Identification and characterization of a chitinase of Stenotrophomonas maltophilia, a bacterium that is antagonistic towards fungal phytopathogens

A rhizosphere strain of Stenotrophomonas maltophilia strain MUJ that is strongly antagonistic towards fungal phytopathogens secretes to the culture medium a single form of active chitinolytic enzyme belonging to family 18 of glycosyl hydrolases. The chitinase was purified by a two-stage procedure embracing fractionation with ammonium sulfate and affinity chromatography. The molecular mass of the purified enzyme determined by SDS-PAGE was approximately 52 kDa. The enzyme demonstrated highest activity at 45°C and pH 6.8. The enzymatic protein showed considerable thermal stability during 2 h incubation at 45°C. The activity of the enzyme was strongly inhibited in the presence of Hg²⁺ and Cu²⁺. By applying mass spectrometry analysis, the peptides derived from the purified chitinase were assigned to amino acid sequences of the type ChiA chitinases synthesized by Stenotrophomonas bacteria. The purified enzyme inhibited the growth of fungal phytopathogens belonging to the genera Fusarium, Rhizoctonia and Alternaria.     

Antibacterial activity of chitin,chitosan and its oligomers prepared from shrimp shell waste

The antimicrobial activities of chito-oligosaccharides against four Gram-positive and seven Gram-negative bacteria were compared to chitosan and chitin with an emphasis on the effects of biopolymer molecular weight (Mv) and degree of deacetylation (DD). Chitin was isolated from shrimp (Parapenaeus longirostris) shell waste by sequential chemical treatments. Chitosan and its oligomers N-acetyl chito-oligosaccharides and chito-oligosaccharides were prepared by deacetylation and chemical hydrolysis, respectively. Chitin exhibited a bacteriostatic effect on Gram-negative bacteria, Escherichia coli ATCC 25922, Vibrio cholerae, Shigella dysenteriae, and Bacteroides fragilis. Chitosan exhibited a bacteriostatic effect on all bacteria tested, except Salmonella typhimurium. The oligomers exhibited a bactericidal effect on all bacteria tested.     

The bacterium Burkholderia gladioli strain CHB101 produces two different kinds of chitinases belonging to families 18 and 19 of the glycosyl hydrolases

Two genes (chiA and chiB) coding for chitanases A and B (ChiA and ChiB) were isolated from the chitinolytic bacterium, Burkholderia gladioli strain CHB101. chiA contains an open reading frame that encodes a protein of 343 amino acids, whereas chiB encodes a protein of 307 amino acids. The deduced amino acid sequence of ChiA showed a high similarity to those of microbial chitinases belonging to family 18 of the glycosyl hydrolases, while ChiB showed significant sequence similarity to plant chitinases and Streptomyces spp. chitinases belonging to family 19.     

Antibacterial activity of chemically defined chitosans: influence of molecular weight, degree of acetylation and test organism

Chitosans, polysaccharides obtained from the exoskeleton of crustaceans, have been shown to exert antibacterial activity in vitro and their use as a food preservative is of growing interest. However, beyond a consensus that chitosan appears to disrupt the bacterial cell membrane, published data are inconsistent on the chemical characteristics that confer the antibacterial activity of chitosan. While most authors agree that the net charge density of the polymer (reflected in the fraction of positively charged amino groups at the C-2 position of the glucosamine unit) is an important factor in antibacterial activity, conflicting data have been reported on the effect of molecular weight and on the susceptibility among different bacterial species to chitosan. Therefore, we prepared batches of water-soluble hydrochloride salts of chitosans with weight average molecular weights (M_w) of 2-224 kDa and degree of acetylation of 0.16 and 0.48. Their antibacterial activity was evaluated using tube inhibition assays and membrane integrity assays (N-Phenyl-1-naphthylamine fluorescence and potassium release) against Bacillus cereus, Escherichia coli, Salmonella Typhimurium and three lipopolysaccharide mutants of E. coli and S. Typhimurium. Chitosans with lower degree of acetylation (F_A = 0.16) were more active than the more acetylated chitosans (F_A = 0.48). No trends in antibacterial action related to increasing or decreasing M_w were observed although one of the chitosans (M_w 28.4 kDa, F_A = 0.16) was more active than the other chitosans, inhibiting growth and permeabilizing the membrane of all the test strains included. The test strains varied in their susceptibility to the different chitosans with wild type S. Typhimurium more resistant than the wild type E. coli. Salmonellae lipopolysaccharide mutants were more susceptible than the matched wild type strain. Our results show that the chitosan preparation details are critically important in identifying the antibacterial features that target different test organisms.     

