Γ-glutamic Acid Research Articles

Preparation of gelatin/poly (γ-glutamic acid) hydrogels with stimulated response by hot-pressing preassembly and radiation crosslinking.

The conventional preparation method of natural polymers is low in strength, which limits the application of multi stimuli responsive hydrogels as intelligent wound dressings. In this work, interpenetrating gelatin and γ-polyglutamic acid (γ-PGA) hydrogels with good biocompatibility, biodegradability and mechanical properties were prepared by hot-pressing pre-gelation of gelatin/γ-PGA and synergistic with gamma irradiation. The effects of radiation crosslinking dosage on gel fraction, swelling ratio, tensile strength and surface morphology were investigated. The biocompatibility of radiation cross-linked hydrogel was evaluated by aseptic test, hemolysis test, cytotoxicity test, delayed hypersensitivity reaction as well as in vivo degradation studies from in vitro to in vivo. The optimized hydrogel irradiated by 25kGy has good water retention and biodegradability, especially the stimulation of temperature, pH value, salt species and concentration. The mechanical strength, biocompatibility and responsive properties of the hydrogel indicate that the intelligent hydrogel prepared by this method is a good hydrogel biomaterial for developing interactive wound dressing.

Application of 3D-FS-SPGR imaging combined synovial fluid GGCX detection in the evaluation of knee osteoarthritis.

Osteoarthritis represents a kind of chronic and degenerative joint disease characterized by articular cartilage injury and osteoproliferation. Osteoarthritis especially poses a serious threat to the elderly patients. At present, the diagnosis of osteoarthritis mainly consists of clinical examination, X-ray examination, magnetic resonance imaging (MRI), and arthroscopy. However, limitations and misdiagnosis are found within the single method. This article intends to investigate the feasibility of assessing the condition of knee osteoarthritis through quantitative analysis of cartilage using nuclear magnetic resonance 3D fast-spin spoiled gradient-recalled echo (NMR 3D-FS-SPGR) imaging and γ-glutamic acid carboxylase (GGCX) detection in synovial fluid. A total of 60 patients with primary knee osteoarthritis were enrolled. All the patients were staged and received 3D-FS-SPGR sequence MRI scan for grading based on scan results and cartilage injury. Cartilage tissues were collected for immunohistochemistry (IHC). The GGCX in cartilage was detected using western blotting to analyze the correlation with arthritis. The condition of articular cartilage injury in arthritis patients was clearly observed using 3D-FS-SPGR sequence. The expression of GGCX was decreased in 46 patients (p < 0.05). The expression of GGCX in synovial fluid was significantly reduced following upstaging (p < 0.05). The sensitivity measured using combined 3D-FS-SPGR imaging and synovial fluid GGCX detection for the evaluation of arthritis condition was significantly higher than that of the single detection method (p < 0.05). Our data showed that the sensitivity of combined detection was obviously higher than single detection for the evaluation of arthritis. The 3D-FS-SPGR combined with synovial fluid GGCX detection could be treated as a promising strategy for arthritis evaluation.

Inhibition of nattokinase against the production of poly (γ-glutamic Acid) in Bacillus subtilis natto.

To study the effect of nattokinse (NK) on the synthesis of poly(γ-glutamic acid) (γ-PGA) in Bacillus subtilis natto. γ-PGA yield significantly decreased as NK was added in the original medium. With the increment of NK dosage, the yield decreased increasingly, but biomass increased instead of decreasing. The fact that cell density triggers the synthesis of γ-PGA is a controversial issue. γ-PGA yield and biomass closely correlate with addition time of NK. The later the addition of NK, the more γ-PGA yield decreased but the more biomass increased. It is concluded that cell hunger is a key factor to trigger the transmission of the cell density signal, and NK may inhibit γ-PGA synthesis by alleviating cell hunger. Besides, NK may reduce γ-PGA yield by degrading extracellular γ-PGA molecules. The study of adding L-glutamate of 0-20g/L to the original medium showed that low concentration of L-glutamate (less than 5g/L) could promote the synthesis of NK and γ-PGA, and thus NK may inhibit γ-PGA synthesis through strengthening substrate competition. NK mainly inhibits γ-PGA synthesis in Bacillus subtilis natto through alleviating cell starvation and strengthening substrate competition, and reduces γ-PGA yield through degrading extracellular γ-PGA molecules.

