In this article, an actively Q-switched multi-wavelength random fiber laser based on a Sagnac loop filter and electro-optic modulator is proposed and demonstrated experimentally. The random distributed feedback media is a section of a 25 km long single-mode fiber. When the pump power is 350 mW, the polarization angle of the Sagnac loop filter can be adjusted by polarization controller to achieve the switching of a single, double, triple, and quadruple channels laser output. In the case of a single laser channel, dual laser channels, and three laser channels output, multiple laser channels can be tuned simultaneously with a fixed wavelength interval. In addition, by changing the waveform of the external signal source, the light and dark pulses can be switched. Owing to the half-open cavity structure and the high gain of the erbium-doped fiber, the laser threshold was reduced to 25 mW, and the light conversion efficiency was 0.67%. The laser is an ideal light source for medical imaging and long-distance sensing.

Advances in ultrahigh-throughput screening technologies for protein evolution.

Inspired by natural evolution, directed evolution randomly mutates the gene of interest through artificial evolution conditions with variants being screened for the required properties. Directed evolution is vital to the enhancement of protein properties and comprises the construction of libraries with considerable diversity as well as screening methods with sufficient efficiency as key steps. Owing to the various characteristics of proteins, specific methods are urgently needed for library screening, which is one of the main limiting factors in accelerating evolution. This review initially organizes the principles of ultrahigh-throughput screening from the perspective of protein properties. It then provides a comprehensive introduction to the latest progress and future trends in ultrahigh-throughput screening technologies for directed evolution.

Optimization of Ultrahigh-Throughput Screening Assay for Protein Engineering of d-Allulose 3-Epimerase.

d-Allulose is the corresponding epimer of d-fructose at the C-3 position, which exhibits a similar taste and sweetness to sucrose. As a low-calorie sweetener, d-allulose has broad application prospects in the fields of medicine, food, and so on. Currently, the production method of d-allulose is mainly the enzymatic conversion of d-fructose by d-allulose 3-epimerase (DAEase). However, the limited specific activity and thermal stability of DAEase restrict its industrial application. Herein, an ultrahigh-throughput screening assay based on the transcription factor PsiR was extensively optimized from the aspects of culture medium components, screening plasmid, and expression host, which enhanced the correction between the fluorescent readout and the enzyme activity. Then, the error-prone PCR (epPCR) library of Clostridium cellulolyticum H10 DAEase (CcDAEase) was screened through the above optimized method, and the variant I228V with improved specific activity and thermal stability was obtained. Moreover, after combining two beneficial substitutions, D281G and C289R, which were previously obtained by this optimized assay, the specific activity of the triple-mutation variant I228V/D281G/C289R reached up to 1.42-fold of the wild type (WT), while its half-life (T1/2) at 60 °C was prolonged by 62.97-fold. The results confirmed the feasibility of the optimized screening assay as a powerful tool for the directed evolution of DAEase.

Tunable and switchable multi-wavelength random distributed feedback fiber laser based on SMF and a cascaded Sagnac ring filter.

A tunable and switchable multi-wavelength random distributed feedback fiber laser based on cascaded Sagnac loops is proposed and experimentally demonstrated. The random distribution feedback of the laser is provided by the Rayleigh scattering generated by the single-mode fiber (SMF). The cascaded Sagnac loops act as a filter and a reflector in the half-open cavity laser. The single-, dual-, three-, and four-wavelength channels can be realized by adjusting the angle of the polarization controller at the pump power of 300 mW. In the single-, dual-, and three-wavelength channels, the wavelength spacing can be maintained, and the laser wavelength position can be changed at the same time. The maximum wavelength tuning ranges of single-, dual-, and three-wavelength outputs are about 4.5 nm, 2.6 nm, and 1 nm, respectively. The proposed multi-wavelength random fiber laser has the advantages of simple structure and low threshold, and it has good application prospects in remote sensing and imaging systems.

Boosting the Heterologous Expression of d-Allulose 3-Epimerase in Bacillus subtilis through Protein Engineering and Catabolite-Responsive Element Box Engineering.

As a natural sweetener with low calories and various physiological activities, d-allulose has drawn worldwide attention. Currently, d-allulose 3-epimerase (DAEase) is mainly used to catalyze the epimerization of d-fructose to d-allulose. Therefore, it is quite necessary to enhance the food-grade expression of DAEase to meet the surging market demand for d-allulose. In this study, initially, the promising variant H207L/D281G/C289R of Clostridium cellulolyticum H10 DAEase (CcDAEase) was generated by protein engineering, the specific activity and the T1/2 of which were 2.24-fold and 13.45-fold those of the CcDAEase wild type at 60 °C, respectively. After that, PamyE was determined as the optimal promoter for the recombinant expression of CcDAEase in Bacillus subtilis, and catabolite-responsive element (CRE) box engineering was further performed to eliminate the carbon catabolite repression (CCR) effect. Lastly, high-density fermentation was carried out and the final activity peaked at 4971.5 U mL-1, which is the highest expression level and could effectively promote the industrial production of DAEase. This research provides a theoretical basis and technical support for the molecular modification of DAEase and its efficient fermentation preparation.

