Biochemical characterization and molecular modification of a zearalenone hydrolyzing enzyme Zhd11D from Phialophora attinorum.

ZEN lactone hydrolase (ZHD) can hydrolyze zearalenone (ZEN) to less or non-toxic product, providing an environment-friendly way for food or feeds-containing ZENs detoxification. Here, a newly identified ZHD from Phialophora attinorum, annotated as Zhd11D, was characterized to exhibit highest activity against ZEN at pH 8.0 and 35 â„ƒ with a specific activity of 304.7 U/mg, which was far higher than most of the reported ZHDs. A nonspecific protein engineering method was introduced through fusing a segment of amphiphilic short peptide S1 at the N-terminus of Zhd11D, resulting in both improved activity (1.5-fold) and thermostability (2-fold at 40 â„ƒ). Biochemical analysis demonstrated that self-aggregation caused by intermolecular interactions between S1 contributed to the improvement of the enzymatic properties of Zhd11D. Additionally, S1-Zhd11D showed a higher hydrolysis rate of ZEN than Zhd11D in peanut oil.

Lycium barbarum polysaccharide-glycoprotein ameliorates ionizing radiation-induced epithelial injury by regulating oxidative stress and ferroptosis via the Nrf2 pathway.

Radiation-induced oral mucositis (RIOM) is considered to be the most common acute side effect of radiation therapy and occurs during intentional or accidental radiation exposure. Antioxidant synthesis agents have been reported to protect against or alleviate the development of mucositis, but the resulting side effects of chemical synthesis agents limit their use in clinical practice. Lycium barbarum polysaccharide-glycoprotein (LBP), a polysaccharide extract of the Lycium barbarum fruit, has superior antioxidant capacity and biosafety and is a potential option for radiation prevention and treatment. Here, we aimed to investigate whether LBP conferred radioprotection against ionizing radiation-induced oral mucosal damage. We found that LBP exerted radioprotective effects in irradiated HaCaT cells, improving cell viability, stabilizing mitochondrial membrane potential, and decreasing cell death. LBP pretreatment reduced oxidative stress and ferroptosis in radioactivity-damaged cells by activating the transcription factor Nrf2 and promoting its downstream targets, such as HO-1, NQO1, SLC7A11, and FTH1. Knockdown of Nrf2 eliminated the protective effects of LBP, implying the essential role of Nrf2 in LBP activity. Additionally, the topical application of LBP thermosensitive hydrogel on rat mucosa resulted in a significant decrease in ulcer size in the irradiated group, suggesting that LBP oral mucoadhesive gel may be a potential tool for the treatment of irradiation. In conclusion, we demonstrated that LBP attenuates ionizing radiation-induced oral mucosa injury by reducing oxidative stress and inhibiting ferroptosis via the Nrf2 signaling pathway. LBP may be a promising medical countermeasure against RIOM.

The N-terminal hydrophobicity modulates a distal structural domain conformation of zearalenone lacton hydrolase and its application in protein engineering.

Zearalenone (ZEN) is one of the most common mycotoxins in maize, wheat, barley, sorghum, rye and other grains. ZEN contamination in feed is an international health issue due to its estrogenicity by competitively binding to estrogen receptors. Enzymatic detoxification of ZEN is superior to physical and chemical methods in terms of safety, environmental impact and preserving nutritional value and palatability, but is hampered by both the currently limited repertoire of detoxifying enzymes and the lack of knowledge about their structure-function relationships. In this study, a ZEN lacton hydrolase candidate (ZHD11C) was identified from thermo-tolerant Fonsecaea multimorphosa CBS 102226, and characterized to be more thermostable than these reported homologues. An intriguing feature of ZHD11C is that the N-terminal hydrophobicity affects its thermal stability and causes conformational change of a domain far from the N-terminal. This finding was successfully applied to enhance the thermostability of the most active ZEN lacton hydrolase ZHD518 through rationally tailoring its N-terminal hydrophobicity. Our results not only provide more insights into the structure-function relationships of ZEN lacton hydrolases, but generate better candidate for bio-decontamination of zearalenone in feed industries.