Covalently Adaptable Hydrogel Based on Hyaluronic Acid and Poly(γ-glutamic acid) for Potential Load-Bearing Tissue Engineering.

Reversibly cross-linked adaptable hydrogels show great advantages in tissue engineering. The dynamic adaptable networks can overcome three-dimensional (3D) physical restrictions to enable normal cellular functions without hydrogel degradation. However, because of the dynamic reversibility, adaptable hydrogels typically exhibit weak mechanical properties and rapid erosion behaviors. Herein, we develop a facile strategy to prepare stable adaptable hydrogels using dynamic covalent chemistry, stable covalent chemistry, and the interpenetrating polymeric network (IPN) strategy. The developed IPN HA/γ-PGA adaptable hydrogels have stable structures, good mechanical properties, enzymatic degradability, and injectability. Compression test indicates that although containing 95% water, the IPN HA/γ-PGA adaptable hydrogels can suffer more than 85% compressive strain and show a fast shape recovery capacity and good antifatigue ability. Benefitting from the good cytocompatibility of functionalized HA and γ-PGA and the mild preparation process of IPN HA/γ-PGA adaptable hydrogels, NIH 3T3 cells can tolerate the 3D encapsulation process and show high cell viability. Therefore, owing to their desirable properties, the developed HA/γ-PGA adaptable hydrogels have great potential applications for load-bearing tissue engineering.

Electrostatic assembly functionalization of poly (γ-glutamic acid) for biomedical antibacterial applications

Poly (γ-glutamic acid) (γ-PGA) has been found widespread applications in biomedical field because of its excellent water solubility, biocompatibility, and bioactivity. Herein, a water-insoluble γ-PGA antibacterial compound is facilely fabricated via one-pot electrostatic assembly of γ-PGA with cationic ethyl lauroyl arginate (ELA). The functionalized γ-PGA compound (γ-PGA-ELA) ethanol solution can facilely produce colorless and transparent coatings on various inorganic, metal, and polymeric substrates, especially for the lumen of slender catheters (length up to 2 m, and inner diameter down to 1 mm). The functionalized γ-PGA coating presents remarkable antibacterial efficacy in vitro and in vivo. In addition, the γ-PGA compound is used as antibacterial additives of polyolefin via melting extrusion, and the as-prepared antibacterial polyolefin demonstrates advantageous antibacterial efficacy. More importantly, the functionalized γ-PGA coating exhibit good hemocompatibility, low cytotoxicity, and satisfactory histocompatibility. The as-proposed γ-PGA compound has a great potential to serve as a safe and multifunctional antibacterial candidate to combat biomedical devices-related infections.

Fabrication of a porous chitosan/poly-(γ-glutamic acid) hydrogel with a high absorption capacity by electrostatic contacts

A porous chitosan (CS)/poly-(γ-glutamic acid) (γ-PGA) hydrogel was prepared by polymerization by electrostatic contacts of CS with γ-PGA without linker. The porosity of the hydrogel remarkably depends on γ-PGA content, pH regulator, and drying way. The optimization of them had given the hydrogel a high swelling capacity of 1398%, 11-fold higher than that of neat CS hydrogel. The hydrogel was applied to recover bovine serum albumin (BSA) from aqueous solution, and its adsorption capacity was influenced by the initial concentration and pH of BSA solution. Based on the studies of kinetics and isotherm, a high equilibrium adsorption capacity (qe = 948 mg/g) and a correlation coefficient of 0.996 were calculated from pseudo-second order kinetic equation, and a high affinitive constant (kF = 472 mL/mg) and a high saturated absorption capacity (qm = 1818.5 mg/g) were observed from Freundlich isotherm equation, indicating the porous hydrogel will be a good absorbent for protein recovery from sewage.

