A multi-tolerant low molecular weight mannanase from Bacillus sp. CSB39 and its compatibility as an industrial biocatalyst

Bacillus sp. CSB39, isolated from popular traditional Korean food (Kimchi), produced a low molecular weight, thermostable mannanase (MnCSB39); 571.14 U/mL using locust bean gum galactomannan as a major substrate. It was purified to homogeneity using a simple and effective two-step purification strategy, Sepharose CL-6B and DEAE Sepharose Fast Flow, which resulted in 25.47% yield and 19.32-fold purity. The surfactant-, NaCl-, urea-, and protease-tolerant monomeric protein had a mass of ∼30 kDa as analyzed by SDS-PAGE and galactomannan zymography. MnCSB39 was found to have optimal activity at pH 7.5 and temperature of 70 °C. The enzyme showed ˃55% activity at 5.0–15% (w/v) NaCl, and ˃93% of the initial activity after incubation at 37 °C for 60 min. Trypsin and proteinase K had no effect on MnCBS39. The enzyme showed ˃80% activity in up to 3 M urea. The N-terminal amino acid sequence, ALKGDGX, did not show identity with reported mannanases, which suggests the novelty of our enzyme. Activation energy for galactomannan hydrolysis was 26.85 kJmol⁻¹ with a K_cat of 142.58 × 10⁴ s⁻¹. MnCSB39 had K_m and V_max values of 0.082 mg/mL and 1099 ± 1.0 Umg⁻¹, respectively. Thermodynamic parameters such as ΔH, ΔG, ΔS, Q₁₀, ΔG_E-S, and ΔG_E-T supported the spontaneous formation of products and the high hydrolytic efficiency and feasibility of the enzymatic reaction, which strengthen its novelty. MnCSB39 activity was affected by metal ions, modulators, chelators, and detergents. Mannobiose was the principal end-product of hydrolysis. Bacillus subtilis CSB39 produced a maximum of 1524.44 U mannanase from solid state fermentation of 1 g wheat bran. MnCSB39 was simple to purify, was active at a wide pH and temperature range, multi-stress tolerant and catalyzes a thermodynamically possible reaction, characteristics that suggests its suitability for application as an industrial biocatalyst.     

Biochemical characteristics of phytases from fungi and the transformed microorganism

Five sources of phytases were used to study their biochemical characteristics. Phytase E was from an original Escherichia coli (E. coli), phytase PI and PG from the transformed Pichia pastoris (P. pastoris) with phytase gene of E. coli, phytase B and R from Aspergillus niger (A. niger). The results showed that the relative phytase activities had no significant changes when temperature was below 60 °C (P>0.05), and then decreased significantly with temperature increasing (P<0.01). The fungal phytase with the phytase gene from A. niger had the higher thermostability than the bacterial phytase with the phytase gene from E. coli; i.e. at 70 °C, 27–58% of phytase activity (compared with 30 °C) was retained for the bacterial phytase, and 73–96% for the fungal phytase; at 90 °C, 20–47% was retained for the bacterial phytase, and 41–52% for the fungal phytase, especially for the most thermostable phytase R (P<0.01). The optimum pH ranges were 3.0–4.5 for the bacterial phytases and 5.0–5.5 for the fungal phytases (P<0.01). When pH levels were 1, 7 and 8, only 3–7% of phytase activity (compared with the maximum phytase activity at a pH point) was retained for both bacterial and fungal phytases. The amount of inorganic P released from soybean meal was significantly increased when the levels of phytase activity in the soybean meal increased from 0 to 1.0 U/g soybean meal (P<0.01), except for phytase PI. The maximum P released was obtained at 1 U/g soybean meal for all five kinds of phytases (P<0.01). The most economical phytase concentration for P released was 0.25 U/g for phytase PI and B, and 0.50–1.0 U/g for phytase PG, E and R. In addition, the linear and non-linear regression models were established to estimate phytase activity and its characteristics very easily and economically.     

