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Fermentation conditions affect pectinase production. Among these conditions, fermentation period is one of the important parameters to be optimized for saving the process costs. However, information regarding whether metabolic regulation plays a role is not available. Here, a Bacillus licheniformis strain DY2 was incubated in submerged fermentation for pectinase production for 48 h. The pectinase activity was assayed every 4 h from 8th h of fermentation. The pectinase activity was low at 8 h, middle at 28 h, and peak at 44 h. Consistently, metabolomics analysis showed that the metabolic shift is related to the fermentation period. The metabolomes exhibited almost of differential metabolites in the enriched metabolic pathways, which were decreased at 28 h and 44 h compared to 8 h in a time-dependent manner. Furthermore, reduced palmitic acid and stearic acid in a time-dependent manner were identified as crucial biomarkers at 48 h. These findings in our work reveal that the reduced fatty acid biosynthesis is required for pectinase yield, which was further demonstrated by addition of inhibitors furfural and triclosan to inhibit pyruvate dehydrogenase and fatty acid biosynthesis, respectively. Our work reveals a potential possibility to elevate pectinase yield through metabolomics modulation.

Pectinase is widely used in numerous industrial fields, including the food, wine, and paper industries. In this work, seven bacteria were isolated from orange peel and their pectinase production activity was assayed. One bacterium (OR-B2) identified as a Bacillus sp. showed the highest enzyme activity towards others. A gene encoding a pectate lyase designed as PelB-B2 in this work was amplified and heterogeneous expressed in E.coli. PelB-B2 was defined as a member of the PelB pectate lyase family after phylogenic tree analysis. 3D model of PelB-B2 was constructed by SWISS-MODEL and PelB-B2 showed conserved para-β structure. After inducing culture and purified by Ni-affinity chromatography, the properties of the purified PelB-B2 were assayed. Optimal pH and temperature for PelB-B2 was pH 8.0 and 50 °C, respectively. PelB-B2 showed excellent pH stability and thermostability. It was stable within pH range 3.0–11.0 and retained more than 51% activity after incubation at 40 °C, 50 °C, or 60 °C for 1 h. Furthermore, we determined that PelB-B2 was a Ca2+-dependent pectinase and the pectin extracted from citrus was the benefit substrate for PelB-B2. The Km and Vmax of PelB-B2 were 1.64 g/L and 232.56 mol/(L min), respectively. The OR-B2 can be a new resource for pectinase production and the PelB-B2 has potential for industrial application. 7 bacteria were isolated from orange peel, namely OR-B1 to OR-B7 and their pectinase activities were assayed. One pectate lyase belongs to PelB family was cloned from OR-B2 and heterogeneous expressed in E. coli. Purified PelB-B2 was further studied with its properties. Effects of pH, temperature, chemicals, substrate on the enzyme activity were assayed and the enzyme kinetic was also measured.

Microbial pectinases are important sources due to the ease of production and unique physicochemical properties. Here, DY2, a strain of Bacillus licheniformis, was identified from 14 strains of bacteria as a pectinase-producing bacterium with good application potential. Optimized carbon sources of submerged fermentation led to the identification of glucose as an ideal carbon source for activity and production of P-DY2, the pectinase produced by DY2. GC-MS based metabolomics was used to explore metabolic mechanisms mediated by glucose, showing the frustrated TCA cycle is necessary to elevate the activity and production of P-DY2. Decreased activity of α-ketoglutaric dehydrogenase and succinate dehydrogenase of DY2 in glucose-treated samples supports the conclusion that P-DY2 production is the TCA cycle-independent. These results reveal a metabolic mechanism of high-activity pectinase mediated by exogenous glucose. These findings highlight the way to understand metabolic mechanisms and promote pectinase yield through metabolomics approach and metabolic modulation, respectively.

Background: Protein glutaminase specifically deamidates glutamine residue in protein and therefore significantly improves protein solubility and colloidal stability of protein solution. In order to improve its preparation efficiency, we exploited the possibility for its secretory expression mediated by twin-arginine translocation (Tat) pathway in Bacillus licheniformis. Results: The B. licheniformis genome-wide twin-arginine signal peptides were analyzed. Of which, eleven candidates were cloned for construction of expression vectors to mediate the expression of Chryseobacterium proteolyticum protein glutaminase (PGA). The signal peptide of GlmU was confirmed that it significantly mediated PGA secretion into media with the maximum activity of 0.16 U/ml in Bacillus subtilis WB600. A mutant GlmU-R, being replaced the third residue aspartic acid of GlmU twin-arginine signal peptide with arginine by site-directed mutagenesis, mediated the improved secretion of PGA with about 40% increased (0.23 U/ml). In B. licheniformis CBBD302, GlmU-R mediated PGA expression in active form with the maximum yield of 6.8 U/ml in a 25-l bioreactor. Conclusions: PGA can be produced and secreted efficiently in active form via Tat pathway of B. licheniformis, an alternative expression system for the industrial-scale production of PGA.How to cite: Niu D, Li C, Wang P, et al. Twin-arginine signal peptide of Bacillus licheniformis GlmU efficiently mediated secretory expression of protein glutaminase. Electron J Biotechnol 2019;42. https://doi.org/10.1016/j.ejbt.2019.10.006 Keywords: Arginine, Aspartic acid, Bacillus licheniformis, Bacillus subtilis, Escherichia coli, Prostaglandins A, Protein glutaminase, Protein sorting signals, Secretion expression, Signal peptide, Tat pathway