Bone-Inspired Tube Filling Decellularized Matrix of Toad Cartilage Provided an Osteoinductive Microenvironment for Mesenchymal Stem Cells to Facilitate the Radius Defect Repair of Rabbit.

Toad bone not only contains the rich cartilage-like matrix but also presents low immunogenicity. It is inferred that decellularized toad bone matrix (dBECM) may provide the more profitable osteoinductive microenvironment for mesenchymal stem cells (MSCs) to promote the repair of bone defects. Herein, a hollow bone-inspired tube is first made from hydroxyapatite (HA) and poly (γ-glutamic acid) (PGA), and then MSCs/dBECM hydrogel is uniformly filled to its central cavity, constructing a biomimetic bone (dBECM + MSCs - PGA + HA). In vitro scratch and transwell experiments show that dBECM hydrogel not only effectively promotes migration and proliferation of MSCs but also induces their osteogenic differentiation. Moreover, the less inflammatory macrophages infiltrate at rat skin after subcutaneously injecting dBECM hydrogel, indicating its low potential for inflammatory attack. After implanting dBECM + MSCs - PGA + HA to critical radius defect of rabbit, X-ray and CT imaging shows that the cortex is effectively regenerated and the medullary cavity recanalization is completed at 20 weeks. Moreover, the expression of Collagen-II and OCN are obviously increased in the defect after implanting dBECM + MSCs - PGA + HA. The therapeutic mechanism of dBECM + MSCs - PGA + HA scaffold are highly associated with the enhanced angiogenesis. Collectively, the biomimetic dBECM + MSCs - PGA + HA scaffold may be a promising strategy to improve radius defect healing efficiency.

Effect of bioglass on in vitro bioactivity and cytocompatibility of biphasic α-tricalcium phosphate/gypsum cements

ABSTRACT In the present study, a novel bioglass (BG)-incorporated α-triphosphate calcium (α-TCP)/gypsum composite cement with poly (γ-glutamic acid) (PGA) aqueous solution as a reactive liquid phase was developed. The effects of BG content on the properties of the PGA modified α-TCP/gypsum composite cement (PGA-Cement) were systematically investigated. Results revealed that the introduction of BG (5–20 wt%) maintained the injectibility and anti-washout ability, but prolonged the setting time to reasonable initial setting time of ~13-24 min and final setting time within ~24-33 min. Moreover, after the dosage of 5 wt% BG, the compressive strength increased from 21.52 ± 1.68 MPa of PGA-Cement to 30.17 ± 1.00 MPa of 5BG-PGA-Cement. Meanwhile, in vitro test indicated that the prepared 5BG-PGA-Cement and 10BG-PGA-Cement had better degradation and bioactivity than 20BG-PGA-Cement. All these results suggest that BG-incorporated α-TCP/gypsum composite cement have broad application prospects in minimally invasive surgery for the treatment of bone defects.

Biodegradable Poly(γ-glutamic acid)@glucose oxidase@carbon dot nanoparticles for simultaneous multimodal imaging and synergetic cancer therapy

It is known that tumor antigens could induce obvious anti-tumor immune responses for efficient cancer immunotherapy when combined with checkpoint blockade. However, the amount of tumor antigens is often limited due to the suppressive tumor microenvironment (TME). Here, a new type of nanomaterial was developed to improve tumor treatment by the combined action of starving therapy/photodynamic therapy (PDT)/photothermal therapy (PTT) and checkpoint-blockade immunotherapy. In detail, the immunoadjuvant nanoagents (γ-PGA@GOx@Mn,Cu-CDs) were fabricated by integrating the gamma-glutamyl transferase (GGT) enzyme-induced cellular uptake polymer-poly (γ-glutamic acid) (γ-PGA), a glucose-metabolic reaction agent - glucose oxidase (GOx), Mn,Cu-doped carbon dots (CDs) as photosensitizer and self-supplied oxygenator nanodots. γ-PGA@GOx@Mn,Cu-CDs nanoparticles (NPs) showed long retention time at the tumor acidic microenvironment and could further target cancer cells. The NPs also displayed both photothermal and photodynamic effects under laser irradiation at 730 nm. Interestingly, the endogenous generation of hydrogen peroxide (H2O2) caused by the nanoreactors could significantly relieve tumor hypoxia and further enhance in vivo PDT. By synergistically combining the NPs-based starving-like therapy/PDT/PTT and check-point-blockade therapy, the treatment efficiency was significantly improved. More importantly, the systematic antitumor immune response would eliminate non-irradiated tumors as well, which is promising for metastasis inhibition.