A multi-responsive targeting drug delivery system for combination photothermal/chemotherapy of tumor.

To achieve efficient delivery and precise release of chemotherapy drugs at tumor sites, an active targeting multi-responsive drug delivery platform was developed. Here, doxorubicin hydrochloride (DOX) was loaded onto polydopamine (PDA), which were coated by the cystamine-modified hyaluronic acid (HA-Cys), designated as DOX@PDA-HA (PDH). The combination of PDA and HA-Cys endowed the nanoplatform photothermal conversion, tumor-targeting, and pH/redox/NIR sensitive drug release capacity. Moreover, HA could be degraded by the excess hyaluronidase (HAase) in the tumor microenvironment (TME), promoting DOX release, and further enhancing the effect of chemotherapy. Experimental results demonstrated PDH good biocompatibility, high loading rate, targeted drug delivery, and efficient tumor cell killing ability. This ingenious strategy based on PDH showed huge potential in photothermal/chemotherapy combination treatment of cancer.

Protective effect of Lycium barbanun glycopeptide on human keratinocytes against radiation-induced oxidative stress damage and its mechanism / 中国生物制品学杂志

@#Objective To investigate the protective effect of Lycium barbanun glycopeptide(LbGP)on human keratinocyte HaCaT cells against radiation-induced cytotoxicity and its mechanism. Methods HaCaT cells were exposed to radiation with linear accelerator(rate 6 Gy/min)at doses of 4,8,12,16,20,24 and 28 Gy respectively,and the cell activity was detected by CCK-8 assay. HaCaT cells were treated with LbGP(0,0. 05,0. 1,0. 5,0. 8,1. 0,1. 5 and 3 mg/mL)for 4 h,exposed to radiation of 12 Gy,and detected for the cell viability by CCK-8 assay. HaCaT cells were divided into control group(without LbGP and radiation),radiation group(radiation of 12 Gy)and LbGP + radiation group(0. 8 mg/mL LbGP for 24 h,radiation of 12 Gy). After irradiation for 1 h,the reactive oxygen species(ROS)production was measured by flow cytometry,the superoxide dismutase(SOD)activity was determined by WST-8 assay and the expression of nuclear factor-E2 related factor 2(Nrf2),p-Nrf2,NADPH quinone oxidoreductase 1(NQO1)and heme oxygenase-1(HO-1)were detected by Western blot;The mRNA transcription levels of Nrf2,HO-1 and NQO1 were detected by qRT-PCR at 1,3 and 5 h after irradiation. Results Radiation of12 Gy induced about 50% cell death,and 0. 8 mg/mL LbGP showed the best protective effect on the activity of irradiated cells. After irradiation for 1 h,compared with the control group,the content of ROS in HaCaT cells increased significantly in radiation group(F = 2. 55,P < 0. 001),the activity of SOD decreased significantly(F = 1. 23,P < 0. 01),the contents of NQO1 and Nrf2 protein had no significant difference(F = 1. 78 and 1. 00,respectively,P > 0. 05),the content of HO-1protein increased significantly(F = 1. 37,P < 0. 05),and the content of p-Nrf2 protein decreased significantly(F = 2. 75,P < 0. 01);Compared with the radiation group,the content of ROS in HaCaT cells of LbGP + radiation group decreased significantly(F = 3. 61,P < 0. 001),the activity of SOD increased significantly(F = 1. 23,P < 0. 05),and the contents of Nrf2,p-Nrf2,HO-1 and NQO1 protein increased significantly(F = 4. 00,2. 25,6. 25 and 1. 27,respectively,P < 0. 05). In addition,the mRNA levels of Nrf2,HO-1 and NQO1 genes in LbGP + radiation group were significantly higher than those in radiation group at 1,3 and 5 h after irradiation(F = 0. 20～36. 00,P < 0. 05). Conclusion LbGP can mitigate oxidative stress damage of HaCaT cells induced by radiation and protect cell activity,which may play a role by activating Nrf2 and increasing the levels of its downstream antioxidant enzymes SOD,HO-1 and NQO1.