A new α-amylase-encoding gene (amySL3) of glycoside hydrolase (GH) family 13 was identified in soda lake isolate Alkalibacterium sp. SL3. The deduced AmySL3 shares high identities (82-98%) with putative α-amylases from the genus Alkalibacterium, but has low identities (

A novel shortening was developed based on oleogels structured by ethylcellulose (EC) polymers. The texture and oil retention ability of EC oleogels were characterized against the level of viscosity of different grades of EC, as well as the rheological properties in relation to the polymer structure in the gel network. EC100, which has an average viscosity of 100 cP, was selected as the most suitable organogelator at 4% (w/w) in combination with base oil (30% degree of saturation by mixing palm stearin and soybean oil) to form the shortening. Triglyceryl monostearate (TMS) was found to be the most effective emulsifier as evidenced by its ability to strengthen air-incorporation ability of the shortening while creating evenly distributed fine crystals in the system. The EC100 shortening was able to create breads with excellent specific volume, indicating its ability to incorporate air bubbles during dough development and to serve as an antifirming agent to create bread with stable soft texture. PRACTICAL APPLICATION: In the present study, we attempted to create a novel shortening by employing oleogels structured by ethylcellulose (EC), the most promising gelation agent to develop gel network capable of replacing solid fat without health concerns. EC oleogels in shortening with detailed characterization of the shortening microstructure in relation to its functional properties was elucidated. The optimal formulation in relation to preservation of gel structure and consistency with enhanced moisture and air retention were also identified.

The purpose of this account is to review the compounds capable of eliciting mitochondria-mediated apoptosis in cancer cells produced by medicinal fungi and plants. The medicinal fungi discussed encompass Cordyceps, Ganoderma species, Coriolus versicolor and Hypsizygus marmoreus. The medicinal plants discussed comprise Astragalus complanatus, Dendrobium spp, Dioscorea spp, Glycyrrhiza spp, Panax notoginseng, Panax ginseng, and Momordica charantia. These compounds have the potential of development into anticancer drugs.

Aflatoxin B1 (AFB1) is a known toxic human carcinogen and can be detoxified by laccases, which are multicopper oxidases that convert several environmental pollutants and toxins. In this study, a new laccase that could catalyze AFB1 degradation was purified and identified from the white-rot fungus Cerrena unicolor 6884. The laccase was purified using (NH4)2SO4 precipitation and anion exchange chromatography, and then identified as Lac 2 through zymogram and UHPLC-MS/MS based on the Illumina transcriptome analysis of C. unicolor 6884. Six putative laccase protein sequences were obtained via functional annotation. The lac 2 cDNA encoding a full-length protein of 512 amino acids was cloned and sequenced to expand the fungus laccase gene library for AFB1 detoxification. AFB1 degradation by Lac 2 was conducted in vitro at pH 7.0 and 45 &deg, C for 24 h. The half-life of AFB1 degradation catalyzed by Lac 2 was 5.16 h. Acetosyringone (AS), Syrinagaldehyde (SA) and [2,2&prime, azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] (ABTS) at 1 mM concentration seemed to be similar mediators for strongly enhancing AFB1 degradation by Lac 2. The product of AFB1 degradation catalyzed by Lac 2 was traced and identified to be Aflatoxin Q1 (AFQ1) based on mass spectrometry data. These findings are promising for a possible application of Lac 2 as a new aflatoxin oxidase in degrading AFB1 present in food and feeds.

Increase of pectinase activity is especially important in fermentation industry. Understanding of the metabolic mechanisms can find metabolic modulation approach to promote high yield of pectinase. Higher activity of pectinase was detected in DY1 than DY2, two strains of Bacillus licheniformis. GC–MS-based metabolomics identified differential metabolome of DY2 compared with DY1, characterizing the increased TCA cycle and biosynthesis of fatty acids. Elevated activity of pyruvate dehydrogenase (PDH), α-ketoglutaric dehydrogenase (KGDH) and succinate dehydrogenase (SDH) showed global elevation of carbon metabolism, which is consistent with the result that lowers glucose in DY2 than DY1. Inhibitors malonate, furfural and triclosan, of PDH, SDH and biosynthesis of fatty acids, promoted pectinase activity, where triclosan increased pectinase activity by 179%. These results indicate that functional metabolomics is an effective approach to understand metabolic mechanisms of fermentation production and provides clues to develop new methods for changing bacterial physiology and production.

