An Oxidant- and Solvent-Stable Protease Produced by <Emphasis Type="Italic">Bacillus cereus</Emphasis> SV1: Application in the Deproteinization of Shrimp Wastes and as a Laundry Detergent Additive

The current increase in amount of shrimp wastes produced by the shrimp industry has led to the need in finding new methods for shrimp wastes disposal. In this study, an extracellular organic solvent- and oxidant-stable metalloprotease was produced by Bacillus cereus SV1. Maximum protease activity (5,900 U/mL) was obtained when the strain was grown in medium containing 40 g/L shrimp wastes powder as a sole carbon source. The optimum pH, optimum temperature, pH stability, and thermal stability of the crude enzyme preparation were pH 8.0, 60 °C, pH 6–9.5, and <55 °C, respectively. The crude protease was extremely stable toward several organic solvents. No loss of activity was observed even after 60 days of incubation at 30 °C in the presence of 50% (v/v) dimethyl sulfoxide and ethyl ether; the enzyme retained more than 70% of its original activity in the presence of ethanol and N,N-dimethylformamide. The protease showed high stability toward anionic (SDS) and non-ionic (Tween 80, Tween 20, and Triton X-100) surfactants. Interestingly, the activity of the enzyme was significantly enhanced by oxidizing agents. In addition, the enzyme showed excellent compatibility with some commercial liquid detergents. The protease of B. cereus SV1, produced under the optimal culture conditions, was tested for shrimp waste deproteinization in the preparation of chitin. The protein removal with a ratio E/S of 20 was about 88%. The novelties of the SV1 protease include its high stability to organic solvents and surfactants. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis. In addition, the enzyme may find potential applications in the deproteinization of shrimp wastes to produce chitin.     

A Solvent-Stable Metalloprotease Produced by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> A2 Grown on Shrimp Shell Waste and Its Application in Chitin Extraction

A solvent-stable protease-producing bacterium was isolated and identified as Pseudomonas aeruginosa A2. The strain was found to produce high level of protease activity when grown in media containing only fresh shrimp waste (FSW) or shrimp waste powder (SWP), indicating that it can obtain its carbon, nitrogen, and salts requirements directly from shrimp waste. Maximum protease activities 17,000 and 12,000 U/mL were obtained with 80 g/L SWP and 135 g/L FSW, respectively. The optimum temperature and pH for protease activity were 60 °C and 8.0, respectively. The crude protease, at different enzyme/substrate (E/S) ratio, was tested for the deproteinization of shrimp waste to produce chitin. The crude enzyme of P. aeruginosa A2 was found to be effective in the deproteinization of shrimp waste. The protein removals after 3 h hydrolysis at 40 °C with an E/S ratio of 0.5 and 5 U/mg protein were about 56% and 85%, respectively. ¹³C CP/MAS-NMR spectral analysis of the chitin prepared by treatment with the crude protease was carried out and was found to be similar to that of the commercial α-chitin. These results suggest that enzymatic deproteinization of shrimp waste by A2 protease could be applicable to the chitin production process.     

Depolymerization of Chitosan with a Crude Cellulase Preparation from <Emphasis Type="Italic">Aspergillus Niger</Emphasis>

A crude cellulase preparation from Aspergillus niger was used to depolymerize chitosan. The depolymerization process was followed by measuring the apparent viscocity and the intrinsic viscosity. The optimum conditions for enzymatic hydrolysis were investigated. On the selected optimum conditions (pH 5.0, temperature 50 °C, and an enzyme to substrate ratio of 1:5), chitosan was hydrolyzed for 1, 4, 8, and 24 h, its viscosity-average molecular weights were 3.49 × 10⁴, 1.18 × 10⁴, 5.83 × 10³, and 1.13 × 10³, respectively. Compared with chitosan having viscosity-average molecular weight of 5.18 × 10⁵ before enzymatic hydrolysis, the crude cellulase preparation had rather apparent effect on depolymerization of chitosan. Through the comparison of different origin of cellulases, the prepared cellulase has good ability of enzymatic hydrolysis. The reproducibility and reversibility for enzymatic hydrolysis was appraised. The data are of value for the production of low-molecular weight chitosans and chitooligomers of medical and biotechnological interest.     