Antioxidative activity of chitosans with varying molecular weights

Antioxidant activity of chitosans of different molecular weights (30, 90 and 120 kDa chitosan) in salmon (Salmo salar) was investigated. The progress of oxidation was monitored by employing the 2-thiobarbituric acid-reactive substances (TBARS) and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assays. In general, all chitosans exhibited antioxidative activities in salmon. The addition of chitosans to salmon reduced lipid oxidation for seven days of storage. The TBARS values of salmon containing chitosan were significantly lower than those of the control (p < 0.01). At 0.2% (w/v) and 0.5% (w/v) concentrations, the TBARS with chitosan addition was decreased by 75% and 45%, respectively, over 15 days. At 1% concentration, the TBARS value with native chitosan addition was decreased by 32% after 15 days of storage. 90 kDa chitosan showed an increased DPPH free radical-scavenging activity with increasing concentration in the range of 0.2–1% (w/v). The free radical-scavenging activity of the 0.2 mM DPPH solution was saturated by 30 kDa chitosan at a concentration of ?0.7% (w/v), resulting in a strong antioxidant activity of approximately 85%. This was comparable to the DPPH free radical-scavenging activity of BHT.     

Preparation,water solubility and rheological property of the N-alkylated mono or disaccharide chitosan derivatives

N-alkylation of chitosan was performed in a mixture of methanol and 1% acetic acid containing different amounts of monosaccharides or disaccharides including glucose, galactose, glucosamine, fructose, lactose, maltose and cellobiose. All the N-alkylated chitosan derivatives with monosaccharides were insoluble in aqueous solution (pH 7), while N-alkylated chitosan derivatives with disaccharides were easily soluble in distilled water, and the N-alkylated chitosan derivatives with lactose were soluble only at high pH. The degree of substitution (DS) of the N-alkylated chitosan derivatives increased with increasing disaccharides levels and with increasing reaction time. The reduced viscosity of the N-alkylated chitosan derivatives with disaccharides decreased with increasing DS. Apparent viscosity and pseudoplasticity of the N-alkylated disaccharide containing derivative solutions generally decreased with increasing DS. Although apparent viscosities of N-alkylated chitosan derivatives with low DS decreased with increase in pH or ionic strength, changes in high DS N-alkylated chitosan derivatives with pH values or ionic strength were not marked.     

Development of a simple capillary electrophoretic determination of glucosamine in nutritional supplements using in-capillary derivatisation with o-phthalaldehyde

A simple capillary electrophoretic method was developed for the determination of glucosamine using in-capillary derivatisation. Glucosamine in commercial products was extracted with purified water. The CE separation was achieved on an uncoated fused-silica capillary using a 20 mM borate buffer (pH 9.2) containing 5 mM o-phthalaldehyde (OPA) and 5 mM 3-mercaptopropionic acid (MPA) at 25 kV, followed by UV detection at 340 nm. The detector response was linear (r² > 0.999) in the concentration range 10–1000 μg/mL. The limit of detection (LOD) was 1.3 mg/g. Spiked glucosamine recoveries at 50 and 100 mg/g level were 95.1% and 104.3%, respectively. The method was applied to 16 commercial products. The concentrations of glucosamine were 109–705 mg/g, and the ratios of detected glucosamine content to the labelled value were 88.8–124%. No significant bias was observed (r² = 0.989, p < 0.01), between results obtained by the proposed CE method and an official colorimetric method (Japanese Health Food & Nutrition Food Association).     