A novel glycosyl hydrolase family 11 xylanase gene, xynMF13A, was cloned from Phoma sp. MF13, a xylanase-producing fungus isolated from mangrove sediment. xynMF13A was heterologously expressed in Pichia pastoris, and the recombinant XynMF13A (rXynMF13A) was purified by Ni-affinity chromatography. The temperature and pH optima of purified rXynMF13A were 45 °C and pH 5.0, respectively. rXynMF13A showed a high level of salt tolerance, with maximal enzyme activity being seen at 0.5 M NaCl and as much as 53% of maximal activity at 4 M NaCl. The major rXynMF13A hydrolysis products from corncob xylan were xylobiose, xylotriose, xylotetraose, and xylopentaose, but no xylose was found. These hydrolysis products suggest an important potential for XynMF13A in the production of xylooligosaccharides (XOs). Furthermore, rXynMF13A had beneficial effects on Chinese steamed bread production, by increasing specific volume and elasticity while decreasing hardness and chewiness. These results demonstrate XynMF13A to be a novel xylanase with potentially significant applications in baking, XOs production, and seafood processing.

Thin polymer films with well-assembled fluorinated groups on their surfaces are not easily achieved via spin-coating film-fabrication methods because the solution solidifies very rapidly during spin-coating, which hinders the fluorinated moieties from segregating and organizing on the film surface. In this contribution, we have proposed a comprehensive strategy toward achieving well-ordered fluorinated thin films surfaces by optimizing the molecular organization at air/liquid interface of the film-formation solutions. To validate such a route, poly(methyl methacrylate) (PMMA) end-capped with several 2-perfluorooctylethyl methacrylate (FMA) units was employed as the model polymer for investigations. The air/solution interfacial structures were optimized by systematically changing the polymer chain structures and properties of the casting solvents. It was found that the polymers that form loosely associated aggregates (e.g., FMA1- ec-PMMA65- ec-FMA1) and a solvent with better solubility to FMA while having not too low surface tension (i.e., toluene) can combine to produce solutions with well-assembled FMA at the interfaces. By spin-coating the solutions with well-organized interfaces, an ultrathin film with perfluorinated groups that were highly oriented toward the film surface was readily achieved, exhibiting surface energies as low as 7.2 mJ/m2, which is among the lowest reported so far for the spin-coated thin films, and a very high F/C ratio (i.e., 0.98).

Laccases (EC 1.10.3.2) have important industrial values in areas such as bioremediation, but they are often inactivated by heavy metal ions in real applications. In this report, laccase from a high laccase-producing Cerrena sp. HYB07 presented resistance to many metal ions except for Ag+, Hg2+, Li+ and Pb2+, the inhibition of which on the laccase were all reversible. The presence of these four cations decreased Remazol Brilliant Blue R decolorization efficiencies catalyzed by the laccase, and the mediator 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) could restore the decolorization efficiency in spite of Hg2+ inhibition. Hg2+ had the lowest IC50 value (0.17 mM) on the laccase activity, followed by Ag+, Li+ and Pb2+. The inhibition type on laccase activity was competitive for Pb2+, noncompetitive for Hg2+, and mixed type for Ag+ and Li+. The inhibition kinetic model of Hg2+ on laccase activity was established by using the kinetic method of substrate reaction. The microscopic forward inhibition rate constant (k+0) of Hg2+was 3.26 × 10−2/s and the microscopic reverse inhibition rate constant (k-0) was 1.36 × 10−3/s, indicating the laccase would be completely inhibited when Hg2+ concentration was sufficiently high because k+0 was much larger than k-0. Furthermore, Hg2+ reduced thermal and pH stability of the laccase. Fluorescence emission spectra demonstrated that Hg2+ had one binding site per laccase protein regardless of pH, and the binding was stronger at pH 6.0 than that at pH 5.0 or 9.0, in accordance with the lowest stability of the enzyme at pH 6.0 with Hg2+. Dynamic simulation was performed to identify the binding sites of each ion on the Lac7 protein.

White-rot fungus Cerrena sp. strain HYB07 produces laccase with high yields and strong decolorization ability. Cerrena laccase was immobilized by preparing cross-linked enzyme aggregates (CLEAs), and ammonium sulfate and glutaraldehyde were the optimal precipitant and cross-linking agent, respectively. An activity recovery rate of 68.1% was obtained at pH 8 after precipitation with (NH 4 ) 2 SO 4 and cross-linking with 30 mM glutaraldehyde for 3 h at 25 oC. Immobilized Cerrena laccase demonstrated improved tolerance to NaCl, metal ions and organic solvents. NaCl was a mixed-type inhibitor of both free and immobilized laccases with 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) as the substrate, and the improvement of NaCl resistance by laccase immobilization was quantified kinetically. Laccase CLEAs had higher inhibition constants K I and K IS compared to free laccase, suggesting that the affinity of NaCl to laccase was attenuated by immobilization. Laccase CLEAs also displayed greater velocity and efficiency in Remazol Brilliant Blue Reactive decolorization in the presence of NaCl than free laccase. Greater differences in the half time ( t 1/2 ) of Remazol Brilliant Blue Reactive decolorization by free and immobilized laccases were observed with increasing NaCl concentrations.