Solvent-Stable Digestive Alkaline Proteinases from Striped Seabream (<Emphasis Type="Italic">Lithognathus mormyrus</Emphasis>) Viscera: Characteristics,Application in the Deproteinization of Shrimp Waste,and Evaluation in Laundry Commercial Detergents

Alkaline proteases from the viscera of the striped seabream (Lithognathus mormyrus) were extracted and characterized. Interestingly, the crude enzyme was active over a wide range of pH from 6.0 to 11.0, with an optimum pH at the range of 8.0–10.0. In addition, the crude protease was stable over a broad pH range (5.0–12.0). The optimum temperature for enzyme activity was 50 °C. The crude alkaline proteases showed stability towards various surfactants and bleach agents and compatibility with some commercial detergents. It was stable towards several organic solvents and retained more than 50% of its original activity after 30 days of incubation at 30 °C in the presence of 25% (v/v) dimethyl sulfoxide, N,N-dimethylformamide, diethyl ether, and hexane. The crude enzyme extract was also tested for shrimp waste deproteinization in the preparation of chitin. The protein removal with a ratio enzyme/substrate of 10 was about 79%.     

Extraction and Characterization of Chitin and Chitosan from Parapenaeus longirostris from Moroccan Local Sources

The contents of the exoskeleton of Parapenaeus longirostris from Moroccan local sources were analyzed and the percentages of inorganic salt, protein, lipid, and chitin were determined. Chitin in the α form was extracted from Parapenaeus longirostris shells by 0.25 M HCl and 1 M NaOH treatment for demineralization and deproteinization, respectively. The obtained chitin was converted into the more useful soluble chitosan. The chemical structure and physico-chemical properties of chitin and chitosan were characterized using Fourier transform-infrared (FT-IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, X-ray diffractometry (XRD), and scanning electron microscopy (SEM). The molecular weight (MW) of chitosan was determined by viscometric methods. The degree of acetylation (DA) of chitin and chitosan was determined by the ¹H NMR technique. To the best of our knowledge this is the first report on the extraction and characterization of chitin and chitosan from Parapenaeus longirostris.     

Synthesis,characterization and antimicrobial activity of maltol functionalized chitosan derivatives

Chitin was isolated from prawn shell powder through demineralization and deproteinization process. Chitosan was synthesized from isolated chitin by deacetylation process and characterized by Fourier Transform Infrared (FTIR) spectra which showed close agreement with commercial chitosan. Physicochemical features such as moisture content, ash content, degree of deacetylation and molecular weight has been measured. The prepared chitosan was found to have comparatively higher molecular weight than the commercial chitosan. Functionalization of NH₂ group of chitosan with C?=?O group of maltol and ethyl maltol by refluxing equimolar quantities of respective ketones was performed. These synthesized derivatives of chitosan were characterized by their FTIR, ¹³C-Nuclear magnetic resonance (NMR) spectroscopy, elemental analysis, X-Ray Diffraction (XRD), Thermogravimetric analysis (TGA) and Differential thermal analysis (DTA) instrumental techniques. Antibacterial screening results of the synthesized chitosan and its derivatives indicate that these compounds are active against Escherichia coli bacteria.     

Cloning of a Heat-Stable Chitin Deacetylase Gene from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> and its Functional Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A gene encoding chitin deacetylase was cloned by polymerase chain reaction from Aspergillus nidulans. Sequencing result showed 40% homology to the corresponding gene from Colletotrichum lindemuthianum. The complete gene contains an open reading frame of 747 nucleotides encoding a sequence of 249 amino acid residues. The chitin deacetylase gene was subcloned into a pET28a expression vector and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a His-bind column. The purified chitin deacetylase demonstrated an activity of 0.77 U ml⁻¹ for the glycol chitin substrates, and its specific activity was 4.17 U mg⁻¹ for it. The optimal temperature and pH of the purified enzyme were 50 °C and 8.0, respectively. When glycol chitin was used as the substrate, K _m was 4.92 mg ml⁻¹, and K _cat showed 6.25 s⁻¹, thus the ratio of K _cat and K _m was 1.27 ml s⁻¹ mg⁻¹. The activity of chitin deacetylase was affected by a range of metal ions and ethylenediaminetetraacetic acid.     