Dynamic bond crosslinked poly(γ-glutamic acid)/Salecan derived hydrogel as a platform for 3D cell culture

Hydrogel platform is considered as a feasible solution for 3D cell culture but suffers from many limitations such as polymers selection and crosslinking methods. Herein, dynamic bond crosslinked hydrogels (marked as PS) with “breathable” channels were designed based on poly(γ-glutamic acid) and Salecan. The adipic dihydrazide grafted poly(γ-glutamic acid) maintains the pH sensitive property while providing functional group for the reversible bond formation. Equilibrium water content, swelling and degradation behavior, morphology, dynamic rheology and cytotoxicity assay were investigated to characterize the hydrogels’ properties. The HeLa cells were encapsulated in PS hydrogels to determine their feasibility as platform for 3D cell culture. All the results revealed PS hydrogels possessed many adjustable properties and excellent biological functions to maintain cell growth and proliferation. Therefore, PS hydrogels can be served as a promising platform for cell culture and also expected to apply in many other medical fields.

Reduction sensitive nanocarriers mPEG-g-γ-PGA/SSBPEI@siRNA for effective targeted delivery of survivin siRNA against NSCLC

Poly γ-glutamic acid (γ-PGA) is attractive due to its desirable biological properties such as nontoxicity, excellent biocompatibility, and minimal immunogenicity. Additionally, γ-PGA could be recognized by γ-glutamyl transpeptidase, which is regarded as a potential biomarker for many tumors. In this study, we have developed a new biodegradable, reduction sensitive, and tumor-specific gene nano-delivery platform consisting of a cationic carrier (SSBPEI) for siRNA condensation, mPEG shell for nanoparticle stabilization, and γ-PGA for accelerated cellular uptake. Disulfide bonds (-SS-) could be reduced specifically in the tumor environment, which is full of reductants such as glutathione reductase. Conjugating polyethylene glycol (PEG) to the γ-PGA led to the formation of mPEG-g-γ-PGA, with a decreased positive charge on the surface of SSBPEI@siRNA and substantially higher stability in an aqueous medium. As a result, mPEG-g-γ-PGA/SSBPEI@siRNA nanoparticles could protect siRNAs from RNase A degradation and release siRNAs in a reduction sensitive way. The multifunctional delivery system was shown to silence the Survivin gene and further promote chemotherapeutic drug-induced apoptosis in the A549 NSCLC cell line efficiently, thereby representing a novel promising platform for the delivery of siRNAs.

Injectable pH-responsive poly (γ-glutamic acid)-silica hybrid hydrogels with high mechanical strength, conductivity and cytocompatibility for biomedical applications

A novel injectable pH-responsive poly (γ-glutamic acid) (PGA)-silica hybrid hydrogel with controlled structural and physicochemical properties has been developed. Hybrid hydrogels are composited of PGA and 3-glycidoxypropyl trimethoxysilane (GPTMS) with different content of silica which is derived from hydrolyzed tetraethylorthosilicate (TEOS). In this hybrid system, GPTMS is employed as a coupling agent and TEOS is used for adjusting the silica content. Effects of the silica content on the morphology, microstructure, mechanical strength, swelling behavior, drug delivery performance, conductivity and cytocompatibility are investigated. Results reveal that the microporous structure, compressive strength, swelling as well as drug delivery behavior, and in vitro cytocompatibility of hybrid hydrogels are effectively tailored by adjusting the silica content. Besides the pH-responsive swelling and drug release behaviors, the hybrid hydrogel possesses good conductivity as well as high sensitivity to external strain. Therefore, the prepared hybrid hydrogels reveal great application potential as promising injectable biomaterials for biomedical applications.