The data presented in this article are related to the research article entitled “Destaining of Coomassie Brilliant Blue R-250-stained polyacrylamide gels with fungal laccase” [1]. Laccase is a class of multicopper oxidases that can catalyze oxidation of recalcitrant dyestuffs. This article describes optimal parameters for destaining of polyacrylamide gels, stained with Coomassie Brilliant Blue R-250, with laccase from basidiomycete Cerrena sp. strain HYB07. Effects of laccase activity, mediator type and concentration, temperature and time on destaining of polyacrylamide gels were evaluated with respect to gel background intensity and protein band signals, and the optimal destaining effects were obtained with 15 U mL−1 laccase and 2 μM ABTS at 37 °C after 2 h. Keywords: Laccase, Destaining, Polyacrylamide gel, Coomassie Brilliant Blue R-250

In the original article.

A variety of fungi, plants, and their different tissues are used in Traditional Chinese Medicine to improve health, and some of them are recommended for dietary therapy. Many of these plants and fungi contain antifungal proteins and peptides which suppress spore germination and hyphal growth in phytopathogenic fungi. The aim of this article is to review antifungal proteins produced by medicinal plants and fungi used in Chinese medicine which also possess anticancer and human immunodeficiency virus-1 (HIV-1) enzyme inhibitory activities.

An enzyme-based method for destaining polyacrylamide gels stained with Coomassie Brilliant Blue R-250 is described. Distilled water supplemented with diluted fermentation broth of a laccase-producing white-rot fungus, Cerrena sp., was used for gel destaining, and a clear gel background was obtained in 2 h at 37 °C. Sensitivity of protein detection was 10 ng. The method did not require organic solvents or changing the destaining solution. Due to simultaneous gel destaining and dye decolorization, the colorless destaining solution can be disposed of directly. Laccase destaining of polyacrylamide gels was simple, efficient, and environmentally friendly.

Laccases are polyphenol oxidases with widespread applications in various industries. In the present study, the laccase from Cerrena sp. HYB07 was immobilized with four methods, namely entrapment in alginate, covalently binding to chitosan as well as formation of cross-linked enzyme aggregates (CLEAs) and magnetic CLEAs (M-CLEAs). The activity recovery rates of the immobilized laccases ranged from 29% to 68%. Immobilization elevated the reaction temperature optimum and reduced substrate specificity, but not necessarily the turnover rate. pH stability of immobilized laccases was improved compared with that of the free laccase, especially at acidic pH values. Thermal inactivation of all laccases followed a simple first-order exponential decay model, and immobilized laccases displayed higher thermostability, as manifested by lower thermal inactivation rate constants and longer enzyme half-life time. Operational stability of the immobilized laccase was demonstrated by decolorization of the triphenylmethane dye malachite green (MG) at 60 °C. MG decolorization with free laccase was accompanied by a shift of the absorption peak and accumulation of a stable, colored intermediate tetradesmethyl MG, probably due to lower thermostability of the free laccase and premature termination of the degradation pathway. In contrast, complete decolorization of MG was achieved with laccase CLEAs at 60 °C.

An agar-degrading bacterium, strain BN3, was isolated from a coastal soil sample collected in Taiwan Strait, China and identified to be a novel species of the genus Microbulbifer. The gene (N3-1) encoding for a novel β-agarase from the isolate was cloned and sequenced. It encoded a mature protein with 274 amino acids and a calculated molecular mass of 34.3 kDa. The deduced amino acid sequence manifested sequence similarity (61–84% identity) to characterized β-agarases in the glycoside hydrolase family 16. The recombinant agarase was hyper-produced extracellularly using Pichia pastoris as the host. After induction in a shake flask for 96 h, the yield of recombinant N3–1 protein reached 0.406 mg/mL, and the enzyme activity attained 502.1 U/mL. The enzyme purified by ion exchange chromatography displayed a specific activity of 6447 U/mg at pH 6.0 and 50 °C. The optimal pH and temperature for agarase activity were approximately 6 and 50 °C, respectively. The pattern of agarose hydrolysis showed that the enzyme was an endo-type β-agarase, capable of hydrolyzing agarose and Gracilaria lemaneiformis, with neoagarobiose and neoagarotetraose as the final main products.