Purification and Characterization of Thermostable Chitinase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> SK-1

Chitinase was purified from the culture medium of Bacillus licheniformis SK-1 by colloidal chitin affinity adsorption followed by diethylamino ethanol-cellulose column chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of chitinase 72 (Chi72) were 72 kDa and 4.62 (Chi72) kDa, respectively. The purified chitinase revealed two activity optima at pH 6 and 8 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from 40 to 70°C, with optimum at 55°C. It was stable for 2 h at temperatures below 60°C and stable over a broad pH range of 4.0–9.0 for 24 h. The apparent K _m and V _max of Chi72 for colloidal chitin were 0.23 mg ml⁻¹ and 7.03 U/mg, respectively. The chitinase activity was high on colloidal chitin, regenerated chitin, partially N-acetylated chitin, and chitosan. N-bromosuccinamide completely inhibited the enzyme activity. This enzyme should be a good candidate for applications in the recycling of chitin waste.     

Production and Characterization of an Alkaline Thermostable Crude Lipase from an Isolated Strain of <Emphasis Type="Italic">Bacillus cereus</Emphasis> C<Subscript>7</Subscript>

A bacterial strain isolated from spoiled coconut and identified as Bacillus cereus was found capable of producing alkaline thermostable extracellular lipase. Optimum temperature, time, and pH for enzyme substrate reaction were found to be 60 °C, 10 min, and 8.0 respectively. Common surfactants except Triton X 100 and cetyltrimethylammonium bromide have no or very little inhibitory effects on enzyme activity. The enzyme was found to be stable in presence of oxidizing agents and protease enzyme. The maximum lipase production was achieved at 30–33 °C, pH 8.0 on 24 h of fermentation using 50 ml medium in a 250-ml Erlenmeyer flask. The superior carbon and nitrogen sources for lipase production were starch (2%) and ammonium sulfate (nitrogen level 21.2 mg/100 ml), peptone (nitrogen level 297 mg/100 ml), and urea (nitrogen level 46.62 mg/100 ml) in combination, respectively. The maximum enzyme activity obtained was 33 ± 0.567 IU/ml.     

<Emphasis Type="Italic">E. coli</Emphasis>, <Emphasis Type="Italic">P. aeruginosa</Emphasis>, and <Emphasis Type="Italic">B. cereus</Emphasis> Bacteria Sterilization Using Afterglow of Non-Thermal Plasma at Atmospheric Pressure

We developed and employed a new geometrical structure of dielectric barrier discharge in atmospheric pressure for bacterial broad spectrum sterilization. We utilized a plasma source having an AC power supply at 50 HZ and 5,400 V (rms value). We prepared suspensions of the Gram-negative bacteria species (Escherichia coli, Pseudomonas aeruginosa) and a Gram-positive of Bacillus cereus with Luria–Bertani broth media up to OD_600 nm = 0.25 of McFarland standard. Afterglow of non-thermal atmospheric pressure plasma treated these suspensions. The influence of the atmospheric plasma afterglow on the species was assayed in different time durations 5, 10, and 15 min. The spectroscopic results of this investigation indicated that the survival reduction of the species can reach to 100% for P. aeruginosa in an exposure time of 10 min, E. coli and B. cereus in an exposure time of 15 min.     