壳聚糖酶高产菌株选育及发酵条件研究

以自筛曲霉CJ2 2 -3为出发株 ,经紫外线和6 0 Co诱变处理 ,获得 1株壳聚糖酶高产菌株CJ2 2 -3 2 6,经正交实验初步优化了其液态产酶培养基 :壳聚糖 1 5 % ,麸皮 2 % ,(NH4 ) 2 SO4 0 2 % ,KH2 PO4 0 2 % ,MgSO4 0 0 5 % ,Tween -80 0 0 5 % ,pH5 5。优化的发酵条件为 :装液量 75mL/ 2 5 0mL三角瓶 ,摇床转速 1 5 0r/min ,发酵温度为 3 0℃ ,发酵时间为 96h。在此条件下 ,CJ2 2 -3 2 6产酶活力为3 0 6U/mL。     

Purification and characterization of hydrolytic and transgalactosyl α-galactosidase from Lactobacillus helveticus ATCC 10797

α-Galactosidase purified from Lactobacillus helveticus ATCC 10797 by fast performance liquid chromatography system using ion exchange and gel-filtration columns showed the K _m of 3.83 mM and V _max of 416.44 µmol/min/mg protein calculated from the substrate p-nitrophenyl-α-d-galactopyranoside. The molecular mass was 188 kDa by gel-filtration, but 90 kDa by SDS-PAGE, indicating a homodimer. The optimum temperature was 37 °C, and the optimum pH was at 6 with an acceptable stability between pH 4 and 8. This enzyme was activated by 10 mM monovalent ions such as K⁺, NH₄ ⁺, Li⁺, and CS⁺, while the activity was inhibited by divalent ions such as Cu²⁺, Zn²⁺, and Fe²⁺. Melibiose was hydrolyzed to glucose and galactose, raffinose to galactose and sucrose, while stachyose to galactose and sucrose. A novel source of α-galactosidase from L. helveticus possessing both hydrolytic activity to eliminate flatulence sugars and transgalactosylation activities to synthesize galacto-oligosaccharides is identified and characterized.     

Purification and some properties of a chitinase from Xanthomonas sp. strain AK

Chitinase B (ChiB) was purified from the culture supernatant of Xanthomonas sp. strain AK by Phenyl-Toyopearl 650M and DEAE-Toyopearl 650M column chromatographies. The purified enzyme preparation gave a single band on SDS-polyacrylamide gel electrophoresis and the molecular weight of ChiB was estimated to be 48,000. The enzyme was optimally active at pH 6.0 and 60 degrees C. N-Terminal amino acid sequence analysis suggested that ChiB is a member of glycosyl hydrolase family 18 and that it is genetically different from ChiA recently reported (Sakka et al., J. Ferment. Bioeng., 86, 527-533, 1998). Immunological analysis suggested that ChiB was the major chitinase species in the culture supernatant of Xanthomonas sp. strain AK and that production of the enzyme was induced by the presence of chitin.     

Preparation of chitosan oligomers and their antioxidant activity

Chitosan oligomers with different molecular weights were prepared by oxidative degradation method involving hydrogen peroxide (H₂O₂) and the combined degradation method using hydrogen peroxide and microwave radiation. Viscosity determination and end group analysis were applied to measure molecular weights of chitosan oligomers. Effects of concentration of H₂O₂ and degradation time on molecular weights of chitosan oligomers were studied. Both methods were effective to prepare chitosan oligomers from the initial chitosan (8.5×10⁵ Da). The degradation process of chitosan will be accelerated with the aid of microwave and degradation time may be reduced. The antioxidant activity of chitosan oligomers was evaluated as radical scavengers against superoxide anion and hydroxyl radical by application of flow injection chemiluminescence technology. Chitosan oligomers A, B, C and D (2300, 3270, 6120, and 15,250 Da) had different antioxidant activity. Among the four chitosan oligomers, oligomer D (15,250 Da) had the lowest scavenging ability against superoxide anion and hydroxyl radicals. For superoxide anion scavenging, the 50% inhibition concentrations (IC₅₀s) of other three oligomers A, B, and C were 5.54, 8.11, and 12.15 mg/mL, respectively. And for hydroxyl radical scavenging the values were 0.4, 0.76, and 1.54 mg/mL, respectively. At the concentration range examined, the maximal inhibiting efficacy of A, B, C, and D were 89, 75, 74, and 41% for superoxide anion, and 71, 65, 51, and 7% for hydroxyl radical. These results indicated that chitosan oligomers with lower molecular weight had better antioxidant activity.     