A novel multi-domain high molecular xylanase coding gene (xynSL3) was cloned from Alkalibacterium sp. SL3, an alkaliphilic bacterial strain isolated from the sediment of soda lake Dabusu. The deduced XynSL3 is composed of a putative signal peptide, three tandem domains of carbohydrate binding module (CBM) family 22, a catalytic domain of glycosyl hydrolase (GH) family 10 and a domain of CBM9. XynSL3 shares the highest identity of 66% to a hypothetical protein from Alkalibacterium sp. AK22 and has low identities (33%-45%) with other functionally characterized xylanases. The gene xynSL3 was expressed heterologously in Escherichia coli and the recombinant enzyme demonstrated some particular characteristics. Purified recombinant XynSL3 (rXynSL3) was highly active and stable over the neutral and alkaline pH ranges from 7.0 to 12.0, with maximum activity at pH 9.0 and around 45% activity at pH 12.0. It had an apparent temperature optimum of 55°C and was stable at 50°C. The rXynSL3 was highly halotolerant, retaining more than 60% activity with 3 M NaCl and was stable at up to a 4 M concentration of NaCl. The hydrolysis products of rXynSL3 from corncob xylan were mainly xylobiose and xylotetraose. The activity of rXynSL3 was enhanced by Ca2+ and it has strong resistance to sodium dodecyl sulfate (SDS). This multi-domain, alkaline and salt-tolerant enzyme has great potential for basic research and industrial applications such as the biobleaching of paper pulp and production of xylo-oligosaccharides (XOS).

A β-mannanase gene (Man5) from Bacillus subtilis BS5 was cloned by PCR and integrated into the genome of Pichia pastoris GS115 via pPIC9 vector. The recombinant Man5 with a molecular mass of 43 kDa was successfully expressed and secreted into the culture medium. After methanol induction in a shake flask for 96 H, the recombinant Man5 protein reached 375 µg/mL in concentration, with an enzyme activity of 892 U/mL. The recombinant Man5 was purified 3.35-fold with 60% yield by using HiTrap DEAE FF and HiTrap Phenyl FF columns. The specific activity of the purified enzyme was 7,978 U/mg. The optimum temperature and pH of the recombinant Man5 were 50 °C and 6.0, respectively. Studies of substrate specificity showed that the optimum substrate for the Man5 was konjac flour, suggesting that it has great potential as an effective additive in the food industry.

Cold-active xylanases are of great interest due to their large potential for application in the food industry. In this study, salt bridges of the eight glycoside hydrolase (GH) family 10 cold-active xylanases reported to date were predicted and the salt bridges specific to the cold-active xylanase XynGR40 were identified. Seven mutants were constructed to disrupt salt bridges specific to XynGR40. The results suggested that five mutants lost their xylanase activity, while the other two mutants, D30N and D83N, displayed different properties when compared with the wild-type XynGR40. First, both mutations showed an obvious decrease in thermostability, with the T1/2 of D30N and D83N at 50 °C being about one half and one sixth of the wild-type, respectively. Second, both D30N and D83N had a higher specific activity than the wild-type, with activities about 13 and 163% higher, respectively. Third, both D30N and D83N had high kcat and Km values, which resulted in a higher catalytic efficiency of the mutant D83N, but a lower catalytic efficiency of the mutant D30N compared to the wild-type. Our results suggested that salt bridges play important roles in both the activity and thermostability of the cold-active xylanase XynGR40. The mutant D83N had a higher kcat and higher relative activity at low temperatures than the wild-type, and is a good candidate for application in the food industry.

Magnetic cross-linked enzyme aggregates (M-CLEAs) were prepared for Cerrena laccase and used in antibiotic treatment. Of the seven antibiotics examined in this study, Cerrena laccase M-CLEAs were most effective in degradation of tetracycline (TC) and oxytetracycline (OTC), followed by ampicillin, sulfamethoxazole and erythromycin. The redox mediator ABTS was not able to improve efficiencies of degradation of TC and OTC. Cerrena laccase at 40U/mL eliminated 100μg/mL TC at pH 6 and 25°C in 48h in the absence of a redox mediator, with over 80% degradation occurring within the first 12h. Laccase treatment also significantly suppressed the antimicrobial activity of TC and OTC. Three TC transformation products, the levels of which initially increased and subsequently decreased during laccase treatment were identified by using LC-TOF MS. A mechanism of laccase-mediated TC oxidation was proposed based on the identified intermediates.

A novel esterase gene (estSL3) was cloned from the Alkalibacterium sp. SL3, which was isolated from the sediment of soda lake Dabusu. The 636-bp full-length gene encodes a polypeptide of 211 amino acid residues that is closely related with putative GDSL family lipases from Alkalibacterium and Enterococcus. The gene was successfully expressed in E. coli and the recombinant protein (rEstSL3) was purified to electrophoretic homogeneity and characterized. rEstSL3 exhibited the highest activity towards pNP-acetate and had no activity towards pNP-esters with acyl chains longer than C8. The enzyme was highly cold-adapted, showing an apparent temperature optimum of 30 °C and remaining approximately 70% of the activity at 0 °C. It was active and stable over the pH range from 7 to 10 and highly salt-tolerant up to 5 M NaCl. Moreover, rEstSL3 was strongly resistant to most tested metal ions, chemical reagents, detergents and organic solvents. Amino acid composition analysis indicated that EstSL3 had fewer proline residues, hydrogen bonds and salt bridges than mesophilic and thermophilic counterparts, but more acidic amino acids and less hydrophobic amino acids when compared with other salt-tolerant esterases. The cold active, salt-tolerant and chemical-resistant properties make it a promising enzyme for basic research and industrial applications.