Production and Stability of Protease from <Emphasis Type="Italic">Candida buinensis</Emphasis>

Cow raw milk from dairy cooperatives was examined for its microbial composition. Among the isolates identified, 17.6% were yeasts. The most frequent genus was Candida, although members belonging to the genera Brettanomyces, Dekkera, and Geotricum were also identified. Although qualitative and quantitative tests for extracellular proteolytic activity were positive for all the species isolated, Candida buinensis showed the highest response (23.5 U/mg); therefore, it was selected for subsequent investigation. The results of fermentations carried out at variable temperature, pH, and soybean flour concentration, according to a 2³ full factorial design, demonstrated that this yeast ensured the highest production of extracellular proteases (573 U/mL) when cultivated at 35 °C, pH 6.5, and using soybean flour concentrations in the range 0.1–0.5% (w/v). The cell-free supernatants showed the highest activity at 25 °C and pH 7.0, and satisfactory stability in the ranges 25–30 °C and pH 7–9. The first-order rate constants of protease inactivation in the cell-free supernatants were calculated at different temperatures from semi-log plots of the residual activity versus time and then used in Arrhenius and Eyring plots to estimate the main thermodynamic parameters of thermoinactivation (E* = 40.0 kJ/mol; ΔH* = 37.3 kJ/mol; ΔS* = −197.5 J/mol K; ΔG* = 101 kJ/mol).     

Determination of Kinetic Parameters in the Biosorption of Cr (VI) on Immobilized <Emphasis Type="Italic">Bacillus cereus</Emphasis> M<Superscript>1</Superscript><Subscript>16</Subscript> in a Continuous Packed Bed Column Reactor

Due to technological advancement, environment suffers from untreated toxic heavy metal bearing effluent coming from different industries. Chromium (VI) is one of those heavy metals having adverse impact on ecological balance, human, and plant health because of its carcinogenic properties. Biosorption is presented as an alternative to traditional technologies which are costly and inefficient for treatment of industrial wastes containing low amount of heavy metals. In this study, bioremediation of Cr (VI) ions by immobilized Bacillus cereus M¹ ₁₆ was investigated in a laboratory scale packed bed up-flow column reactor. The effect of important parameters, such as the inlet flow rate, influent concentration, and effective bed height, has been studied. External mass transfer, surface adsorption, and intrabead mass transfer were also studied to conclude the rate limiting step for removal of Cr (VI) and to determine the process parameters which are important for biosorption optimization. The external mass transfer coefficient was calculated at different flow rates (6.51 × 10⁻² to 7.58 × 10⁻² cm/min). Using the model, the surface adsorption rate constant (k _ad) and the intrabead mass transfer coefficient (k _i) were predicted as 0.0267 × 10⁻³ and 0.7465 × 10⁻³ l/g/min, respectively. Both are much lower than the external mass transfer coefficient (k _e). The surface adsorption phenomenon is acting as the rate-limiting step due to its high resistance for removal of Cr (VI).     

Recovery and Characterization of a Serine Collagenolytic Extract from Snow Crab (<Emphasis Type="Italic">Chionoecetes opilio</Emphasis>) By-products

Sequential acidic precipitation followed by a single chromatographic step (gel filtration) allowed the recovery of a collagenolytic fraction containing several proteases from by-products of snow crab (Chionoecetes opilio). The partial purification was particularly efficient to recover tryptic (purification fold = 1,352.5; yield = 110%) but also chymotryptic, elastolytic, and collagenolytic activities. A temperature of 40 °C and pH 8.0–8.5 were optimal for enzyme activity, which was stable for 2 h under these conditions. Calcium was not required for stability and thus activity. The isoelectric points of the protein components ranged from 3.7 to 4.6. Zymography revealed 29 and 48 kDa major components and others from 22 to 56 kDa. Enzymes were inhibited by PMSF and TLCK but were insensitive to TPCK. In view of these properties, the proteases likely belong to the serine collagenase group. Inhibition by EDTA could be due to a mechanism other than Ca²⁺ chelation. Using a food system (ground fish), the fraction was more proteolytic than a commercial bacterial protease, suggesting potential applications in enzymatic hydrolysis processes.     