Antibacterial and antibiofilm activities of novel antimicrobial peptide DP7 against the periodontal pathogen Porphyromonas gingivalis.

AIMS: Accumulating evidence suggests that Porphyromonas gingivalis is closely associated with the development of various chronic inflammatory diseases, particularly periodontitis. This study investigated the antibacterial activity and action mechanism of a novel antimicrobial peptide (AMP), DP7, against P. gingivalis. METHODS AND RESULTS: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for DP7 were determined via a broth microdilution method, revealing an MIC of 8 µg ml-1 and MBC of 32 µg ml-1 . Growth inhibition and killing assays confirmed the bactericidal effect of DP7, and treatment with DP7 at MBC eliminated P. gingivalis within 8 h. DP7 had a low cytotoxic effect against human cells. Transmission electron microscopy revealed that DP7 destroyed the bacterial membrane, and confocal laser scanning microscopy revealed its inhibitory effect on P. gingivalis biofilms. Quantitative reverse transcription-polymerase chain reaction revealed DP7-mediated inhibition of several virulence factor genes, partially explaining its antibacterial mechanism. CONCLUSIONS: DP7, a novel AMP with low mammalian cytotoxicity, inhibits both planktonic and biofilm forms of P. gingivalis by destroying the bacterial membrane and reducing virulence factor gene expression. SIGNIFICANCE AND IMPACT OF THE STUDY: DP7 has potential clinical application in the prevention and treatment of P. gingivalis-associated diseases.

A review of biomimetic scaffolds for bone regeneration: Toward a cell-free strategy.

In clinical terms, bone grafting currently involves the application of autogenous, allogeneic, or xenogeneic bone grafts, as well as natural or artificially synthesized materials, such as polymers, bioceramics, and other composites. Many of these are associated with limitations. The ideal scaffold for bone tissue engineering should provide mechanical support while promoting osteogenesis, osteoconduction, and even osteoinduction. There are various structural complications and engineering difficulties to be considered. Here, we describe the biomimetic possibilities of the modification of natural or synthetic materials through physical and chemical design to facilitate bone tissue repair. This review summarizes recent progresses in the strategies for constructing biomimetic scaffolds, including ion-functionalized scaffolds, decellularized extracellular matrix scaffolds, and micro- and nano-scale biomimetic scaffold structures, as well as reactive scaffolds induced by physical factors, and other acellular scaffolds. The fabrication techniques for these scaffolds, along with current strategies in clinical bone repair, are described. The developments in each category are discussed in terms of the connection between the scaffold materials and tissue repair, as well as the interactions with endogenous cells. As the advances in bone tissue engineering move toward application in the clinical setting, the demonstration of the therapeutic efficacy of these novel scaffold designs is critical.

[Levels and Risk Assessment of Short and Medium-Chain Chlorinated Paraffins in Soil from Paper Mill Area].

Short-chain chlorinated paraffins are persistent organic pollutants, and chlorinated paraffins were widely used as sizing agent in the paper industry. In order to investigate the levels and risk assessment of short-chain and medium-chain chlorinated paraffins in the paper mill plant, the surface soil and soil of different depths were collected.The concentrations, congener group profiles of short-chain chlorinated paraffins (SCCPs) and medium-chain chlorinated paraffins (MCCPs) in soil were determined by two-dimensional gas chromatography coupled with electron capture-negative ion mass spectrometry. The SCCPs and MCCPs concentrations were 42-3853 ng·g-1 and 34-2091 ng·g-1. The chlorine contents were 59.9%-61.9% and 48.7%-52.8%. The concentrations of SCCPs and MCCPs were different in the soil collected in different sampling site. The concentration of SCCPs and MCCPs were relatively higher in soil of sewage treatment area and coating area. The CP levels in soil from the paper mill plant were at a high level compared with those in other regions. C10Cl6-7 and C14-15Cl5 were the main congener groups in most soil samples. The results of principal component analysis showed that the CP52 commercial products may be sources of SCCPs and MCCPs in the soil. The risk quotient (RQ) for SCCPs and MCCPs were assessed in soil of paper mill plant. The results showed that the RQ values for SCCPs in soil ranged from 0.01 to 0.73 which are the medium risk, and the RQ values for MCCPs in soil ranged from 0 to 0.07, which are the low risk. The human exposure values of children and adults are lower than TDI[10 µg·(kg·d)-1] in both cases. The health risks caused by non-dietary exposure under paper mill area are low.

