Statistical optimization of solid state fermentation conditions for the enhanced production of thermoactive chitinases by mesophilic soil fungi using response surface methodology and their application in the reclamation of shrimp processing by-products

The mesophilic strains Aspergillus flavus CFR 10 and Fusarium oxysporum CFR 8 are potent producers of extracellular thermoactive chitinases (endo-chitinase and β-N-acetylhexosaminidase). Chitinases have a wide range of applications in many areas including reclamation of seafood processing chitinous by-products. In the present study, the interactive effects of four fermentation conditions on thermoactive chitinase production by solid state fermentation (SSF) using commercial wheat bran (CWB) was investigated employing response surface methodology (RSM). Further, these chitinases were applied for the preparation of N-acetyl chitooligosaccharides from shrimp chitin. Statistical optimization resulted in the production (unit/g initial dry substrate, U/g IDS) of 19.8 endo-chitinase and 649.0 β-N-acetylhexosaminidase activity by A. flavus CFR 10, and 17.5 endo-chitinase and 319.9 β-N-acetylhexosaminidase activity by F. oxysporum CFR 8. Activity of crude endo-chitinase and β-N-acetylhexosaminidase were found to be optimum at 62?±?1 °C in a wide pH range. Hydrolysis of colloidal chitin with crude chitinases produced the maximum N-acetyl chitooligosaccharides yield (mmol/l) of 10.4?±?0.28 at 6 h and 10.2?±?0.01 at 30 h post-reaction initiation, respectively, by the enzymes of A. flavus CFR 10 and F. oxysporum CFR 8. HPLC analysis revealed the presence of N-acetyl chitooligosaccharides with N-acetyl chitotriose as the main end product of the colloidal chitin hydrolysis. These results indicate the potential of mesophilic A. flavus CFR 10 and F. oxysporum CFR 8 in the production of thermoactive chitinases employing the economical SSF process using CWB as an ideal substrate, as well as the potential of these chitinases for the reclamation of abundant shrimp processing by-products and production of defined N-acetyl chitooligosaccharides.     

Optimization, purification and characterization of novel thermostable, haloalkaline, solvent stable protease from Bacillus halodurans CAS6 using marine shellfish wastes: a potential additive for detergent and antioxidant synthesis

A protease producing marine bacterium, Bacillus halodurans CAS6 isolated from marine sediments, was found to produce higher enzyme by utilizing shrimp shell powder. Optimum culture conditions for protease production were 50 °C, pH 9.0, 30 % NaCl and 1 % shrimp shell powder (SSP) and the protease purified with a specific activity of 509.84 U/mg. The enzyme retained 100 % of its original activity even at 70 °C, pH 10.0 and 30 % NaCl for 1 h. The purified protease exhibited higher stability when treated with ionic, non-ionic (72–94 %) and commercial detergents (76–88 %), and organic solvents (88–126 %). Significant blood stain removal activity was found with the enzyme in washing experiments. The culture supernatant supplemented with 1 % SSP showed 93.67 ± 2.52 % scavenging activity and FT-IR analysis of the reaction mixture confirmed the presence of antioxidants such as cyclohexane and cyclic depsipeptide with aliphatic amino groups. These remarkable qualities found with this enzyme produced by Bacillus halodurans CAS6 could make this as an ideal candidate to develop the industrial process for bioconversion of marine wastes and antioxidant synthesis.     

Optimization of protease production by Streptomyces sp. A6 using statistical approach for reclamation of shellfish waste

Protease producing Streptomyces sp. A6 was isolated from intertidal zone of the coast of Diu (Gujarat, India). Plackett–Burman method was applied to identify important factors (shrimp waste, FeCl₃, ZnSO₄ and pH) influencing protease production by Streptomyces sp. A6. Further optimization was done by response surface methodology using central composite design. The concentrations of medium components for higher protease production as optimized using the above approach were (g l^?1): Shrimp waste, 14; FeCl₃, 0.035; ZnSO₄, 0.065 and pH, 8.0. This statistical optimization approach led to production of 129.02 ± 2.03 U ml^?1 of protease which was 4.96 fold higher compared to that obtained using the unoptimized medium. The protease production was scaled to 3 l in a 5-l bench fermenter using optimized medium which further increased the production by 63.4%. Deproteinization and chitin recovery obtained at the end of fermentation was 85.12 ± 4.7 and 70.58 ± 1.33%, respectively. The present study is the first report on statistical optimization of medium components for production of protease by Streptomyces species using cheaper raw material such as shrimp waste. The study also explored the possibility Streptomyces sp. A6 for reclamation of shrimp wastes.     