Enhancement of the degradation capacity of IsPETase for PET plastic degradation by protein engineering.

The enormous waste of polyethylene terephthalate (PET) plastic has a great negative impact on the ecological environment because of its chemical inertia. To reduce the environmental threat posed by PET plastic, researchers gradually concentrate on the biodegradation of PET plastic. In this study, DuraPETaseN233C/S282C/H214S/S245R (DuraPETase-4M) was designed through protein engineering, which can be used to improve the efficiency of PET plastic biodegradation. Based on the DuraPETase, a pair of disulfide bonds (N233C/S282C) was added to improve the thermal stability. Meanwhile, the key region flexibility adjustment (H214S) was proposed to enhance the biodegradation capacity of PET plastic. Additionally, protein surface electrostatic charge optimization (S245R) was adopted to improve the binding ability between enzyme and PET plastic. Based on molecular dynamic simulations (MDs), the rationality of the design was further verified. This study provides a strategy for obtaining high-efficiency PET degradation mutants and a new possibility of environmentally friendly plastic degradation.

Enhanced biodegradation activity towards poly(ethyl acrylate) and poly(vinyl acetate) by anchor peptide assistant targeting.

The paper industry is one of the most important basic raw material pillar industries. With the decrease of forest wood resources, the recycling of wastepaper has drawn increasingly attention. However, the stickies generated in the process of wastepaper recycling will flocculate and deposite in the pulp, resulting in production accidents and inferior product quality. The biological enzymatic method, with the advantages of high efficiency, specificity, and pollution-free, can prevent the flocculation of the stickies by enzymatically hydrolyzing the ester bond of the stickies components. Previous studies have demonstrated that cutinase (EC 3.1.1.74) had the ability to degrade polyester components of stickies. Meanwhile, relevant studies have shown that anchor peptides possessed the ability to bind polyester. Herein, the cutinase from Humicola insolens (HiC) was fused with Escherichia coli anchor peptide OMP25, the enzymatic properties of the fusion protein HiC-OMP25 and its degradation efficiency of the stickies model substrate, poly(ethyl acrylate) (PEA) and poly(vinyl acetate) (PVAc), as well as stickies sediment were determined. All of the results demonstrated that OMP25 efficiently enhanced the degradation ability of HiC.

Enhancement of PET biodegradation by anchor peptide-cutinase fusion protein.

With the increasing production of polyethylene terephthalate (PET) plastic products, the problem of PET waste has become a serious threat to ecosystem. PET enzymatic biodegradation, due to its environmental friendliness and sustainability, has gradually attracted attention. As a multifunctional hydrolase, cutinase (EC 3.1.1.74) can not only degrade fatty acid esters, soluble synthetic esters, and emulsified triglycerides, but also exhibit potential for PET degradation. In order to enhance the PET degradation activity of cutinase, we functionally screened several PET binding domains, e.g. carbohydrate binding module, anchor peptide, and hydrophobin, that promote the absorption of enzyme to PET substrate, selected Dermaseptin SI (DSI) and fused it onto the N-terminus of Thermobifida fusca cutinase mutant D204C/E253C (Tfuc2), and finally achieved the PET degradation rate up to 57.9% at 70 °C for 96 h, which was 22.7-fold of that of Tfuc2 itself. These results indicate that the fusion of PET binding domain is a promising strategy to enhance PET enzymatic degradation.

[Characterization of Humicola insolens cutinase-tachystatin A2 fusion protein and its application in treatment of recycled paper stickies].

With the decrease of forest timber resources, the recycling of waste paper has received increasing attention. However, the stickies produced in the process of waste paper recycling may negatively affect the production of recycled paper. The biological decomposition of stickies, which has the advantages of high efficiency, high specificity and pollution-free, is achieved mainly through the enzymatic cleavage of the ester bond in the stickies components to prevent flocculation. Cutinase is a serine esterase that can degrade some components of the stickies. Previous research indicated that the anchor peptide tachystatin A2 (TA2) is able to bind polyurethane. In this study, the cutinase HiC derived from Humicola insolens was used to construct a fusion protein HiC-TA2 by megaprimer PCR of the whole plasmid (MEGAWHOP). The enzymatic properties and the degradation efficiency of the fusion protein on poly(ethyl acrylate) (PEA), a model substrate of stickies component, were determined. The results showed that the degradation efficiency, the size decrease of PEA particle, and the amount of ethanol produced by HiC-TA2 were 1.5 times, 6.8 times, and 1.4 times of that by HiC, respectively. These results demonstrated that TA2 improved the degradation efficiency of HiC on PEA. This study provides a useful reference for biological decomposition of stickies produced in the process of recycled paper production.