Adsorption of volatile benzene series compounds by surface-modified glass fibers: kinetics, thermodynamic adsorption efficiencies, and mechanisms.

The presence of volatile benzene series compounds (VBSCs) in the environment is continually increasing, with the potential for negative effects on human health. It is therefore important to develop new materials for the adsorption of these compounds using various modification techniques. Glass fibers are a promising adsorbent for VBSCs and offer a number of advantages. In the present work, the surfaces of glass fibers were modified using hydrogen peroxide, a sodium hydroxide solution, or Piranha solution (a mixture of concentrated sulfuric acid and hydrogen peroxide). The adsorption characteristics of the resulting specimens were investigated, employing 10 volatile benzene-based compounds, and the activated glass fibers showed significantly improved adsorption efficiencies. The fibers activated with the Piranha solution were further modified with a triethoxysilyl benzene compound to obtain an aryl-modified material that demonstrated enhanced adsorption of aniline, salicylaldehyde, benzyl alcohol, and xylene relative to that obtained from a combination of polyurethane foam and XAD-2 resin. The adsorption efficiency of benzyl alcohol by these aryl glass fibers was found to be as high as 93% and the adsorption mechanism is believed to be associated with hydrogen bonding and π-π conjugation. This study provides a reliable technique for the quantification of VBSCs and a basis for the evaluation of various adsorption materials.

[Expression and characterization of ß-N-acetylglucosaminidases from Bacillus coagulans DSM1 for N-acetyl-ß-D glucosamine production].

ß-N-acetylglucosaminidases (NAGases) can convert natural substrates such as chitin or chitosan to N-acetyl-ß-D glucosamine (GlcNAc) monomer that is wildly used in medicine and agriculture. In this study, the BcNagZ gene from Bacillus coagulans DMS1 was cloned and expressed in Escherichia coli. The recombinant protein was secreted into the fermentation supernatant and the expression amount reached 0.76 mg/mL. The molecular mass of purified enzyme was 61.3 kDa, and the specific activity was 5.918 U/mg. The optimal temperature and pH of the BcNagZ were 75 °C and 5.5, respectively, and remained more than 85% residual activity after 30 min at 65 °C. The Mie constant Km was 0.23 mmol/L and the Vmax was 0.043 1 mmol/(L·min). The recombinant BcNagZ could hydrolyze colloidal chitin to obtain trace amounts of GlcNAc, and hydrolyze disaccharides to monosaccharide. Combining with the reported exochitinase AMcase, BcNagZ could produce GlcNAc from hydrolysis of colloidal chitin with a yield over 86.93%.

Enhanced Low Molecular Weight Poly-γ-Glutamic Acid Production in Recombinant Bacillus subtilis 1A751 with Zinc Ion.

Poly-γ-glutamic acid (γ-PGA) is a novel biodegradable polyamide material. Microbial fermentation is the only way to produce γ-PGA, but the molecular weight of γ-PGA varied depending on different strains and culture conditions used. The molecular weight of γ-PGA is a main factor affecting the utilization of γ-PGA. It is urgent to find an efficient way to prepare γ-PGA with specific molecular weight, especially low molecular weight. Bacillus subtilis ECUST is a glutamate-dependent strain that produces γ-PGA. In this study, a recombinant B. subtilis harboring the γ-PGA synthase gene cluster pgsBCAE of our preciously identified γ-PGA-producing B. subtilis ECUST was constructed. Assay of γ-PGA contents and properties showed that recombinant B. subtilis 1A751-pBNS2-pgsBCAE obtained the ability to synthesize γ-PGA with low molecular weight (about 10 kDa). The excessive addition of glutamate inhibited the γ-PGA synthesis, while the addition of Zn2+ could promote the synthesis of γ-PGA by increasing the transcription of pgsB but had no effect on the molecular weight of synthesized γ-PGA. Under optimized conditions, γ-PGA produced by recombinant B. subtilis 1A751-pBNS2-pgsBCAE increased from initial 0.54 g/L to 3.9 g/L, and the glutamate conversion rate reached 78%. Recombinant B. subtilis 1A751-pBNS2-pgsBCAE has the potential for efficient preparation of low molecular weight γ-PGA.