Enzymatically Cross-Linked Poly(γ-glutamic acid) Hydrogel with Enhanced Tissue Adhesive Property.

Enzymatic cross-linking of polymer-catechol conjugates in the presence of horseradish peroxidase (HRP) and H2O2 has emerged as an important method to fabricate in situ-forming, injectable hydrogels. Subsequently, tissue adhesion studies using catechol-containing polymers were extensively reported. However, because of the presence of numerous variables such as polymer concentration, oxidizing agent/enzyme, and stoichiometry, the design of the polymer with optimized tissue adhesive property is still challenging. In this study, a poly(γ-glutamic acid) (γ-PGA)-dopamine (PGADA) conjugate was synthesized, and in situ hydrogels were fabricated via enzymatic cross-linking of a catechol moiety. To optimize the tissue adhesive property of the PGADA hydrogel, the effect of various factors, such as polymer concentration, catechol substitution degree (DS), HRP concentration, and H2O2 content, on the gelation behavior and mechanical strength was investigated. The gelation behavior of PGADA hydrogels was characterized using a rheometer and rotational viscometer. Also, the possibility of its use as a tissue adhesive was examined by evaluating the tissue adhesion strength in vitro and ex vivo.

Poly(γ-glutamic acid)-based electrospun nanofibrous mats with photodynamic therapy for effectively combating wound infection.

Pathogenic bacterial infections associated with wound healing progress usually result in serious complications. Herein, biocompatible and antimicrobial electrospun nanofibrous mats with photodynamic therapy (PDT) effect were fabricated to accelerate the infected wound healing. The nanofibrous mats were fabricated by co-electrospining of polyanionic poly(γ-glutamic acid) (γ-PGA) and cationic photosensitizer 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetra (p-toluenesulfonate) (TMPyP) in aqueous solution and stabilized by the chemical crosslinking. The as-prepared nanofibrous mats can not only confer the moist microenvironment to the wound bed, but also provide potent bactericidal activity upon visible light irradiation by releasing the cytotoxic reactive oxygen species (ROS). The antibacterial assay in vitro showed that they can effectively eradicate the board-spectrum bacteria at a relatively low loading dose of TMPyP (e.g., 0.1wt%). Meanwhile, those nanofibrous mats showed good biocompatibility with no obvious adverse effects on mammalian cells and red blood cells (RBCs). The animal test in vivo suggested that the restrained inflammatory reaction and better wound healing could be achieved upon timely and effective antibacterial treatment with negligible local toxicities. This biocompatible and antibacterial γ-PGA-TMPyP nanofibrous mat may show great potential in practical infection-resistant applications, particularly for wound dressing applications.

Chelation of zinc(II) with poly(γ-glutamic acid) in aqueous solution: kinetics, binding constant, and its antimicrobial activity

The binding of zinc(II) ions onto γ-pga was studied in aqueous solution as a function of contact time, solution pH, initial concentration of metal ion, and solution temperature, respectively. IR and 13C NMR spectra of γ-pga and Zn(γ-pga) complex revealed binding of zinc(II) with carboxylate and amide groups on γ-pga. The strong interaction between zinc(II) ions and γ-pga occurred at pH 5. The binding kinetic data followed a pseudo-second-order kinetic model. The equilibrium binding data were fitted well by Langmuir isotherm model. The maximum amount of zinc(II) ions bound to γ-pga was estimated to be 13.97 mmol/g at 30 °C and 20.58 mmol/g at 45 °C. The values of activation energy (Ea 18.23 kJ/mol), changes in free energy (∆G°), enthalpy (∆H°), and entropy (∆S°) indicate that the formation of Zn(γ-pga) complex is an endothermic spontaneous physisorption process. The antimicrobial activity of Zn(γ-pga) complex was examined against a gram-negative bacteria (i.e., Escherichia coli) and a gram-positive bacteria (i.e., Bacillus subtilis). The IC50 values of Zn(γ-pga) complex were estimated to be (0.76 ± 0.03) mmol/L for E. coli and (0.94 ± 0.02) mmol/L for B. subtilis, respectively. Therefore, Zn(γ-pga) complex can be used as an antimicrobial agent against gram-positive and gram-negative microorganisms.