Evaluation of food grade antioxidant formulation for sustained antifungal,antiaflatoxigenic and insecticidal activities on peanut conditioned at different water activities

The aim of this study was to investigate antifungal and insecticidal activity of two microencapsulated antioxidants: 2(3)-tert-butyl-4 hydroxyanisole (BHA) and 2,6-di(tert-butyl)-p-cresol (BHT) against Aspergillus section Flavi and Oryzaephilus surinamensis (L.), a vector carrier of aflatoxigenic fungi on stored peanuts. Susceptibility of Aspergillus section Flavi, insects, and aflatoxin B₁ accumulation in sterile peanut kernels conditioned at two different water activities (a_w) (0.83 a_w and 0.95 a_w) was determined with different doses of antioxidant formulations (10, 20 and 30 mM) during 45 days. Moreover, Aspergillus section Flavi isolation frequency from live and dead insects was evaluated. The BHA formulation completely inhibited Aspergillus section Flavi development regardless of a_w and doses assayed. Antifungal effect of microencapsulated BHT was highly dependent on a_w, with 86–100% fungal inhibition at 20 and 30 mM, at the lowest a_w (0.83 a_w) and at the end of the experiment. No aflatoxin accumulation was detected in samples treated with the BHA formulation. In general, low levels of Aspergillus section Flavi were detected in dead insects. Our results show efficacy for 45 days, in addition microencapsulated BHT could be an alternative to control peanut pests in dry kernels.     

Inhibition of Bacillus cereus spore outgrowth and multiplication by chitosan

Bacillus cereus is an endospore-forming bacterium able to cause food-associated illness. Different treatment processes are used in the food industry to reduce the number of spores and thereby the potential of foodborne disease. Chitosan is a polysaccharide with well-documented antibacterial activity towards vegetative cells. The activity against bacterial spores, spore germination and subsequent outgrowth and growth (the latter two events hereafter denoted (out)growth), however, is poorly documented. By using six different chitosans with defined macromolecular properties, we evaluated the effect of chitosan on Bacillus cereus spore germination and (out)growth using optical density assays and a dipicolinic acid release assay. (Out)growth was inhibited by chitosan, but germination was not. The action of chitosan was found to be concentration-dependent and also closely related to weight average molecular weight (M_w) and fraction of acetylation (F_A) of the biopolymer. Chitosans of low acetylation (F_A = 0.01 or 0.16) inhibited (out)growth more effectively than higher acetylated chitosans (F_A = 0.48). For the F_A = 0.16 chitosans with medium (56.8 kDa) and higher M_w (98.3 kDa), a better (out)growth inhibition was observed compared to low M_w (10.6 kDa) chitosan. The same trend was not evident with chitosans of 0.48 acetylation, where the difference in activity between the low (19.6 kDa) and high M_w (163.0 kDa) chitosans was only minor. In a spore test concentration corresponding to 10²-10³ CFU/ml (spore numbers relevant to food), less chitosan was needed to suppress (out)growth compared to higher spore numbers (equivalent to 10⁸ CFU/ml), as expected. No major differences in chitosan susceptibility between three different strains of B. cereus were detected. Our results contribute to a better understanding of chitosan activity towards bacterial spore germination and (out)growth.     

Purification and characterization of a new endo-β-1,3-glucanase exhibiting a high specificity for curdlan for production of β-1,3-glucan oligosaccharides

Endo-β-1,3-glucanase (Endo23) was purified from a Trichoderma reesei GIMCC 3.498 fermentation broth using anion exchange and 2-stage size exclusion chromatography. Purification of 44.5× and a 12% recovery yield of enzyme activity were achieved. The Mw and isoelectric point were estimated to be 24 kDa and 3.85 using SDS-PAGE and IEF, respectively. The highest substrate specificity was observed for water-insoluble curdlan. The optimal conditions for hydrolyzing curdlan were pH 5.0 and 50°C. The main hydrolytic products were glucobiose and glucotriose. Minor amounts of glucose and glucotetraose were detected. Hg²⁺, Fe²⁺, Fe³⁺, and Sn²⁺ inhibited the hydrolysis activity of Endo23 at 5 and 50 mM. K⁺ slightly promoted Endo23 activity. Endo23 belongs to the category EC3.2.1.39. The peptide sequences of Endo23 showed identity with conserved sequences that typically exist in β-1,3-glucanases of the glycoside hydrolase family. The Endo23 sequence was partially similar to a hypothetical lignocellulase from Penicillium oxalicum 114-2.     