A Bacillus pumilus originated ß-N-acetylglucosaminidase for chitin combinatory hydrolysis and exploration of its thermostable mechanism.

ß-N-acetylglucosaminase (NAGase) plays pivotal roles in industrial applications. Here, a GH3 family NAGase encoding gene from Bacillus pumilus was cloned and expressed in Escherichia coli. The optimal temperature and pH of the recombinant BpNagZ were 70 °C and 6.0, respectively, and kept more than 40% residual activity at 70 °C for 30 min. Metal ions such as Zn2 +, Cu2+, Cd2+, Mg2+, and Ca2+, even chelating agent, EDTA had slight effects on the activity of BpNagZ, indicating that BpNagZ was not a metal-dependent enzyme. Compared with the homology protein, BsNagZ from B. subtilis, the thermostability and activity of BpNagZ improved significantly. Structural simulation and sequence alignment showed that the increases in secondary structure, the content of proline and valine, the number of hydrogen bond were the main factors affecting the thermostability of BpNagZ. Mutation analysis also verified that four prolines in the BpNagZ had obvious effects on the thermostability. Combinatory hydrolysis of colloidal chitin with acidic mammalian exochitinase (AMcase) and BpNagZ showed the maximum combinatory efficiency of GlcNAc can reach 87% during 2.25 h. These biochemical characteristics indicated that BpNagZ was a thermostable enzyme with high activity in industrial application.

A new cold-active and alkaline pectate lyase from Antarctic bacterium with high catalytic efficiency.

Cold-active enzymes have become attractive biocatalysts in biotechnological applications for their ability to retain high catalytic activity below 30 °C, which allows energy reduction and cost saving. Here, a 1041 bp gene pel1 encoding a 34.7 KDa pectate lyase was cloned from a facultatively psychrophilic Antarctic bacterium Massilia eurypsychrophila and heterologously expressed in Escherichia coli. PEL1 presented the highest 66% identity to the reported mesophilic pectate lyase PLXc. The purified PEL1 exhibits the optimum temperature and pH of 30 °C and 10 toward polygalacturonic acid, respectively. PEL1 is a cold-active enzyme that can retain 60% and 25% relative activity at 10 °C and 0 °C, respectively, while it loses most of activity at 40 °C for 10 min. PEL1 has the highest specific activity (78.75 U mg-1) than all other reported cold-active pectinase, making it a better choice for use in industry. Based on the detailed sequence and structure comparison between PEL1 and PLXc and mutation analysis, more flexible structure and some loop regions may contribute to the cold activity and thermal instability of PEL1. Our investigations of the cold-active mechanism of PEL1 might guide the rational design of PEL1 and other related enzymes.

CRISPR-assisted multi-dimensional regulation for fine-tuning gene expression in Bacillus subtilis.

Fine-tuning of gene expression is crucial for protein expression and pathway construction, but it still faces formidable challenges due to the hierarchical gene regulation at multiple levels in a context-dependent manner. In this study, we defined the optimal targeting windows for CRISPRa and CRISPRi of the dCas9-α/ω system, and demonstrated that this system could act as a single master regulator to simultaneously activate and repress the expression of different genes by designing position-specific gRNAs. The application scope of dCas9-ω was further expanded by a newly developed CRISPR-assisted Oligonucleotide Annealing based Promoter Shuffling (OAPS) strategy, which could generate a high proportion of functional promoter mutants and facilitate the construction of effective promoter libraries in microorganisms with low transformation efficiency. Combing OAPS and dCas9-ω, the influences of promoter-based transcription, molecular chaperone-assisted protein folding and protease-mediated degradation on the expression of amylase BLA in Bacillus subtilis were systematically evaluated, and a 260-fold enhancement of BLA production was obtained. The success of the OAPS strategy and dCas9-ω for BLA production in this study thus demonstrated that it could serve as a powerful tool kit to regulate the expression of multiple genes multi-directionally and multi-dimensionally in bacteria.