Hierarchical 0D-2D bio-composite film based on enzyme-loaded polymeric nanoparticles decorating graphene nanosheets as a high-performance bio-sensing platform

Herein, we developed a hierarchical bio-composite sensing film by facile one-step electro-deposition of 0D enzyme-polymer nanoparticles (NPs) with 2D graphene oxide nanosheets as conductive supports and nanofillers, based on which an effective and robust enzymatic biosensor platform was constructed. Horseradish peroxidase (HRP) as a model enzyme was co-assembled with a photo-cross-linkable polypeptide of 2-hydroxyethyl methacrylate modified poly(γ-glutamic acid) (γ-PGA-HEMA), generating hybrid HRP@γ-PGA-HEMA nanoparticles (HRP@PGH NPs). Then HRP@PGH NPs and graphene oxide nanosheets (GO NSs) were simultaneously electrodeposited onto the electrode surface, obtaining a hierarchical 0D-2D bio-composite film. After subsequent electrochemical reduction of GO NSs into graphene nanosheets (GNSs) and following photo-cross-linking, the resultant nanostructured HRP@PGH/GNSs sensing film was successfully applied to construct an enzymatic biosensor for hydrogen peroxide (H2O2). The biosensor exerted high sensitivity, fast response, and good stability for H2O2 sensing. Satisfactory results were also demonstrated for its practical application in human serum samples, suggesting a promising application potential in biomedical diagnostics. The one-step generated 0D-2D bio-composite sensing film demonstrates synergetic effects from both the soft nanoparticles and hard conductive nanosheets, which would enlighten the innovative construction of composite nanomaterials and nanoarchitectonics for bio-sensing systems.

Poly(N-Isopropyl-Acrylamide)/Poly(γ-Glutamic Acid) Thermo-Sensitive Hydrogels Loaded with Superoxide Dismutase for Wound Dressing Application.

IntroductionChronic trauma repair is an important issue affecting people’s healthy lives. Thermo-sensitive hydrogel is injectable in situ and can be used to treat large-area wounds. In addition, antioxidants play important roles in promoting wound repair.MethodsThe purpose of this research was to prepare a novel thermo-sensitive hydrogel-poly(N-isopropyl-acrylamide)/poly(γ-glutamic acid) (PP) loaded with superoxide dismutase (SOD) to improve the effect for trauma treatment. The micromorphology of the hydrogel was observed by scanning electron microscope and the physical properties were measured. The biocompatibility of hydrogel was evaluated by MTT experiment, and the effect of hydrogel on skin wound healing was evaluated by in vivo histological staining.ResultsGelling behavior and differential scanning calorimeter outcomes showed that the PP hydrogels possessed thermo-sensitivity at physiological temperature and the phase transformation temperature was 28.2°C. The high swelling rate and good water retention were conducive to wound healing. The activity of SOD in vitro was up to 85% at 10 h, which was advantageous to eliminate the superoxide anion. MTT assay revealed that this hydrogel possessed good biocompatibility. Dressings of PP loaded with SOD (SOD-PP) had a higher wound closure rate than other treatments in vivo in diabetic rat model.DiscussionThe SOD-PP thermo-sensitive hydrogels can effectively promote wound healing and have good application prospects for wound repair.