High-level production of biologically active chemokines in Escherichia coli

The chemokines eotaxin-1 (CCL11) and eotaxin-2 (CCL24), belonging to the CC chemokines family, play key roles in the inflammatory response, allergic asthma and other diseases. When expressed in Escherichia coli, chemokines are prone to form inclusion bodies devoid of biological activity, and it is hard to refold them properly. Here an expression and purification protocol for high-level production of soluble and biologically active CCL11 and CCL24 in E. coli has been established. A final yield of 8.7 mg/l for CCL11 and 3.9 mg/l for CCL24 has been obtained and the purified proteins were characterized with SDS-PAGE, mass spectrometry and circular dichroism. High binding affinity of purified chemokines with CC chemokine receptor type 3 (CCR3) has been confirmed with surface plasmon resonance (SPR) and the K_D values are 3.7 × 10⁻⁷ M and 3.0 × 10⁻⁷ M, respectively, for CCL11 and CCL24. This report provides a straightforward strategy for the efficient production of soluble and biologically active chemokines in E. coli.     

Optimal conditions for enhanced β-mannanase production by recombinant Aspergillus sojae

Optimization of the growth conditions for maximum β-mannanase production in shake flasks by using recombinant Aspergillus sojae ATCC11906 (AsT1) was carried out by Box–Behnken design of response surface methodology. The highest β-mannanase activity on the fourth day of cultivation at 30 °C was obtained as 363 U/ml in the optimized medium consisting of 7% sugar beet molasses, 0.43% NH₄NO₃, 0.1% K₂HPO₄ and 0.05% MgSO₄ (by weight per volume) at 207 rpm. On the sixth day of cultivation under the optimized conditions, the highest β-mannanase activity was achieved as 482 U/ml which is 1.4-fold of 352 U/ml activity found on glucose medium previously.     

Microbial surface displaying formate dehydrogenase and its application in optical detection of formate

In the present work, NAD⁺-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD⁺, and the production of NADH can be detected spectrometrically at 340 nm. After induction for 24 h in Luria-Bertani medium containing isopropyl-β-d-thiogalactopyranoside, over 80% of MBP-INP-FDH fusion protein present on the surface of E. coli cells. The cell-surface-displayed FDH showed optimal temperature of 50 °C and optimal pH of 9.0. Additionally, the cell-surface-displayed FDH retained its original enzymatic activity after incubation at 4 °C for one month with the half-life of 17 days at 40 °C and 38 h at 50 °C. The FDH activity could be inhibited to different extents by some transition metal ions and anions. Moreover, the E. coli cells expressing FDH showed different tolerance to solvents. The recombinant whole cell exhibited high formate specificity. Finally, the E. coli cell expressing FDH was used to assay formate with a wide linear range of 5–700 μM and a low limit of detection of 2 μM. It is anticipated that the genetically engineered cells may have a broad application in biosensors, biofuels and cofactor regeneration system.     

Selection and characterization of mannanase-producing bacteria useful for the formation of prebiotic manno-oligosaccharides from copra meal

In order to select bacterial strains effectively secreting mannanase activity for the production of prebiotic mannooligosaccharides, a two-step screening procedure was performed. Enriched cultures from isolation medium containing copra meal were primary screened on an isolation agar medium containing 1% locust bean gum (LBG), which resulted in 48 mannanase-producing bacterial isolates with significant clearing zones on the mannan-containing agar. However, only nine isolates showed appreciable mannanase activities against copra meal in their culture supernatants (0.054–0.185 U/mg of protein) as determined in a standard assay based on the detection of reducing sugars released from this substrate. The isolates CW2-3 and ST1-1 displayed the highest activity against LBG and copra meal, respectively. Copra mannan hydrolysates that were obtained by using crude mannanase from these nine isolates were further used for a secondary screening towards a growth-enhancing activity on Lactobacillus reuteri and inhibitory activity against Escherichia coli as well as Salmonella Enteritidis, resulting in 0.09–2.15 log CFU/ml enhancing activity and low inhibitory activity of 0.46–1.78 log CFU/ml as well as 0.37–1.72 log CFU/ml, respectively. The hydrolysate of CW2-3 mannanase showed the highest enhancing activity of 2.15 log CFU/ml while isolate ST1-1 was most effective with respect to growth inhibition against E. coli E010 and S. Enteritidis S003 with 0.76 and 1.61 log CFU/ml, respectively. Based on morphological, physical, biochemical and genetics properties, isolates CW2-3 and ST1-1 were identified as Klebsiella oxytoca and Acinetobacter sp., respectively. Crude mannanase activity from these two strains was characterized preliminarily. The pH optima of mannanase activity from Klebsiella oxytoca CW2-3 and Acinetobacter sp. ST1-1 were 7 and 6, respectively. The enzymes were stable at 4°C over a pH range of 3–6 and 3–10, respectively.     