High-level expression of ß-N-Acetylglucosaminidase BsNagZ in Pichia pastoris to obtain GlcNAc.

ß-N-Acetylglucosaminidases (NAGase) can remove N-acetylglucosamine (GlcNAc) from the non-reducing end of chitin or chitosan. GlcNAc has many important physiological functions in organism, which can be used for the treatment of rheumatoid arthritis clinically and be used as food antioxidant, infant food additive and diabetic sweetener. Thus, it is very important to develop genetic-engineering strains with high-yield NAGase to hydrolyze chitin into GlcNAc. Here, the NAGase gene of Bacillus subtilis 168 (BsnagZ) was synthesized according to the codon bias of Pichia pastoris and expressed in P. pastoris. The expression level of BsNagZ in P. pastoris increased over the induced time and the highest activity reached 0.76 U/mL at the 7th day. The recombinant BsNagZ was purified for characterization. The optimal temperature and pH are 60 °C and 6.0, respectively. It can both keep over 80% activities after pre-incubation at 55 °C for one hour and at 4 °C for 12 h from pH 4.5 to 10.0. To further improve the expression level of BsNagZ, a recombinant strain with four copy BsnagZs was screened using a high concentration of zeocin. The highest BsNagZ activity reached 3.2 U/mL at the 12th day, which was fourfold higher than that of single-copy strain. Combined with commercial chitinase CtnSg, GlcNAc can be produced by recombinant BsNagZ when used colloidal chitin as the substrate. Our study highlights that the NAGase was first successfully expressed in P. pastoris and GlcNAc can be produced via NAGase hydrolyzing the colloidal chitin.

Increasing thermal stability of glutamate decarboxylase from Escherichia. coli by site-directed saturation mutagenesis and its application in GABA production.

Gamma-amino butyric acid (GABA) is an important bio-product used in pharmaceuticals, functional foods, and a precursor of the biodegradable plastic polyamide 4 (Nylon 4). Glutamate decarboxylase B (GadB) from Escherichia. coli is a highly active biocatalyst that can convert l-glutamate to GABA. However, its practical application is limited by the poor thermostability and only active under acidic conditions of GadB. In this study, we performed site-directed saturation mutagenesis of the N-terminal residues of GadB from Escherichia coli to improve its thermostability. A triple mutant (M6, Gln5Ile/Val6Asp/Thr7Gln) showed higher thermostability, with a 5.6 times (560%) increase in half-life value at 45 °C, 8.7 °C rise in melting temperature (Tm) and a 14.3 °C rise in the temperature at which 50% of the initial activity remained after 15 min incubation (T1550), compared to wild-type enzyme. Protein 3D structure analysis showed that the induced new hydrogen bonds in the same polypeptide chain or between polypeptide chains in E. coli GadB homo-hexamer may be responsible for the improved thermostability. Increased thermostability contributed to increased GABA conversion ability. After 12 h conversion of 3 mol/L l-glutamate, GABA produced and mole conversion rate catalyzed by M6 whole cells was 297 g/L and 95%, respectively, while those by wild-type GAD was 273.5 g/L and 86.2%, respectively.

Improved extracellular expression and high-cell-density fed-batch fermentation of chitosanase from Aspergillus Fumigatus in Escherichia coli.