Microwave‐assisted degradation of chitosan with hydrogen peroxide treatment using Box‐Behnken design for enhanced antibacterial activity

Low molecular mass (MM) chitosan with high degree of deacetylation (DDA) has excellent bioactivity including antioxidant, antibacterial and encapsulation properties. In this work, to reduce the MM of chitosan, microwave‐assisted heating treatment (MAHT) conditions were investigated using three factors at three levels Box‐Behnken design (BBD). Microwave heating (MH) time, H₂O₂ concentration and solid‐to‐liquid ratio significantly affected the DDA and MM of chitosan. The antibacterial activities of chitosan before and after degradation were investigated based on minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The results showed that a second‐order polynomial equation fitted the observed values using multiple regression analysis and had a high coefficient of determination (R² = 0.9591 and 0.9161 for the DDA and MM of chitosan, respectively). An optimisation study was performed using Derringer's desirability function methodology, and the optimal conditions were 80‐s MH time, 1.5% H₂O₂ concentration and 1:40 solid‐to‐liquid ratio. The MIC and MBC of chitosan before and after degradation against Escherichia coli and Salmonella typhimurium were 0.031 and 0.063 mg mL^?1, and 0.25 and 0.125 mg mL^?1, respectively. The optimised DDA and MM of chitosan were 90.58 ± 2.04% and 124.25 ± 14.36 kDa, respectively, which significantly reduces the use of oxidant reagent.     

An extra-cellular alkaline metallolipase from Bacillus licheniformisMTCC 6824: Purification and biochemical characterization

An extra-cellular lipase produced by Bacillus licheniformis MTCC 6824 was purified to homogeneity by ammonium sulphate fractionation, ethanol/ether precipitation, dialysis, followed by anion-exchange chromatography on Amberlite IRA 410 (Cl⁻ form) and gel exclusion chromatography on Sephadex G 100 using Tris–HCl buffer (pH 8.0). The crude lipase extract had an activity of 41.7 LU/ml of culture medium when the bacterium was cultured for 48 h at 37 °C and pH 8.0 with nutrient broth supplemented with sardine oil as carbon source. The enzyme was purified 208-fold with 8.36% recovery and a specific activity of 520 LU/mg after gel exclusion chromatography. The pure enzyme is a monomeric protein and has an apparent molecular mass of 74.8 kDa. The lipase had a V_max and K_m of 0.64 mM/mg/min and 29 mM, respectively, with 4-nitro phenylpalmitate as a substrate, as calculated from the Lineweaver–Burk plot. The lipase exhibited optimum activity at 45 °C and pH 8.0, respectively. The enzyme had half-lives (T_1/2) of 82 min at 45 °C, and 48 min at 55 °C. The catalytic activity was enhanced by Ca²⁺ (18%) and Mg²⁺ (12%) at 30 mM. The lipase was inhibited by Co²⁺, Cu²⁺, Zn²⁺, Fe² even at low concentration (10 mM). EDTA, at 70 mM concentration, significantly inhibited the activity of lipase. Phenyl methyl sulfonyl fluoride (PMSF, 70 mM) completely inactivated the original lipase. A combination of Ca²⁺ and sorbitol induced a synergistic effect on the activity of lipase with a significantly high residual activity (100%), even after 45 min, as compared to 91.5% when incubated with Ca²⁺ alone. The lipase was found to be hydrolytically resistant toward triacylglycerols with more double bonds.     