A comparative study of different dyes for the detection of proteomes derived from Escherichia coli and MDCK cells: Sensitivity and selectivity

We compared sensitivity and selectivity of five dyes for detection of 2D PAGE-resolved proteins derived from Escherichia coli and MDCK cells. The sensitivity of these dyes was in the following order: SYPRO Ruby > Deep Purple > CBB-G250 > CBB-R250 > Colloidal Gold. Also, we report herein for the first time the application of Colloidal Gold (which is commonly used for staining proteins on blotted membranes) for in-gel staining of proteins. For E. coli, most of the dyes preferably detected proteins with pI range of 4.0–6.9, whereas Deep Purple preferably detected proteins with less acidic range (pI 5.0–7.9). For MDCK cells, while other dyes preferably stained proteins at pI range of 5.0–7.9, Colloidal Gold preferably stained more basic proteins (pI 7.0–9.9). This preferential staining property of Colloidal Gold to basic proteins was confirmed in SDS-PAGE-separated lysozyme (pI 9.4), compared to calmodulin (pI 4.0) and albumin (pI 6.0). These data provide useful information to select appropriate dyes for gel-based proteomic analysis of individual samples.     

Electrochemical analysis of autodisplayed adrenodoxin (Adx) on the outer membrane of E. coli

In this work, adrenodoxin (Adx) was expressed on the outer membrane of E. coli by autodisplay and then the iron–sulfur cluster was incorporated into apo-Adx by an anaerobic reconstitution process. For the determination of the redox potentials of the iron–sulfur clusters of the autodisplayed Adx, E. coli cells with autodisplayed Adx were immobilized on a gold electrode modified with a self-assembled monolayer of mercaptoundecanoic acid (MUA). From the repeated cyclic voltammetry (CV) analysis, the E. coli (10 mM HEPES buffer, pH 7.0) with autodisplayed Adx showed significant changes in shape with an oxidation peak at + 0.4 V (vs. Ag/AgCl) and a reduction peak at − 0.3 V (vs. Ag/AgCl) after the reconstitution process for the incorporation of the iron–sulfur cluster. From the repeated CV analysis in the reduction and oxidation potential ranges, the iron–sulfur clusters of the autodisplayed Adx were observed to undergo reversible redox reactions via direct electron transfer to the MUA-modified gold electrode.     

Enhanced mannanase production by submerged culture of Aspergillus niger NCH-189 using defatted copra based media

Copra and other mannans including locust bean gum, guar gum, konjac flour, copra and defatted copra were used to produce extracellular mannanase by shaken flask cultures of Aspergillus niger NCH-189 in this study. The best carbon source for mannanase production was defatted copra, which provided more nitrogen source and mannan content. The peak mannanase activity at 28 U ml⁻¹ was obtained on the day 3 at 30 °C, which was four times of those obtained from other carbon sources. Presence of oil in copra depressed the mannanase production of the fungus and the amount should be less than 3% (w/w). The copra suspension could be sequentially treated by boiling and refrigeration, followed by using n-hexane to remove copra oil.     

Intestinal Lactobacillus sp. is associated with some cellular and metabolic characteristics of blood in elderly people

The higher counts or particular groups (Firmicutes/Bacteroidetes) of intestinal microbiota are related to host metabolic reactions, supporting a balance of human ecosystem. We further explored whether intestinal lactobacilli were associated with some principal cellular and metabolic markers of blood in 38 healthy >65-year-old persons. The questionnaire, routine clinical and laboratory data of blood indices as much as the oxidized low-density lipoprotein (ox-LDL) and baseline diene conjugates in low-density lipoprotein (BDC-LDL) of blood sera were explored. The PCR-based intestinal Lactobacillus sp. composition and counts of cultivable lactobacilli (LAB) were tested. The facultative heterofermentative lactobacilli (Lactobacillus casei and Lactobacillus paracasei) were the most frequent (89 and 97%, respectively) species found, while Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus reuteri were present in almost half of the elderly persons. The number of species simultaneously colonizing the individuals ranged from 1 to 7 (median 4). In elderly consuming probiotics the LAB counts were significantly higher than in these not consuming (median 7.8, range 4.2–10.8 vs. median 6.3, range 3.3–9.7 log cfu/g; p = 0.005), adjusted (OR = 1.71, CI95 1.04–2.82; p = 0.035) for age and body mass index (BMI). The colonization by L. acidophilus was negatively related (r = ?0.367, p = 0.0275) to L. reuteri, staying significant after adjusting for age, sex and BMI (OR = 0.16, CI95 0.04–0.73; p = 0.018). However, the blood glucose concentration showed a tendency for a negative correlation for colonization with Lactobacillus fermentum (r = ?0.309, p = 0.062) adjusted for BMI (Adj. R² = 0.181; p = 0.013) but not for age and sex. The higher white blood cells (WBC) count was positively related (r = 0.434, p = 0.007) to presence of Lactobacillus reuteri adjusted for age, sex and BMI (Adj. R² = 0.193, p = 0.027). The lower values of ox-LDL were predicted by higher counts of cultivable lactobacilli adjusted by sex, age and BMI (r = ?0.389, p = 0.016; Adj. R² = 0.184 p = 0.029). In conclusion, the pilot study of elderly persons shows that the intestinal lactobacilli are tightly associated with WBC count, blood glucose and content of ox-LDL which all serve as risk markers in pathogenesis of inflammation, metabolic syndrome and cardiovascular disease (CVD).     