Exiguobacterium himgiriensis sp. nov. a novel member of the genus Exiguobacterium, isolated from the Indian Himalayas

The taxonomic position of an orange coloured bacterium, strain K22–26^T isolated from a soil sample was studied using a polyphasic approach. The organism had phenotypic and chemotaxonomic properties consistent with its allocation into the genus Exiguobacterium. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain K22–26^T belongs to the genus Exiguobacterium and was related to Exiguobacterium aurantiacum DSM 6208^T (99.0 %) Exiguobacterium mexicanum DSM 16483^T (98.6 %), Exiguobacterium aquaticum (98.6 %), Exiguobacterium aestuarii DSM 16306^T (98.1 %), Exiguobacterium profundum DSM 17289^T (98.1 %) and Exiguobacterium marinum DSM 16483^T (97.9 %), whereas sequence similarity values with respect to other Exiguobacterium species with validly published names were between 92.5–94.0 %. The major polar lipids detected were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The major menaquinone was determined to be MK-7 (83 %) whereas MK-8 (11 %) and MK-6 (6 %) occur in smaller amounts. The peptidoglycan of the strain was found to contain l-lysine as the diagnostic diamino acid. The major fatty acids detected were iso C_13:0 (11.2 %), anteiso C_13:0 (15.4 %), iso C_15:0 (13.2 %) and iso C_17:0 (16.1 %). However, analysis of the DNA–DNA relatedness confirmed that strain K22–26^T belongs to a novel species. The G + C content of the strain K22–26^T was determined to be 50.1 mol %. The novel strain was distinguished from closely related type species of the genus Exiguobacterium using DNA–DNA relatedness and phenotypic data. Based on these differences, the strain K22–26^T should be classified as a novel species of the genus Exiguobacterium, for which the name Exiguobacterium himgiriensis sp. nov. strain K22–26^T (= MTCC 7628^T = JCM 14260^T) is proposed.     

Production and characterization of haloalkaline protease from ascidian-associated <Emphasis Type="Italic">Virgibacillus halodenitrificans</Emphasis> RSK CAS1 using marine wastes

A marine ascidian-associated bacterium, Virgibacillus halodenitrificans RSK CAS1, was optimized for protease production by response surface methodology using marine waste as substrate. The central composite design was employed, and the optimal medium constituents for maximum protease production (1461.11 U/ml) were determined to be shrimp shell powder (15.32 g/l), casein (5.37 g/l), MgSO₄ (3.0 g/l) and NaCl (55.31 g/l). The protease was purified from the culture supernatant to homogeneity in a three-step procedure consisting of ammonium sulfate precipitation, ion exchange chromatography (DEAE-cellulose column) and gel-filtration chromatography (Sephadex G-75 column), resulting in a 8.7-fold-change in purified protein. This protein had a specific activity of 1,086.78 U/mg and a molecular weight of 21 kDa. It exhibited optimal activity at 50 °C, pH 9 and 25 % NaCl. The significant stability of this protein at higher levels of salt, metal ions, organic solvents and commercial detergents and at higher, temperature, as well as its application as a cleaning additive in blood stain removal, suggests its possible use the laundry detergent industry.     

