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.

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.

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.

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.

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.

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.

Lycium barbarum Ameliorates Oral Mucositis via HIF and TNF Pathways: A Network Pharmacology Approach.

BACKGROUND: Oral mucositis is the most common and troublesome complication for cancer patients receiving radiotherapy or chemotherapy. Recent research has shown that Lycium barbarum, an important economic crop widely grown in China, has epithelial protective effects in several other organs. However, it is unknown whether or not Lycium barbarum can exert a beneficial effect on oral mucositis. Network pharmacology has been suggested to be applied in "multi-component-multi-target" functional food studies. The purpose of this study is to evaluate the effect of Lycium barbarum on oral mucositis through network pharmacology, molecular docking and experimental validation. AIM: To explore the biological effects and molecular mechanisms of Lycium barbarum in the treatment of oral mucositis through network pharmacology and molecular docking combined with experimental validation. METHODS: Based on network pharmacology methods, we collected the active components and related targets of Lycium barbarum from public databases, as well as the targets related to oral mucositis. We mapped protein- protein interaction (PPI) networks, performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment, and constructed a 'components-disease-targets' network and 'components- pathways-targets' network using Cytoscape to further analyse the intrinsic molecular mechanisms of Lycium barbarum against oral mucositis. The affinity and stability predictions were performed using molecular docking strategies, and experiments were conducted to demonstrate the biological effects and possible mechanisms of Lycium barbarum against oral mucositis. RESULTS: A network was established between 49 components and 61 OM targets. The main active compounds were quercetin, beta-carotene, palmatine, and cyanin. The predicted core targets were IL-6, RELA, TP53, TNF, IL10, CTNNB1, AKT1, CDKN1A, HIF1A and MYC. The enrichment analysis predicted that the therapeutic effect was mainly through the regulation of inflammation, apoptosis, and hypoxia response with the involvement of TNF and HIF pathways. Molecular docking results showed that key components bind well to the core targets. In both chemically and radiation-induced OM models, Lycium barbarum significantly promoted healing and reduced inflammation. The experimental verification showed Lycium barbarum targeted the key genes (IL-6, RELA, TP53, TNF, IL10, CTNNB1, AKT1, CDKN1A, HIF1A, and MYC) through regulating the HIF and TNF signaling pathways, which were validated using the RT-qPCR, immunofluorescence staining and western blotting assays. CONCLUSION: In conclusion, the present study systematically demonstrated the possible therapeutic effects and mechanisms of Lycium barbarum on oral mucositis through network pharmacology analysis and experimental validation. The results showed that Lycium barbarum could promote healing and reduce the inflammatory response through TNF and HIF signaling pathways.

A single-step mixing cloning method for assembly of lentiviral short hairpin RNA expression vectors for gene silencing.

Lentiviral expression vectors encoding short hairpin RNA (shRNA) are widely used for RNAi-based gene silencing in mammalian cells. However, current methods for the construction of shRNA expression vectors require multiple steps, which are expensive, time-consuming, and error-prone. Here, we developed a single-step mixing cloning method for the generation of lentiviral shRNA expression vectors. With this method, a pair of short oligonucleotides (∼50 nt) is required and a lentiviral shRNA vector can be constructed with only one step. This method has been used to construct 30 lentiviral shRNA expression vectors successfully.

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.

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.

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.

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.

Improved method for constructing plant amiRNA vectors with blue-white screening and MAGIC.

Artificial microRNA (amiRNA) technology is a novel tool in reverse genetic research for discovering or validating gene functions in plants. A convenient cloning strategy has been developed to construct plant amiRNA vectors based on lacO reconstruction and mating-assisted, genetically-integrated cloning (MAGIC). The amiRNA precursor fragment was generated by PCR and inserted into a small donor plasmid through reconstruction of integrated lacO sequence. Blue recombinants were selected on plates containing X-gal and the efficiency of successful clones was 100%. The amiRNA expression cassette was transferred from the donor plasmid to the recipient plasmid p1301-gfp through MAGIC and an amiRNA expression plasmid was created. More than 40 plant amiRNA vectors were generated through this method, one of which was transformed into Arabidopsis thaliana and the target gene was silenced efficiently. The approach will be useful for amiRNA expression vectors construction in plants.

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.

[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%.

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.

[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.

Constructing recombinant herpesvirus BAC vectors with mating-assisted genetically integrated clone method.

The large capacity of pseudorabies virus (PRV) for foreign DNA and broad host range make it a prospective tool for the preparation of vaccines and agents of gene and tumour therapy. Here we introduced a cloning strategy that facilitates construction of recombinant PRV-BAC vectors based on mating-assisted genetically integrated clone (MAGIC). The target gene was cloned into a small conditionally replicating donor plasmid, followed by shuffling to a recipient PRV-BAC plasmid in vivo of Escherichia coli through MAGIC. The average efficiency of successful clones was 89%. Moreover, permanent integration of unwanted sequences was avoided.

Molecular cloning and characterization of an acetylcholinesterase cDNA in the brown planthopper, Nilaparvata lugens.

A full cDNA encoding an acetylcholinesterase (AChE, EC 3.1.1.7) was cloned and characterized from the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae). The complete cDNA (2467 bp) contains a 1938-bp open reading frame encoding 646 amino acid residues. The amino acid sequence of the AChE deduced from the cDNA consists of 30 residues for a putative signal peptide and 616 residues for the mature protein with a predicted molecular weight of 69,418. The three residues (Ser242, Glu371, and His485) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds are completely conserved, and 10 out of the 14 aromatic residues lining the active site gorge of the AChE are also conserved. Northern blot analysis of poly(A)+ RNA showed an approximately 2.6-kb transcript, and Southern blot analysis revealed there likely was just a single copy of this gene in N. lugens. The deduced protein sequence is most similar to AChE of Nephotettix cincticeps with 83% amino acid identity. Phylogenetic analysis constructed with 45 AChEs from 30 species showed that the deduced N. lugens AChE formed a cluster with the other 8 insect AChE2s. Additionally, the hypervariable region and amino acids specific to insect AChE2 also existed in the AChE of N. lugens. The results revealed that the AChE cDNA cloned in this work belongs to insect AChE2 subgroup, which is orthologous to Drosophila AChE. Comparison of the AChEs between the susceptible and resistant strains revealed a point mutation, Gly185Ser, is likely responsible for the insensitivity of the AChE to methamidopho in the resistant strain.

Cell-surface display of the active mannanase in Yarrowia lipolytica with a novel surface-display system.

A novel surface-display system was constructed using the cell-wall anchor protein Flo1p from Saccharomyces cerevisiae, the mannanase (man1) from Bacillus subtilis fused with the C-terminus of Flo1p and the 6xHis tag was inserted between Flo1p and man1. The fusion protein was displayed on the cell surface of Yarrowia lipolytica successfully, and it was confirmed by immunofluorescence. In succession, the surface-displayed mannanase was characterized. The optimum catalytic conditions for the recombinant mannanase were 55 degrees C at pH 6.0, and it exhibited high stability against pH variation. The highest activity of the recombinant mannanase reached 62.3 IU/g (dry cell weight) after the recombinant was cultivated for 96 h in YPD medium [1% (w/v) yeast extract/2% (w/v) peptone/2% (w/v) glucose]. To our knowledge, the present paper is the first to report that high-activity mannanase is displayed on the cell surface of Y. lipolytica with Flo1p.