Preparation of biopolymer film from chitosan modified with lipid fraction

Films formed from polysaccharides, as chitosan, present a high permeability in water vapour. In order to increase resistance to water vapour for chitosan‐based films, different lipid fractions were incorporated into a filmogenic matrix: fish and vegetable oils, stearic and oleic acids. The chitosan showed a molecular weight of 150 kDa and a deacetylation degree of 86 ± 1%. Results showed that incorporation of different lipid fractions decreased the water vapour permeability (WVP) (1.3–1.8 g mm m^?2 day^?1 kPa^?1) as compared with pure chitosan film (3.8 g mm m^?2 day^?1 kPa^?1). A higher reduction in WVP (65%) was found with the addition of refined fish oil to the continuous matrix of the films than with the addition of refined rice oil, oleic or stearic acid (50–60%). However, pure chitosan films showed better tensile strength (TS = 33 MPa) and elongation percentage (E = 18%) than lipids fraction–chitosan films (7–19 MPa and 7–13%, respectively).     

Production of fungal lipases using wheat bran and soybean bran and incorporation of sugarcane bagasse as a co-substrate in solid-state fermentation

Fungal strains were screened for lipase producing activities and 10 strains were classified as good producers. Aspergillus sp., Fusarium sp., and Penicillium sp. exhibited the highest activities when fermented in wheat bran (WB) and soybean bran (SB). No fungal growth was observed using sugarcane bagasse (CB). An experimental design was applied to incorporate CB into the fermentation process for lipase production by Aspergillus sp. and Penicillium sp., and to evaluate the best moisture content for the substrate. Strains studied achieved maximum lipase activities with 25% CB combined with 75% WB or SB at 40% moisture content. The highest lipase activities were observed for WB and SB, and for SB combined with CB using Aspergillus sp. Fermentation of 96 h was the optimum period for enzyme production.     

A NaCl-stable serine proteinase from Virgibacillus sp. SK33 isolated from Thai fish sauce

An extracellular proteinase from Virgibacillus sp. SK33, isolated from 1 month-old fish sauce, was purified to electrophoretic homogeneity, using hydrophobic interaction chromatography and hydroxyapatite with purification fold of 2.5 and 7% yield. The anomalous molecular weight (MW) of 19 kDa was obtained from SDS–PAGE, whereas a MW of 33.7 kDa was determined by MALDI-TOF. Optimum conditions for catalytic activity were 55 °C and pH 7.5. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferentially hydrolysed Suc-Ala-Ala-Pro-Phe-AMC, indicating a serine proteinase with subtilisin-like characteristics. K_m and k_cat of the purified proteinase were 27 μM and 12 s⁻¹, respectively. Proteinase activity, toward both synthetic and anchovy substrates, increased with NaCl up to 25%. The proteinase exhibited high stability in both the absence and presence of NaCl up to 25%. Approximately 2.5-fold increase in activity was observed in the presence of divalent cations, including Ca²⁺, Mg²⁺ and Sr²⁺ at 100 mM. MALDI-TOF MS and LC–ESI-MS/MS analyses, as well as N-terminal sequences, revealed that the purified enzyme did not match microbial proteinases in the database, indicating it to be a novel proteinase.     

Purification and characterization of an alkaline pectin lyase produced by a newly isolated Brevibacillus borstelensis (P35) and its applications in fruit juice and oil extraction

An alkaline pectin lyase (PNL) (EC 4.2.2.10) secreted by Brevibacillus borstelensis P35 (GenBank Number: FJ417406) was purified using ammonium sulfate fractionation, anion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-150. The pH and temperature optima of the enzyme were found to be 8.0 and 60 °C. The enzyme does not loose activity up to 60 °C if exposed for 1 h. The values of K _m and V _max of the enzyme were 0.625 mg/mL and 126.32 s^?1, respectively. The molecular weight was found to be 36 ± 01 kDa. The presence of 10 mM concentration of Ca²⁺, Cu²⁺, Mn²⁺, Mg²⁺, Zn²⁺, Hg²⁺, Fe²⁺ and EDTA, l-cystein, ascorbic acid significantly enhanced the PNL of the purified enzyme. In the course of the laboratory trials, it was demonstrated that PNL from B. borstelensis (P35) could be successfully applied to the production and clarification of fruit juice and oil extraction.