Production of xylanase by an alkaline-tolerant marine-derived Streptomyces viridochromogenes strain and improvement by ribosome engineering

Xylanase is the enzyme complex that is responsible for the degradation of xylan; however, novel xylanase producers remain to be explored in marine environment. In this study, a Streptomyces strain M11 which exhibited xylanase activity was isolated from marine sediment. The 16S rDNA sequence of M11 showed the highest identity (99 %) to that of Streptomyces viridochromogenes. The xylanase produced from M11 exhibited optimum activity at pH 6.0, and the optimum temperature was 70 °C. M11 xylanase activity was stable in the pH range of 6.0–9.0 and at 60 °C for 60 min. Xylanase activity was observed to be stable in the presence of up to 5 M NaCl. Antibiotic-resistant mutants of M11 were isolated, and among the various antibiotics tested, streptomycin showed the best effect on obtaining xylanase overproducer. Mutant M11-1(10) isolated from 10 μg/ml streptomycin-containing plate showed 14 % higher xylanase activities than that of the wild-type strain. An analysis of gene rpsL (encoding ribosomal protein S12) showed that rpsL from M11-1(10) contains a K88R mutation. This is the first report to show that marine-derived S. viridochromogenes strain can be used as a xylanase producer, and utilization of ribosome engineering for the improvement of xylanase production in Streptomyces was also first successfully demonstrated.     

Production of microsclerotia by Brazilian strains of Metarhizium spp. using submerged liquid culture fermentation

We investigated the potential production and desiccation tolerance of microsclerotia (MS) by Brazilian strains of Metarhizium anisopliae (Ma), M. acridum (Mc) and M. robertsii (Mr). These fungi were grown in a liquid medium containing 16 g carbon l^?1 with a carbon:nitrogen ratio of 50:1. One hundred milliliters cultures were grown in 250 ml Erlenmeyer flasks in a rotary incubator shaker at 28 °C and 200 rpm for 5 days. Five-day-old MS were harvested, mixed with diatomaceous earth (DE) and air-dried for 2 days at 30 °C. The air-dried MS–DE granular preparations were milled by mortar + pestle and stored in centrifuged tubes at either 26 or ?20 °C. Desiccation tolerance and conidia production were assessed for dried MS granules by measuring hyphal germination after incubation for 2 days on water agar plates at 26 °C and for conidia production following 7 days incubation. Yields of MS by all strains of Metarhizium were 6.1–7.3 × 10⁶ l^?1 after 3 days growth with maximum MS yields (0.7–1.1 × 10⁷ l^?1) after 5 days growth. No differences in biomass accumulation were observed after 3 days growth, whereas Ma-CG168 showed the highest biomass accumulation after 5 days growth. Dried MS–DE preparations of all fungal strains were equally tolerant to desiccation (≥93 % germination) and the highest conidia production was obtained by MS granules of Mc-CG423 (4 × 10⁹ conidia g^?1). All MS granules showed similar stability after storage at either 26 or ?20 °C for 3.5 months.     

Production and characterization of a solvent-tolerant protease from a novel marine isolate Bacillus tequilensis P15

Thirty-six proteolytic bacteria were isolated from the Jakhau coast, Kutch, India, amongst which isolate P15 identified as Bacillus tequilensis (JQ904626) was found to produce an extracellular solvent-- and detergent-tolerant protease (116.69?±?0.48 U/ml) and was selected for further investigation. Deoiled Jatropha seedcake (JSC) was found to be a suitable substrate for protease production under submerged condition. Upon optimization of process parameters following one-factor-at-a-time approach, an overall 6.4-fold (860.27?±?18.48 U/ml) increase in protease production was achieved. The maximum protease yield was obtained using a medium containing 2 % (w/v) deoiled JSC as substrate (pH of 8.0) upon 36 h of fermentation at 30 °C. The optimum temperature and pH for activity of B. tequilensis P15 protease was found to be 50 °C and 8.0, respectively. The enzyme exhibited a half-life of 190 min at 50 °C, which was enhanced to 270 min in presence of 5 mM Ca²⁺. The enzyme exhibited significant stability in almost all the solvents tested in the range of log P _ow varying from 8.8 to ?0.76. The enzyme activity was strongly inhibited by PMSF at 5 mM concentration, whereas the presence of EDTA (5 mM) and pCMB (5 mM) enhanced enzyme activity by 20.9 and 13.7 %, respectively. The enzyme was also found to be stable in the presence of surfactants, commercial detergents and bleach-oxidant (H₂O₂). This protease was demonstrated to be effective in removal of blood stains from fabrics, dehairing of hide, and stripping off the gelatin from used photographic films.     