In this paper, the relationship between the surface structures of spin-coated fluorinated polymer films and their corresponding film-formation solution/air interface structures was investigated. Film-forming poly(n-alkyl methacrylate) end-capped with 2-perfluorooctylethyl methacrylate (FMA; PFMAy-ec-PnAMAx-ec-PFMAy) was synthesized via a controlled/living atom-transfer radical polymerization (ATRP) technique. The structures both at solution interface and on the spin-coated film surface for these polymers were studied by X-ray photoelectron spectroscopy (XPS), sum frequency spectroscopy (SFG), and surface tension measurements. The results showed that, with increasing polymerization degree of PnAMA, the fluorinated moieties in PFMAy-ec-PnAMAx-ec-PFMAy adsorbed at the solution/air interface were gradually completely replaced by PnAMA segments, resulting in an increase in corresponding solution surface tension until it was equal to that of poly(n-alkyl methacrylate) solution. Additionally, it was observed for...

A superoxide dismutase (SOD) was purified from seeds of the Chinese black soybean( Glycine max cv ). The purification procedure comprised ammonium sulfate precipitation, ion-exchange chromatography on CM-Sephadex C-50, affinity chromatography on Affi-gel blue gel and HP LC on DEAE 650-C. The final enzyme was purified 15.40 fold with a specific activity of 4619.3 U/mg protein and a recovery of enzyme was 29.41% from (NH 4 ) 2 SO 4 precipitation. The SDS-PAGE of the purified protein exhibited a molecular mass of 31.0 kDa and could be depolymerized spontaneously to a band at about 14.5 kDa in non-reducing conditions, and a diffusion band at about 15.5 kDa in reducing conditions, indicating a double-strand protein with intra-molecular disulfide bridges. The isoelectric point was determined to be 5.6, indicating it's an acidic protein. The enzyme is a type of copper plus zinc superoxide dismutase (Cu, Zn-SOD) because it was greatly inhibited by hydrogen peroxide, insensitive to chloroform–ethanol, and the maximum ultraviolet absorbance was found to be 266 nm. Thermostability experiments showed that the activity of the purified enzyme was stable after exposure to temperatures below 50 °C, and the activity could be well maintained between pH 6.0 and 8.0. Moreover, the SOD exerted an obvious protective effect against oxidative stress of UVC radiation in normal human liver cell strain L-02 cells.

A novel lysozyme was isolated from the Canadian cranberry beans ( Phaseolus vulgaris ) and its enzymatic properties, antifungal and antibacterial activities were investigated. The isolation procedure involved aqueous extraction, ammonium sulfate precipitation, and chromatography purification including affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-Sephadex, and high performance liquid chromatography (HPLC) on Mono S. The enzyme exhibited a molecular mass of 15.6 kDa in SDS-polyacrylamide gel electrophoresis both under reducing and non-reducing conditions, indicating that it was a monomeric protein. The 15 N-terminal amino acid sequences were determined to be KVDMPGRFALAMQSH, showing some homology to hen egg white lysozyme. The p I was determined to be 9.6 by isoelectric focusing electrophoresis. The specific activity of the lysozyme was 652 U/mg at pH 5.5 and 40 °C. The lysozyme exhibited a pH optimum at pH 5.5 and a temperature optimum at 50 °C. It exerted a potent inhibitory action toward fungal species including Alternaria alternata ( Fr .) Keissl , Pythium aphanidermatum , Fusarium oxysporum and Botrytis cinerea , in addition to an antibacterial action against Staphylococcus aureus . However, it showed no effect on Gram-negative bacteria. The enzyme was thermostable below 58 °C in both enzymatic reaction and antifungal activity. The present findings further the research on leguminous lysozyme with potentially exploitable significance.

New fluorinated copolymers of poly(methyl methacrylate)-b-poly(butyl methacrylate) or poly(n-octadecyl methacrylate) end-capped with 2-perfluorooctylethyl methacrylate (PMMA(x)-b-PBMA(y)-ec-PFMA(z) or PMMA(x)-b-PODMA(y)-ec-PFMA(z)) were synthesized by living atom transfer radical polymerization. Thin films made of PMMA(230)-b-PODMA(y)-ec-PFMA(1) were characterized by differential scanning calorimetry, angle-resolved X-ray photoelectron spectroscopy and X-ray diffraction. These films were found to exhibit robust surface segregation of the end groups. Furthermore, the fluorine enrichment factor at the film surface was found to increase linearly with increasing degree of polymerization of poly(n-octadecyl methacrylate) and its increasing fusion enthalpy in the second block, which enhances the segregation of the fluorinated moieties.