Biochemical properties of a β-mannanase and a β-xylanase produced by Ceriporiopsis subvermispora during biopulping conditions

One mannanase and one of the three xylanases produced by Ceriporiopsis subvermispora grown on Pinus taeda wood chips were characterized. A combination of ion exchange chromatography and SDS-PAGE data revealed the existence of a high-molecular-weight mannanase of 150 kDa that was active against galactoglucomannan and xylan. Its activity was optimal at pH 4.5. The K_m value with galactoglucomannan as substrate was 0.50 mg ml^?1. One xylanase with molecular mass of 79 kDa was also purified and characterized. Its activity was optimal at 60 °C and pH 8.0. Its K_m value with birchwood xylan as substrate was 1.65 mg ml^?1. Both the mannanase and the 79 kDa xylanase displayed relatively high activity on carboxymethyl cellulose. The sensitivity of the xylanase and mannanase to various salts was evaluated. None of the tested salts inhibited the xylanase, but Mn⁺², Fe⁺³, and Cu⁺² were strong inhibitors for the mannanase.     

Rational design,synthesis and biological evaluation of 1,3,4-oxadiazole pyrimidine derivatives as novel pyruvate dehydrogenase complex E1 inhibitors

On the basis of previous study on 2-methylpyrimidine-4-ylamine derivatives I, further synthetic optimization was done to find potent PDHc-E1 inhibitors with antibacterial activity. Three series of novel pyrimidine derivatives 6, 11 and 14 were designed and synthesized as potential Escherichia coli PDHc-E1 inhibitors by introducing 1,3,4-oxadiazole-thioether, 2,4-disubstituted-1,3-thiazole or 1,2,4-triazol-4-amine-thioether moiety into lead structure I, respectively. Most of 6, 11 and 14 exhibited good inhibitory activity against E. coli PHDc-E1 (IC₅₀ 0.97–19.21 μM) and obvious inhibitory activity against cyanobacteria (EC₅₀ 0.83–9.86 μM). Their inhibitory activities were much higher than that of lead structure I. 11 showed more potent inhibitory activity against both E. coli PDHc-E1 (IC₅₀ < 6.62 μM) and cyanobacteria (EC₅₀ < 1.63 μM) than that of 6, 14 or lead compound I. The most effective compound 11d with good enzyme-selectivity exhibited most powerful inhibitory potency against E. coli PDHc-E1 (IC₅₀ = 0.97 μM) and cyanobacteria (EC₅₀ = 0.83 μM). The possible interactions of the important residues of PDHc-E1 with title compounds were studied by molecular docking, site-directed mutagenesis, and enzymatic assays. The results indicated that 11d had more potent inhibitory activity than that of 14d or I due to its 1,3,4-oxadiazole moiety with more binding position and stronger interaction with Lsy392 and His106 at active site of E. coli PDHc-E1.     

Activity of recombinant GST in Escherichia coli grown on glucose and glycerol

We have investigated production, solubility and activity of recombinant glutathione-S-transferase (GST) expressed in Escherichia coli BL21 grown in defined media with glucose or glycerol as carbon source. GST was predominantly expressed as a soluble protein on both carbon sources, and 83–84% was found in the supernatant after cell lysis. In cultures grown on glucose, only 32 ± 9% of the GST was active, while 76 ± 13% of the GST was active in cultures grown on glycerol. This shows that glycerol has the potential to increase the activity of soluble GST in E. coli cultures in vivo.     