Conversion and degradation of shellfish wastes by <Emphasis Type="Italic">Serratia</Emphasis> sp. TKU016 fermentation for the production of enzymes and bioactive materials

A chitosanase and a protease were purified from the culture supernatant of Serratia sp. TKU016 with shrimp shell as the sole carbon/nitrogen source. The molecular masses of the chitosanase and protease determined by SDS–PAGE were approximately 65 and 53 kDa, respectively. The chitosanase was inhibited completely by Mn²⁺, but the protease was enhanced by all of tested divalent metals. The optimum pH, optimum temperature, pH stability, and thermal stability of the chitosanase and protease were (pH 7, 50°C, pH 6–7, <50°C) and (pH 8–10, 40°C, pH 5–10, <50°C), respectively. SDS (2 mM) had stimulatory effect on TKU016 protease activity. The result demonstrates that TKU016 protease is SDS-resistant protease and probably has a rigid structure. Besides, TKU016 culture supernatant (2% SPP) incubated for 2 days has the highest antioxidant activity, the DPPH scavenging ability was about 76%. With this method, we have shown that shrimp shell wastes can be utilized and it’s effective in the production of enzymes, antioxidants, peptide and reducing sugar, facilitating its potential use in biological applications and functional foods.     

Production of tyrosinase by Auricularia auricula using low cost fermentation medium

Low cost fermentation media using agricultural by-products (wheat bran extract, rice bran extract and soybean meal extract) as a major nutrient source, were evaluated for the production of tyrosinase from the fungus Auricularia auricula in submerged culture. In single-factor experiments, three components (wheat bran extract, casein and CuSO₄) were chosen to further optimize medium composition using response surface methodology (RSM). The central composite experimental results showed the following optimum medium composition: wheat bran extract 36.0 %, casein 1.1 g/l and CuSO₄ 0.13 g/l. Under these conditions, the highest tyrosinase activity was 17.22 U/ml, which was 2.1 fold higher than that obtained using the non-optimized medium. The present study is the first to report the statistical optimization of medium composition for production of tyrosinase by A. auricula using cheaper wheat bran extract as a major nutrient source. These results might provide a reference for the development of a cost-effective medium for commercial production of tyrosinase.     

Cross-Species Induction and Enhancement of Antimicrobial Properties in Response to Gamma Irradiation in Exiguobacterium sp. HKG 126

A gram positive, extreme haloalkaliphilic, radioresistant bacterium was isolated from mangrove region of Kerala (India) which was characterized as Exiguobacterium sp. HKG-126 using morphological, physiological, biochemical and molecular characterization. Present investigation was undertaken to examine Exiguobacterium sp. as a potential source of broad-spectrum antimicrobial activity and enhancement in this activity was observed due to cross-species/cross-genera induction and also in response to high dose of gamma (γ) irradiation. Individual studies on the antimicrobial activity of all the co-cultivated bacterial strains before and after mixed culture fermentation, showed excellent enhancement in antimicrobial activity of Exiguobacterium sp. against a variety of clinical pathogens. To the best of our knowledge, this is the first report showing existence of an extremely high radioresistant strain of (up to 15 kGy) Exiguobacterium sp.     

Plant growth regulator induced phytochemical and antioxidant variations in micropropagated and acclimatized Eucomis autumnalis subspecies autumnalis (Asparagaceae)