A β-glucosidase gene (bglI) from Trichoderma reesei was cloned into the pPIC9 vector and integrated into the genome of Pichia pastoris GS115. Under the control of the methanol-inducible alcohol oxidase (AOX) promoter and using Saccharomyces cerevisiae secretory signal peptide (α-factor), the recombinant β-glucosidase was expressed and secreted into the culture medium. The maximum recombinant β-glucosidase activity achieved was 60 U/ml, and β-glucosidase expression reached 0.3 mg/ml. The recombinant 76 kDa β-glucosidase was purified 1.8-fold with 26% yield and a specific activity of 197 U/mg. It was optimally active at 70°C and pH 5.0.

The effects of solvents, fluorinated monomer content and film-formation methods on the surface structures of random copolymers composed of methyl methacrylate (MMA) and 2-perfluorooctylethyl methacrylate (FMA) were investigated by contact angle goniometry, X-ray photoelectron spectroscopy, sum frequency generation (SFG) vibrational spectroscopy and surface tension measurement. It is found that, with cyclohexanone as the solvent, there is a critical FMA content of 9 mol%, below which the copolymer films by spin coating have a more surface segregation extent of fluorinated moieties than those by solution casting; above which the copolymer films by solution casting have a more surface segregation extent of fluorinated moieties than those by spin coating. However, with toluene as solvent, the critical FMA content lowers down to 3 mol%. We believe that the solvent nature and the content of fluorinated moieties in the random copolymer have the great effect because the combined effect of these two factors can determine the random copolymer chain conformations and their thermodynamic dominating factors in the solution and at the solution–air interface. A thermodynamic analysis combining the entropic and enthalpic effects is suggested to explain the observed phenomenon. This research is believed to obtain an enhanced understanding of the surface formation mechanism of the polymer films and thus demonstrate how to promote the segregation of fluorinated moieties at the polymer film surfaces.

Leguminous plants have formed a popular subject of research owing to the abundance of proteins and peptides with important biological activities that they produce. The antifungal proteins and peptides have been purified from a number of leguminous species. However, research continues to discover novel antifungal plant-produced peptides and proteins are being needed, specially those novel ones with both antifungal activity and other significant bioactivities. The objective of this study was to isolate a novel peptide from Phaseolus limensis. A 6.8 kDa peptide designated Limyin, with both antifungal and antiproliferative activity, was isolated from the large lima bean (P. limensis) legumes. The isolation procedure consisted of extraction, precipitation, affinity chromatography on Affi-gel blue gel, ion chromatography on SP-Toyopearl, and gel filtration on Superdex 75. Its N-terminal sequence was determined to be KTCENLATYYRGPCF, showing high homology to defensin and defensin precursors from plants. It potently suppressed mycelial growth in Alternaria alternata, Fusarium solani, and Botrytis cinerea. Its antifungal activity was stable up to 80 degrees C. It showed antiproliferative activity towards tumor cells including human liver hepatoma cells Bel-7402 and neuroblastoma cells SHSY5Y. However, it had no effect on bacteria Staphylococcus aureus and Salmonella. The present findings make a significant addition of the research on leguminous plants.

A novel laccase gene Lac1 and its cDNA were cloned from a white-rot fungus Cerrena sp. and characterized. The 1554-bp cDNA of Lac1 encoded a mature protein with 497 amino acids, preceded by a signal peptide of 20 amino acids. An unconventional intron splice site and incomplete splicing variants of Lac1 were observed. Lac1 was heterologously expressed in the yeast host Pichia pastoris, and a maximal laccase activity of 6.3UmL(-1) in the fermentation broth was achieved after fermentation for 9 days. The recombinant protein rLac1 was purified, and its enzymatic properties and functional characteristics were investigated. When ABTS was used as the substrate, the enzyme was most active at pH 3.5 and 55°C, and stable at pH 4-10 and 20-60°C. The Km and kcat values of rLac1 toward ABTS were 28.9 μM and 332.4s(-1), respectively. Furthermore, rLac1 was tolerant to common metal ions up to 100mM concentration and capable of decolorizing structurally different dyes in the absence of a redox mediator. Hence, Lac1 may be useful for industrial applications, such as dye decolorization and bioremediation.

Malachite green (MG) was decolorized by laccase (LacA) of white-rot fungus Cerrena sp. with strong decolorizing ability. Decolorization conditions were optimized with response surface methodology. A highly significant quadratic model was developed to investigate MG decolorization with LacA, and the maximum MG decolorization ratio of 91.6% was predicted under the conditions of 2.8 U mL(-1) LacA, 109.9 mg L(-1) MG and decolorization for 172.4 min. Kinetic studies revealed the Km and kcat values of LacA toward MG were 781.9 mM and 9.5 s(-1), respectively. UV-visible spectra confirmed degradation of MG, and the degradation mechanism was explored with liquid chromatography-mass spectrometry (LC-MS) analysis. Based on the LC-MS spectra of degradation products, LacA catalyzed MG degradation via two simultaneous pathways. In addition, the phytotoxicity of MG, in terms of inhibition on seed germination and seedling root elongation of Nicotiana tabacum and Lactuca sativa, was reduced after laccase treatment. These results suggest that laccase of Cerrena was effective in decolorizing MG and promising in bioremediation of wastewater in food and aquaculture industries.