Boron-assisted dual-crosslinked poly (γ-glutamic acid) hydrogels with high toughness for cartilage regeneration

Although mesenchymal stem cells (MSCs) encapsulated with bionic scaffolds have been widely used in treating trauma induced cartilage defects, challenges still persist for these hydrogel scaffolds to create suitable shelters for the MSCs and guide their behaviors. In this work, novel biomimetic hydrogel scaffolds were prepared by thiol-ene Michael addition between glycidyl methacrylate (GMA)-modified poly (γ-glutamic acid) (γ-PGA-GMA) and DL-1,4-Dithiothreitol (DTT) for cartilage tissue engineering. Sodium tetraborate decahydrate was added into the system to connect with DTT through hydrogen-bond interaction and served as catalyst for thiol-ene Michael addition to strengthen the intensity of the hydrogel. The hydrogels could be compressed to nearly a 90% strain, with 0.95 MPa compression stresses which was better than that of most hydrogels in mechanical property. Additionally, this hydrogel has other properties: fast and controlled gel-forming speed, adjustable swelling ration, suitable interior structure and so on. Above all, hydrogels have excellent cyto/tissue-compatibility. Cells cultured in hydrogels in vitro exhibited good proliferation and adhesion abilities and the hydrogels scaffolds contained stem cells immensely accelerated the regeneration of auricular cartilage of rabbits in vivo versus control group. The overall results approved that this bionic hydrogel may be a promising biomaterial for cartilage regeneration in the future.

Construction and application of a dual promoter system for efficient protein production and metabolic pathway enhancement in Bacillus licheniformis

Promoter plays the critical role in regulating gene transcription, and dual-promoter has received the widespread attentions due to its high efficiency and continuity, here, we want to construct an efficient dual-promoter for protein production and metabolic pathway enhancement. Firstly, our results indicated that P43 promoter efficiently transcribed at logarithmic period, while the σB-type promoters (PylB, PgsiB, PykzA) were active at stationary phase. Then, several dual promoters were constructed by coupling these σB-type promoters with P43, and the attained dual-promoter PykzA-P43 showed the best performance, which led to 1.72-, 3.46- and 1.85-fold increases of green fluorescence intensity, red fluorescence intensity and α-amylase activity, compared with those of the recognized strong promoter P43, respectively. Furthermore, α-amylase activity was further increased to 389.65 U/mL by 32.20 % via optimizing sigma factor binding sites (-10 and -35 boxes) of PykzA-P43, attaining the optimized dual promoter Pdual3. Finally, Pdual3 was applied in metabolic pathway enhancement, and the yields of Poly γ-glutamic acid, acetoin and 2, 3-butanediol were respectively improved by 82.01 %, 17.09 % and 99.39 %. Our results indicated that dual-promoter significantly enhanced gene expression, and this study provided an energetic dual-promoter Pdual3 for efficient protein production and metabolic pathway enhancement in Bacillus licheniformis.

Optimized production of poly (γ-glutamic acid) (γ-PGA) using Bacillus licheniformis and its application as cryoprotectant for probiotics.

Bacteria produce poly (γ-glutamic acid) (γ-PGA), a polymer of l- or d-glutamic acid, as a defense response and have gained importance due to their applications in food and pharmaceutical industry. In the present investigation, production of γ-PGA using cost-effective carbon substrate, characterization of the produced polymer, and its application as cryoprotectant for selected freeze-dried probiotic bacteria were investigated. Central composite rotatable design of response surface methodology was used to study the main and the interactive effects of medium components: rice bran and casein peptone concentration. Rice bran at 35% (w/v) and casein peptone at 7.5% (w/v) were found to be optimal at an initial pH of 7.5, and incubation temperature of 37°C for 48 H produced 8.2 g/L γ-PGA on dry weight basis. The thermal properties such as melting temperature, heat of fusion, and thermal stability were also studied. Ten percent (w/v) of γ-PGA with 10 percent of sodium alginate (w/v) protected viability of Bifidiobacterium bifidum NCDC 235 and B. adolescentis NCDC 236 during freeze drying at -80˚C for 48 H. The γ-PGA synthesized by the reported bacterium with GRAS status is suitable for food and biomedical applications.