Eucomis species is a valuable plant with both medicinal and horticultural potential. The current study evaluated the role of plant growth regulator (PGR) on growth, phytochemicals, and antioxidant activity in Eucomis autumnalis subspecies autumnalis. Five cytokinins including topolins and benzyladenine (BA) at 2 µM in combination with varying (0–15 µM) concentrations of naphthalene acetic acid (NAA) were tested. In vitro regenerants were acclimatized in the greenhouse for 4 months. Highest number of shoots (9 shoots/explant) was observed with 15 µM NAA alone or when combined with BA. Acclimatized plants derived from the 15 µM NAA treatment had the highest number of roots, largest leaf area and biggest bulbs. While applied PGRs increased the iridoids and condensed tannins in the in vitro regenerants, total phenolics and flavonoids were higher in the PGR-free treatment. Among the in vitro regenerants, 5 µM NAA and 2 µM BA treatments produced the best antioxidant activity in the DPPH (55 %) and beta-carotene (88 %) test systems, respectively. A remarkable carry-over effect of the PGR was conspicuous in the phytochemical levels and antioxidant activity of the 4-month-old plants. In addition to the optimized micropropagation protocols, the current findings present a promising potential for manipulating the type and concentration of applied PGRs to improve phytochemical production and hence medicinal value in E. autumnalis subspecies autumnalis.     

Effects of culture medium and auxins on growth of adventitious root cultures of Cuphea aequipetala Cav. and their ability to produce antioxidant compounds

Cuphea aequipetala Cav. (Lythraceae), a species highly valued for its medicinal properties, is threatened in the wild. To provide an alternative source of material for production of bioactive compounds, we established adventitious root cultures of C. aequipetala and determined their phenolic compounds contents and antioxidant activity. Cultures were initiated from root tips of in vitro C. aequipetala plantlets and were grown in B5 or SH culture medium containing either indole butyric acid (IBA) or α-naphthalene acetic acid at 0, 5 or 10 µM. The maximum root biomass (1.6 g/L dry mass (DM) per L medium) was recorded after 14 days of growth in B5 + 5 µM IBA. Roots in B5 medium remained green, whereas they tended to oxidize in SH medium. The highest contents of total phenolic compounds (9.1 ± 0.1 µg gallic acid equivalents/g DM) and flavonoids (37.5 ± 0.7 µg quercetin equivalents/g DM) were in roots grown in B5 + 5 µM IBA after 14 days of growth. Root cultures accumulated mainly flavan-3-ols, whereas roots or leaves from whole plants accumulated mainly flavonols. We analyzed the antioxidant properties of root extracts using in vitro assays. Roots grown in B5 medium showed stronger free-radical scavenging activity than that of roots grown in SH medium. Our results show that adventitious root cultures of C. aequipetala are a promising system for research on antioxidant compounds biosynthesis and for scaled-up production of useful biological materials.     

Fatty Acid Characterization and Biodiesel Production by the Marine Microalga <Emphasis Type="Italic">Asteromonas gracilis</Emphasis>: Statistical Optimization of Medium for Biomass and Lipid Enhancement

Lipid production is an important indicator for evaluating microalgal species for biodiesel production. In this study, a new green microalga was isolated from a salt lake in Egypt and identified as Asteromonas gracilis. The main parameters such as biomass productivity, lipid content, and lipid productivity were evaluated in A. gracilis, cultivated in nutrient-starved (nitrogen, phosphorous), and salinity stress as a one-factor-at-a-time method. These parameters in general did not vary significantly from the standard nutrient growth media when these factors were utilized separately. Hence, response surface methodology (RSM) was assessed to study the combinatorial effect of different concentrations of the abovementioned factor conditions and to maximize the biomass productivity, lipid content, and lipid productivity of A. gracilis by determining optimal concentrations. RSM optimized media, including 1.36 M NaCl, 1 g/L nitrogen, and 0.0 g/L phosphorus recorded maximum biomass productivity, lipid content, and lipid productivity (40.6 mg/L/day, 39.3%, and 15.9 mg/L/day, respectively) which agreed well with the predicted values (40.1 mg/L/day, 43.6%, and 14.6 mg/L/day, respectively). Fatty acid profile of A. gracilis was composed of C16:0, C16:1, C18:0, C18:3, C18:2, C18:1, and C20:5, and the properties of fuel were also in agreement with international standards. These results suggest that A. gracilis is a promising feedstock for biodiesel production.     