Chitosanase (CSN) from Aspergillus fumigatus has good thermal stability, wide pH range duration, and effective hydrolysis for chitosan. Inhere, CSN was successfully expressed in Escherichia coli followed by extracellular secretion under the guidance of an N-terminal signal peptide PelB, which effectively prompted its secretion out of E. coli cells. To facilitate its later purification, N-terminal or C-terminal 6xHis epitope tag was added to the PelB-CSN protein complex. Our results indicated that PelB-CSN without 6xHis-tag (PelB-CSN) or with N-terminal 6xHis-tag (PelB-CSN-N) can both be effectively secreted into the medium, while CSN with 6xHis-tag anchored at C-terminus was expressed as inclusion bodies. Process optimization strategies were further developed to improve the secretion efficiency of recombinant PelB-CSN-N in E. coli. Under the induction of 10 g/L lactose in shake-flask culture, the extracellular activity of CSN reached 6015 U/mL at 25 °C in TB medium containing 1 % glycine. Moreover, a fed-batch fermentation strategy for high-cell-density cultivation was applied in a 5-L fermenter, increasing the extracellular CSN activity to 14,000 U/mL in 2-day fermentation with the optimal addition of lactose and glycine.

Truncation of the unique N-terminal domain improved the thermos-stability and specific activity of alkaline α-amylase Amy703.

High pH condition is of special interest for the potential applications of alkaline α-amylase in textile and detergent industries. Thus, there is a continuous demand to improve the amylase's properties to meet the requirements set by specific applications. Here we reported the systematic study of modular domain engineering to improve the specific activity and stability of the alkaline α-amylase from Bacillus pseudofirmus 703. The specific activity of the N-terminal domain truncated mutant (N-Amy) increased by ~35-fold with a significantly improved thermo-stability. Kinetic analysis demonstrated that the Kcat and Kcat/Kmof N-Amy were enhanced by 1300-fold and 425.7-fold, respectively, representing the largest catalytic activity improvement of the engineered α-amylases through the methods of domain deletion, fusion or swapping. In addition, different from the wild-type Amy703, no exogenous Ca(2+) were required for N-Amy to maintain its full catalytic activity, implying its superior potential for many industrial processes. Circular dichroism analysis and structure modeling revealed that the increased compactness and α-helical content were the main contributors for the improved thermo-stability of N-Amy, while the improved catalytic efficiency was mainly attributed by the increased conformational flexibility around the active center.

[Expression and characterization of a bispecific antibody targeting TNF-α and ED-B containing fibronectin].

To enhance the specificity of anti-TNF-α single chain Fv antibody (TNF-scFv) to inflamed site, we constructed a bispecific antibody BsDb that targets TNF-α and ED-B-containing fibronectin (B-FN) by covalently linking TNF-scFv and the anti-ED-B scFv L19 at the gene level via a flexible peptide linker deriving from human serum albumin. BsDb was successfully secreted from Pichia pastoris as functional protein, identified by immunoblotting, and purified to homogeneity with affinity chromatography. BsDb retained the immunoreactivity of its original antibodies TNF-scFv and L19, and showed a marked gain in antigen-binding affinity and in TNF-α-neutralizing ability, when compared to TNF-scFv and L19 that were produced in Escherichia coli. In the adjuvant-induced arthritis (AIA) mice model, BsDb showed selective accumulation and retention in the inflamed paws but rapid clearance from blood, resulting in high arthritic paw to blood ratios. These data indicate that BsDb is endowed with high specificity to inflamed site and low toxicity to normal tissues and holds great potential for in vivo application for the targeted therapy of RA and other chronic inflammatory diseases.

Secretory expression of a bispecific antibody targeting tumor necrosis factor and ED-B fibronectin in Pichia pastoris and its functional analysis.

Specific targeting of tumor necrosis factor (TNF)-α antagonist to the inflamed site could increase its efficacy and reduce side-effects. Here, we constructed a bispecific diabody (BsDb) that targets TNF-α and ED-B-containing fibronectin, a fibronectin isoform specifically expressed in the pannus of the inflamed synovium in rheumatoid arthritis. BsDb was secreted from Pichia pastoris as functional protein and was purified to homogeneity. BsDb could simultaneously bind to human TNF-α and B-FN and neutralize TNF-α action. Additionally, BsDb showed a significant gain both in the antigen-binding affinity and in TNF-α-neutralizing ability as compared to its original antibodies, L19 and anti-TNF-α scFv, which were produced in E. coli. BsDb was constructed and was endowed with enhanced bioactivities and improved production processing. Therefore, it holds great potential for in vivo applications.