The Technology of Extracting from Waste Mycelia

Chitin is one of the most abundant natural resources. chitosan is deacetylated from chitin. As natural organisms, chitosan is easier to be decomposed with organisms and eatable. So chitosan is wildly used in biology, medicine, foodstuff, cosmetics and so on[1,2] Chitin is a sort of natural glucosamine compound with wealthy resources, but a large amount of chitin is prepared from crab shell and crayfish shell. Some research works have carried on the preparation of chitosan from other resources, such as silkworm pupa, waste mycelia etc.[3,4]     

Ethanol Production from Cashew Apple Bagasse: Improvement of Enzymatic Hydrolysis by Microwave-Assisted Alkali Pretreatment

In this work, the potential of microwave-assisted alkali pretreatment in order to improve the rupture of the recalcitrant structures of the cashew able bagasse (CAB), lignocellulosic by-product in Brazil with no commercial value, is obtained from cashew apple process to juice production, was studied. First, biomass composition of CAB was determined, and the percentage of glucan and lignin was 20.54 ± 0.70% and 33.80 ± 1.30%, respectively. CAB content in terms of cellulose, hemicelluloses, and lignin, 19.21 ± 0.35%, 12.05 ± 0.37%, and 38.11 ± 0.08%, respectively, was also determined. Results showed that, after enzymatic hydrolysis, alkali concentration exerted influence on glucose formation, after pretreatment with 0.2 and 1.0 mo L⁻¹ of NaOH (372 ± 12 and 355 ± 37 mg g_glucan⁻¹) when 2% (w/v) of cashew apple bagasse pretreated by microwave-assisted alkali pretreatment (CAB-M) was used. On the other hand, pretreatment time (15–30 min) and microwave power (600–900 W) exerted no significant effect on hydrolysis. On enzymatic hydrolysis step, improvement on solid percentage (16% w/v) and enzyme load (30 FPU g_CAB-M⁻¹) increased glucose concentration to 15 g L⁻¹. The fermentation of the hydrolyzate by Saccharomyces cerevesiae resulted in ethanol concentration and productivity of 5.6 g L⁻¹ and 1.41 g L⁻¹ h⁻¹, respectively.     

Growth Inhibition and Induction of Stress Protein,GroEL, of <Emphasis Type="Italic">Bacillus cereus</Emphasis> Exposed to Antibacterial Peptide Isolated from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> SC-8

This study was conducted to investigate the antibacterial effect of BSAP-254 on Bacillus cereus with the induced stress proteins. The BSAP-254 is an antimicrobial peptide isolated from soybean-fermenting bacteria, Bacillus subtilis SC-8. It had a narrow spectrum of activity against B. cereus group. The growth inhibitory effect of BSAP-254 (50 μg/mL) reduced the population of B. cereus from >10⁸ to 10⁴ colony-forming units per milliliter within 30 min. In B. cereus exposed to BSAP-254, 14 intracellular proteins were differentially expressed as determined by 2-DE coupled with MS. Of the differentially expressed proteins identified, the stress protein GroEL, which is heat shock protein, was induced in B. cereus exposed to antibacterial peptide.     

Preparation and release characteristics of cisplatin albumin microspheres containing chitin and treated with chitosan

Cisplatin (CDDP) containing albumin microspheres and microcapsules incorporating biodegradable macromolecules, chitin and chitosan, were prepared, and their CDDP content and releasing ability and susceptibility to various enzymes were examined. Chitin was incorporated during preparation of the microspheres, while chitosan was used to treat preformed microspheres. CDDP content was remarkably increased by chitin; when chitin was incorporated at a concentration of 1.5%, the CDDP content of the microspheres was found to be 16.2% (1.8 times that with no addition of chitin). CDDP release was suppressed by chitin and chitosan. The 50% CDDP release time was about 1.5 h when no chitin was added, but about 16 h was required when chitin was incorporated into the microspheres at a concentration of 1.5%. Chitin and chitosan suppressed the decomposition by protease. The microspheres treated with 70% deacetylated chitosan showed the greatest susceptibility to lysozyme. In conclusion, CDDP release can be controlled by the use of chitin or chitosan, and the microspheres should show no immunogenicity in vivo because of their susceptibility to lysozyme.     