Production of green biocellulose nanofibers by Gluconacetobacter xylinus through utilizing the renewable resources of agriculture residues

The present study demonstrates the ability to produce green biocellulose nanofibers using the renewable resources of agriculture residues. Locally grown wheat straws (WS) were hydrolyzed under different conditions. Their hydrolysates were utilized to produce the nanofibers in separate hydrolysis fermentation process by Gluconacetobacter xylinus strain bacterium. Highest biocellulose production of ~10.6 g/L was achieved with samples that were enzymatically hydrolyzed. Moreover, acidic hydrolyzed WS produced up to 9.7 g/L, with total sugar concentrations in culture media of 43 g/L. Generally, enzymatic hydrolysis of WS resulted in more total sugar concentration than the acidic hydrolysis (i.e., 52.12 g/L), while water hydrolysis produced the least. This can be related to utilizing Xylanase in addition to Cellulase and Beta-glucosidase that helps to hydrolyse WS dry basis of cellulose and hemicelluloses. Sugar mixtures produced under all hydrolysis conditions were mainly composed of glucose and xylose with average percentages of 56 and 28 %, respectively. Acidic hydrolysis at higher acid concentration, as well as soaking WS in the acidic solution for longer time, improved the total sugar concentration in the culture media by 18 %. Conducting thermal treatment at more intense conditions of higher temperature or heating time improved the total sugar produced with acidic hydrolysis. These conditions, however, resulted in further production of furfural, which considerably affected bacterial cells proliferation. This resulted in lowest sugar consumption in the range of 62–64 % that affected final BC production.     

Both Decrease in ACL1 Gene Expression and Increase in ICL1 Gene Expression in Marine-Derived Yeast Yarrowia lipolytica Expressing INU1 Gene Enhance Citric Acid Production from Inulin

In this study, some of the ATP-citrate lyase genes (ACL1) were deleted and the copy number of the iso-citrate lyase gene (ICL1) was increased in the marine-derived yeast Yarrowia lipolytica SWJ-1b displaying the recombinant inulinase. It was found that lipid content and iso-citric acid in the transformant 30 obtained were greatly reduced and citric acid production was greatly enhanced. It was also found that the ACL1 gene expression and ATP-citrate lyase activity in the transformant 30 were declined and the ICL1 gene expression and iso-citrate lyase activity were promoted. During the 2-l fermentation, 84.0 g/l of citric acid and 1.8 g/l of iso-citric acid in the fermented medium were attained from 10.0 % of inulin by the transformant 30 within 214 h. The results showed that only 0.36 % of the residual reducing sugar and 1.0 % of the residual total sugar were left in the fermented medium, suggesting that 89.6 % of the total sugar was used for citric acid production and cell growth by the transformant 30.     

Medium Optimization for Improved Production of Dihydrolipohyl Dehydrogenase from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> PAD-91 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Dihydrolipohyl dehydrogenase (DLD) is a FAD-dependent enzyme that catalyzes the reversible oxidation of dihydrolipoamide. Herein, we report medium optimization for the production of a recombinant DLD with NADH-dependent diaphorase activity from a strain of Bacillus sphaericus PAD-91. The DLD gene that consisted of 1413 bp was expressed in Escherichia coli BL21 (DE3), and its enzymatic properties were studied. The composition of production medium was optimized using one-variable-at-a-time method followed by response surface methodology (RSM). B. sphaericus DLD catalyzed the reduction of lipoamide by NAD⁺ and exhibited diaphorase activity. The molecular weight of enzyme was about 50 kDa and determined to be a monomeric protein. Recombinant diaphorase showed its optimal activity at temperature of 30 °C and pH 8.5. K _m and V _max values with NADH were estimated to be 0.025 mM and 275.8 U/mL, respectively. Recombinant enzyme was optimally produced in fermentation medium containing 10 g/L sucrose, 25 g/L yeast extract, 5 g/L NaCl and 0.25 g/L MgSO₄. At these concentrations, the actual diaphorase activity was calculated to be 345.0 ± 4.1 U/mL. By scaling up fermentation from flask to bioreactor, enzyme activity was increased to 486.3 ± 5.5 U/mL. Briefly, a DLD with diaphorase activity from a newly isolated B. sphaericus PAD-91 was characterized and the production of recombinant enzyme was optimized using RSM technique.     