[The application of carbohydrate binding module-Thermobifida fusca cutinase fusion protein in polyethylene terephthalate degradation].

With the development of global economy, the dramatically increased production of polyethylene terephthalate (PET) plastics has led to a remarkably increased amount of plastic waste. PET waste can be treated by landfill, incineration, or biodegradation. While landfilling and incineration may cause secondary pollution, biodegradation has since received increased attentions due to its environmental friendliness. Recent studies have indicated that the carbohydrate binding module (CBM) can effectively enhance the binding of PET degrading enzymes to PET, and consequently increasing PET degradation rate. Here we constructed a fusion protein BaCBM2-Tfuc containing the BaCBM2 from Bacillus anthraci and the cutinase Tfuc from Thermobifida fusca, by megaprimer PCR of whole plasmids (MEGAWHOP). Notabaly, the PET film degradation efficiency (at 60 ℃) of BaCBM2-Tfuc was 2.8 times that of Tfuc. This study may provide technical support for constructing fusion proteins capable of efficiently degrading PET.

Accelerated biodegradation of polyethylene terephthalate by Thermobifida fusca cutinase mediated by Stenotrophomonas pavanii.

Polyethylene terephthalate (PET) is a general plastic that produces a significant amount of waste due to its non-biodagradable properties. We obtained four bacteria (Stenotrophomonas pavanii JWG-G1, Comamonas thiooxydans CG-1, Comamonas koreensis CG-2 and Fulvimonas soli GM-1) that utilize PET as a sole carbon source through a novel stepwise screening and verification strategy. PET films pretreated with S. pavanii JWG-G1 exhibited weight loss of 91.4% following subsequent degradation by Thermobifida fusca cutinase (TfC). S. pavanii JWG-G1 was able to colonize the PET surface and maintain high cell viability (over 50%) in biofilm, accelerating PET degradation. Compared with PET films with no pretreatment, pretreatment with S. pavanii JWG-G1 caused the PET surface to be significantly rougher with greater hydrophilicity (contact angle of 86.3 ± 2° vs. 96.6 ± 2°), providing better opportunities for TfC to contact and act on PET. Our study indicates that S. pavanii JWG-G1 could be used as a novel pretreatment for efficiently accelerating PET biodegradation by TfC.

Synergistic biodegradation of poly(ethylene terephthalate) using Microbacterium oleivorans and Thermobifida fusca cutinase.

Poly(ethylene terephthalate) (PET) is a major source of plastic pollution. Biodegradation technologies are of paramount interest in reducing or recycling PET waste. In particular, a synergistic microbe-enzyme treatment may prove to be a promising approach. In this study, a synergistic system composed of Microbacterium oleivorans JWG-G2 and Thermobifida fusca cutinase (referred to as TfC) was employed to degrade bis(hydroxyethyl) terephthalate (BHET) oligomers and a high crystalline PET film. A novel degradation product that was obtained by M. oleivorans JWG-G2 treatment alone was identified as ethylene glycol terephthalate (EGT). With the addition of TfC as a second biocatalyst, the highest synergy degrees for BHET oligomers and PET film degradation were 2.79 and 2.26, respectively. The largest amounts of terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) (47 nM and 330 nM, respectively) were detected after combined treatment of PET film with M. oleivorans JWG-G2 at 5 × 103 µL/cm2 and TfC at 120 µg/cm2, and the degree of PET film surface destruction was more significant than those produced by each treatment alone. The presence of extracellular PET hydrolases in M. oleivorans JWG-G2, including three carboxylesterases, an esterase and a lipase, was predicted by whole genome sequencing analysis, and a predicted PET degradation pathway was proposed for the synergistic microbe-enzyme treatment. The results indicated that synergistic microbe-enzyme treatment may serve as a potentially promising tool for the future development of effective PET degradation. KEY POINTS: • An ecofriendly synergistic microbe-enzyme PET degradation system operating at room temperature was first introduced for degrading PET. • A novel product (EGT) was first identified during PET degradation. • Potential PET hydrolases in M. oleivorans JWG-G2 were predicted by whole genome sequencing analysis.