Gene Cloning,Overexpression, and Characterization of a Xylanase from <Emphasis Type="Italic">Penicillium</Emphasis> sp. CGMCC 1669

A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques. The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml⁻¹. After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40°C, was stable at acidic buffers of pH 4.5–9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and α-chymotrypsin). The specific activity, K _m, and V _max for oat spelt xylan substrate was 7,988 U mg⁻¹, 22.2 mg ml⁻¹, and 15,105.7 μmol min⁻¹ mg⁻¹, respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications.     

A Superoxide Dismutase Purified from the Rhizome of <Emphasis Type="Italic">Curcuma aeruginosa</Emphasis> Roxb. as Inhibitor of Nitric Oxide Production in the Macrophage-like RAW 264.7 Cell Line

Superoxide dismutase (SOD, EC 1.15.1.1) is a metalloenzyme or antioxidant enzyme that catalyzes the disproportionation of the harmful superoxide anionic radical to hydrogen peroxide and molecular oxygen. Due to its antioxidative effects, SOD has long been applied in medicinal treatment, cosmetic, and other chemical industries. Fifteen Zingiberaceae plants were tested for SOD activity in their rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Curcuma aeruginosa were found to contain a significant level of SOD activity. The SOD enzyme was enriched 16.7-fold by sequential ammonium sulfate precipitation, diethylaminoethyl cellulose ion exchange, and Superdex 75 gel filtration column chromatography. An overall SOD yield of 2.51 % with a specific activity of 812.20 U/mg was obtained. The enriched SOD had an apparent MW of 31.5 kDa, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a pH and temperature optima of 4.0 and 50 °C. With nitroblue tetrazolium and riboflavin as substrates, the K _m values were 57.31 ± 0.012 and 1.51 ± 0.014 M, respectively, with corresponding V _max values of 333.7 ± 0.034 and 254.1 ± 0.022 μmol min⁻¹ mg protein⁻¹. This SOD likely belongs to the Fe- or Mn-SOD category due to the fact that it was insensitive to potassium cyanide or hydrogen peroxide inhibition, but was potentially weakly stimulated by hydrogen peroxide, and stimulated by Mn²⁺and Fe²⁺ ions. Moreover, this purified SOD also exhibited inhibitory effects on lipopolysaccharide-induced nitric oxide production in cultured mouse macrophage cell RAW 264.7 in a dose-dependent manner (IC₅₀ = 14.36 ± 0.15 μg protein/ml).     

Effects of Radiofrequency Electromagnetic Wave Exposure from Cellular Phones on the Reproductive Pattern in Male Wistar Rats

The present study investigates the effect of free radical formation due to mobile phone exposure and effect on fertility pattern in 70-day-old male Wistar rats (sham exposed and exposed). Exposure took place in Plexiglas cages for 2 h a day for 35 days to mobile phone frequency. The specific absorption rate was estimated to be 0.9 W/kg. An analysis of antioxidant enzymes glutathione peroxidase (P < 0.001) and superoxide dismutase (P < 0.007) showed a decrease, while an increase in catalase (P < 0.005) was observed. Malondialdehyde (P < 0.003) showed an increase and histone kinase (P = 0.006) showed a significant decrease in the exposed group. Micronuclei also show a significant decrease (P < 0.002) in the exposed group. A significant change in sperm cell cycle of G₀–G₁ (P = 0.042) and G₂/M (P = 0.022) were recorded. Generation of free radicals was recorded to be significantly increased (P = 0.035). Our findings on antioxidant, malondialdehyde, histone kinase, micronuclei, and sperm cell cycle are clear indications of an infertility pattern, initiated due to an overproduction of reactive oxygen species. It is concluded that radiofrequency electromagnetic wave from commercially available cell phones might affect the fertilizing potential of spermatozoa.