Identification and molecular characterization of a C-type lectin-like protein from Chinese shrimp (Fenneropenaeus chinensis)

A C-type lectin-like protein was cloned and characterized from the Chinese shrimp Fenneropenaeus chinensis, and named as FcCTL. The results indicated that the full length cDNA of 859 bp had an open reading frame encoded a polypeptide of 220 amino acids with one carbohydrate-recognition domain, six conserved Cys and one key motif EPGD. The theoretical molecular weight and pI of mature protein was 25.3 kDa and 5.4. Sequence comparison of the deduced amino acid sequence of FcCTL showed varied identity of 26–34, 34, 31 and 30 % with those of F. chinensis, Portunus trituberculatus, Tetraodon nigroviridis, Penaeus monodon, respectively. qRT-PCR analysis indicated that FcCTL was expressed highest in hepatopancreas of normal shrimp, and it’s expression was up-regulated in hepatopancreas and gills post white spot syndrome virus challenge. The purified recombinant FcCTL showed higher antimicrobial activity against Gram-positive bacteria than against Gram-negative bacteria and fungi. And the hemagglutinating activity of rFcCTL could be completely inhibited by GlcNAc (5 μg/ml), LPS (2.5 μg/ml), d-galactose (100 mM) and maltose (100 mM). These data suggested that FcCTL might play an important role in shrimp immune and would be helpful to better understand the innate immunity mechanism of shrimp.     

Production of 3-O-xylosyl quercetin in Escherichia coli

Quercetin, a flavonol aglycone, is one of the most abundant flavonoids with high medicinal value. The bioavailability and pharmacokinetic properties of quercetin are influenced by the type of sugars attached to the molecule. To efficiently diversify the therapeutic uses of quercetin, Escherichia coli was harnessed as a production factory by the installation of various plant and bacterial UDP-xylose sugar biosynthetic genes. The genes encoding for the UDP-xylose pathway enzymes phosphoglucomutase (nfa44530), glucose-1-phosphate uridylyltransferase (galU), UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) were overexpressed in E. coli BL21 (DE3) along with a glycosyltransferase (arGt-3) from Arabidopsis thaliana. Furthermore, E. coli BL21(DE3)/?pgi, E. coli BL21(DE3)/?zwf, E. coli BL21(DE3)/?pgi?zwf, and E. coli BL21(DE3)/?pgi?zwf?ushA mutants carrying the aforementioned UDP-xylose sugar biosynthetic genes and glycosyltransferase and the galU-integrated E. coli BL21(DE3)/?pgi host harboring only calS8, calS9, and arGt-3 were constructed to enhance whole-cell bioconversion of exogeneously supplied quercetin into 3-O-xylosyl quercetin. Here, we report the highest production of 3-O-xylosyl quercetin with E. coli BL21 (DE3)/?pgi?zwf?ushA carrying UDP-xylose sugar biosynthetic genes and glycosyltransferase. The maximum concentration of 3-O-xylosyl quercetin achieved was 23.78 mg/L (54.75 μM), representing 54.75 % bioconversion, which was an ~4.8-fold higher bioconversion than that shown by E. coli BL21 (DE3) with the same set of genes when the reaction was carried out in 5-mL culture tubes with 100 μM quercetin under optimized conditions. Bioconversion was further improved by 98 % when the reaction was scaled up in a 3-L fermentor at 36 h.     

Bio-ethanol Production from Green Onion by Yeast in Repeated Batch

Considered to be the cleanest liquid fuel, bio-ethanol can be a reliable alternative to fossil fuels. It is produced by fermentation of sugar components of plant materials. The common onions are considered to be a favorable source of fermentation products as they have high sugar contents as well as contain various nutrients. This study focused on the effective production of ethanol from Green onion (Allium fistulosum L.) by the yeast “Saccharomyces cerevisiae” in repeated batch. The results showed that the total sugar concentration of onion juice was 68.4 g/l. The maximum rate of productivity, ethanol yield and final bio-ethanol percentage was 7 g/l/h (g ethanol per liter of onion juice per hour), 35 g/l (g ethanol per liter of onion juice) and 90 %, respectively.