Laccases (EC 1.10.3.2) are a class of multi-copper oxidases with important industrial values. A basidiomycete strain Cerrena sp. HYB07 with high laccase yield was identified. After cultivation in the shaking flask for 4 days, a maximal activity of 210.8 U mL(-1) was attained. A 58.6-kDa laccase (LacA) with 7.2% carbohydrate and a specific activity of 1952.4 U mg(-1) was purified. 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) was the optimal substrate, with Km and kcat being 93.4 µM and 2468.0 s(-1), respectively. LacA was stable at 60°C, pH 5.0 and above, and in organic solvents. Metal ions Na+, K+, Ca2+, Mg2+, Mn2+, Zn2+ enhanced LacA activity, while Fe2+ and Li+ inhibited LacA activity. LacA decolorized structurally different dyes and a real textile effluent. Its gene and cDNA sequences were obtained. Putative cis-acting transcriptional response elements were identified in the promoter region. The high production yield and activity, robustness and dye decolorizing capacity make LacA and Cerrena sp. HYB07 potentially useful for industrial and environmental applications such as textile finishing and wastewater treatment.

Lyophylized antler powder was hydrolyzed by pepsin and trypsin separately and also simultaneously to give hydrolysates with special physical activities. Complete hydrolysis peptides with MW lower than 1 x 10(3) were collected for assay of angiotensin I-converting enzyme (ACE) inhibitory activity, antioxidant activity and proliferative activity toward UMR-106 osteoblast cells. The results of the experiments revealed that all hydrolysates exhibited potent hydroxyl radical scavenging activity with an IC50 value less than 1 mg/ml which was much lower than the value of 5.5 g x L(-1) for vitamin C. The peptic and peptic tryptic hydrolysates demonstrated strong angiotensin I-converting enzyme (ACE) inhibitory activity. The tryptic hydrolysate increased the proliferation of the UMR-106 cells by 73.43%. The results verified the traditional use of antler in bone-strengthening, anti-aging. The exploratory studies on the ACE inhibitory activity of antler hydrolysates indicated that the hydrolysates might be potentially useful in prevention and treatment of hypertension. Further purification of peptides contributing to the antioxidant activity, angiotensin I-converting enzyme-inhibitory activity and proliferative activity toward osteoblasts from antler hydrolysates is warranted.

Living organisms produce a myriad of molecules to protect themselves from fungal pathogens. This review focuses on antifungal proteins from plants and mushrooms, many of which are components of the human diet or have medicinal value. Plant antifungal proteins can be classified into different groups comprising chitinases and chitinase-like proteins, chitin-binding proteins, cyclophilin-like proteins, defensins and defensin-like proteins, deoxyribonucleases, embryo-abundant protein-like proteins, glucanases, lectins, lipid transfer proteins, peroxidases, protease inhibitors, ribonucleases, ribosome-inactivating proteins, storage 2S albumins, and thaumatin-like proteins. Some of the aforementioned antifungal proteins also exhibit mitogenic activity towards spleen cells, nitric oxide inducing activity toward macrophages, antiproliferative activity toward tumor cells, antibacterial activity, and inhibitory activity toward HIV-1 reverse transcriptase. In contrast to the large diversity of plant antifungal proteins, only a small number of mushroom antifungal proteins have been reported. Mushroom antifungal proteins are distinct from their plant counterparts in N-terminal sequence. Nevertheless, some of the mushroom antifungal proteins have been shown to inhibit HIV-1 reverse transcriptase activity and tumor cell proliferation.

A novel lysozyme exhibiting antifungal activity and with a molecular mass of 14.4kDa in SDS-polyacrylamide gel electrophoresis was isolated from mung bean (Phaseolus mungo) seeds using a procedure that involved aqueous extraction, ammonium sulfate precipitation, ion exchange chromatography on CM-Sephadex, and high-performance liquid chromatography on POROS HS-20. Its N-terminal sequence was very different from that of hen egg white lysozyme. Its pI was estimated to be above 9.7. The specific activity of the lysozyme was 355U/mg at pH 5.5 and 30 degrees C. The lysozyme exhibited a pH optimum at pH 5.5 and a temperature optimum at 55 degrees C. It is reported herein, for the first time, that a novel plant lysozyme exerted an antifungal action toward Fusarium oxysporum, Fusarium solani, Pythium aphanidermatum, Sclerotium rolfsii, and Botrytis cinerea, in addition to an antibacterial action against Staphylococcus aureus.