A Novel Pseudomonas geniculata AGE Family Epimerase/Isomerase and Its Application in d-Mannose Synthesis.

d-mannose has exhibited excellent physiological properties in the food, pharmaceutical, and feed industries. Therefore, emerging attention has been applied to enzymatic production of d-mannose due to its advantage over chemical synthesis. The gene age of N-acetyl-d-glucosamine 2-epimerase family epimerase/isomerase (AGEase) derived from Pseudomonas geniculata was amplified, and the recombinant P. geniculata AGEase was characterized. The optimal temperature and pH of P. geniculata AGEase were 60 °C and 7.5, respectively. The Km, kcat, and kcat/Km of P. geniculata AGEase for d-mannose were 49.2 ± 8.5 mM, 476.3 ± 4.0 s-1, and 9.7 ± 0.5 s-1·mM-1, respectively. The recombinant P. geniculata AGEase was classified into the YihS enzyme subfamily in the AGE enzyme family by analyzing its substrate specificity and active center of the three-dimensional (3D) structure. Further studies on the kinetics of different substrates showed that the P. geniculata AGEase belongs to the d-mannose isomerase of the YihS enzyme. The P. geniculata AGEase catalyzed the synthesis of d-mannose with d-fructose as a substrate, and the conversion rate was as high as 39.3% with the d-mannose yield of 78.6 g·L-1 under optimal reaction conditions of 200 g·L-1d-fructose and 2.5 U·mL-1P. geniculata AGEase. This novel P. geniculata AGEase has potential applications in the industrial production of d-mannose.

In Situ Monitoring of Membrane Protein Insertion into Block Copolymer Vesicle Membranes and Their Spreading via Potential-Assisted Approach.

Synthosomes are polymer vesicles with transmembrane proteins incorporated into block copolymer membranes. They have been used for selective transport in or out of the vesicles as well as catalysis inside the compartments. However, both the insertion process of the membrane protein, forming nanopores, and the spreading of the vesicles on planar substrates to form solid-supported biomimetic membranes have been rarely studied yet. Herein, we address these two points and, first, shed light on the real-time monitoring of protein insertion via isothermal titration calorimetry. Second, the spreading process on different solid supports, namely, SiO2, glass, and gold, via different techniques like spin- and dip-coating as well as a completely new approach of potential-assisted spreading on gold surfaces was studied. While inhomogeneous layers occur via traditional methods, our proposed potential-assisted strategy to induce adsorption of positively charged vesicles by applying negative potential on the electrode leads to remarkable vesicle spreading and their further fusion to form more homogeneous planar copolymer films on gold. The polymer vesicles in our study are formed from amphiphilic copolymers poly(2-methyl oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl oxazoline) (PMOXA-b-PDMS-b-PMOXA). Engineered variants of the transmembrane protein ferric hydroxamate uptake protein component A (FhuA), one of the largest ß-barrel channel proteins, are used as model nanopores. The incorporation of FhuA Δ1-160 is shown to facilitate the vesicle spreading process further. Moreover, high accessibility of cysteine inside the channel was proven by linkage of a fluorescent dye inside the engineered variant FhuA ΔCVFtev and hence preserved functionality of the channels after spreading. The porosity and functionality of the spread synthosomes on the gold plates have been examined by studying the passive ion transport response in the presence of Li+ and ClO4- ions and electrochemical impedance spectroscopy analysis. Our approach to form solid-supported biomimetic membranes via the potential-assisted strategy could be important for the development of new (bio-) sensors and membranes.

Highly efficient production of Clostridium cellulolyticum H10 D-psicose 3-epimerase in Bacillus subtilis and use of these cells to produce D-psicose.

BACKGROUND: D-Psicose 3-epimerase (DPEase) catalyzes the isomerization of D-fructose to the rare sugar D-psicose, which may help prevent obesity, reduce blood sugar and blood fat, and inhibit intra-abdominal fat accumulation. RESULTS: In this study, the DPEase of Clostridium cellulolyticum H10 was expressed in the food-grade host Bacillus subtilis. Optimization of the culture medium during shake-flask experiments yielded a DPEase activity of 314 U/mL. The optimal medium included 20 g/L peptone, 15 g/L corn steep powder, 5 g/L glycerol, and 1 mM Ca2+. Controlling the carbon source concentration was important because elevated concentrations can result in catabolite metabolic suppression (CCR). To avoid CCR and increase DPEase expression, we developed a fed-batch strategy in a 3.6-L fermenter. We altered the ratio of carbon source to nitrogen source (C/N) in the feeding medium and employed a constant feeding rate (6 g/L/h). This strategy improved the DPEase activity to 2246 U/mL (7.8 g/L), which is almost 15 times higher than that observed in the original shake-flask cultures. Finally, we used the DPEase-expressing B. subtilis cells to produce D-psicose from D-fructose, and a 28% conversion yield was achieved with these cells, demonstrating their potential use in D-psicose production. CONCLUSIONS: This is the first report to enhance recombinant DPEase production in B. subtilis using efficient and convenient fermentation strategy, and the DPEase yield is three times higher than the highest yield reported to date. The recombinant B. subtilis cells were further used in the efficient synthesis of D-psicose. This study provides a basis for the industrial production of D-psicose.