Structure and urovirulence characteristics of the fecal Escherichia coli population among healthy women

The fecal Escherichia coli population structure may influence the occurrence and etiology of extraintestinal infection, but is poorly understood. Accordingly, fecal E. coli from 39 healthy women (30 putative colonies per subject) were characterized for clonal identity, urinary tract infection-associated virulence traits, and phylogenetic background. The 120 unique E. coli clones (mean, three per sample) were distributed by phylogenetic group as follows: A (33%), D (31%), B1 (19%), and B2 (17%). However, 36% of women carried ≥1 clone from group B2, and 87% had clones from groups B2 and/or D. Of the B2 clones, 90% were from pauciclonal fecal samples (≤4 clones), compared with 47% and 52% of A and B1 clones (P = .001 and P = .007, respectively). Group B2 and D clones more often were dominant within the source sample than group A and B1 clones (60% vs. 41%: P = .05). Dominant clones exhibited higher virulence scores than non-dominant clones (mean 4.4 vs. 3.1: P = .015). In multilevel regression models, pauciclonal sample, B2, and clonal prevalence significantly predicted virulence score. In conclusion, within the intestinal E. coli population, virulence-associated traits, clonal prevalence, and low fecal clonal diversity are related. Virulence-associated traits of group B2/D E. coli may enhance fitness within the gut, thereby increasing strains’ likelihood of causing extraintestinal infection.     

Stereoselective synthesis of l-homophenylalanine using the carbamoylase method with in situ racemization via N-acylamino acid racemase

N-Acylamino acid racemase (NAAAR) gene of Deinococcus radiodurans BCRC12827 was cloned into expression vector pQE30 to generate pQE-naaar and expressed in recombinant Escherichia coli JM109. The expressed enzyme purified from the crude cell extract of IPTG-induced E. coli JM109 (pQE-naaar) exhibited high racemization activity to N-carbamoyl-l-homophenylalanine (NCa-l-HPA) and N-carbamoyl-d-homophenylalanine (NCa-d-HPA) with specific activities of 1.91 U/mg protein and 1.31 U/mg protein, respectively. To develop a recombinant E. coli whole cell system for the conversion of racemic NCa-HPA to l-homophenylalanine (l-HPA), naaar gene from D. radiodurans and l-N-carbamoylase (LNCA) gene from Bacillus kaustophilus BCRC11223 were cloned and coexpressed in E. coli cells. Recombinant cells treated with 0.5% toluene at 30 °C for 30 min exhibited enhanced NAAAR and LNCA activities, which are about 20- and 60-fold, respectively, higher than those of untreated cells. Using toluene-permeabilized recombinant E. coli cells, a maximal productivity of 7.5 mmol l-HPA/l h with more than 99% yield could be obtained from 150 mmol racemic NCa-HPA. Permeabilized cells also showed considerable stability in the bioconversion process using 10 mmol racemic NCa-HPA as substrate, no significantly decrease in conversion yield for l-HPA was found in the eight cycles.     

Development of a recombinant Escherichia coli-based biocatalyst to enable high styrene epoxidation activity with high product yield on energy source

A highly active recombinant whole-cell biocatalyst, Escherichia coli pETAB2/pG-KJE1, was developed for the efficient production of (S)-styrene oxide from styrene. The recombinant E. coli overexpressed styAB the genes of styrene monooxygenase of Pseudomonas putida SN1 and coexpressed the genes encoding chaperones (i.e., GroEL–GroES and DnaK–DnaJ–GrpE). The styrene monooxygenases were produced to ca. 40% of the total soluble proteins, enabling the whole-cell activity of the recombinant of 180 U/g CDW. The high StyAB activity in turn appeared to direct cofactors and molecular oxygen to styrene epoxidation. The product yield on energy source (i.e., glucose) reached ca. 40%. In addition, biotransformation in an organic/aqueous two-liquid phase system allowed the product to accumulate to 400 mM in the organic phase within 6 h, resulting in an average specific and volumetric productivity of 6.4 mmol/g dry cells/h (106 U/g dry cells) and 67 mM/h (1110 U/L_aq), respectively, under mild reaction conditions. These results indicated that the high productivity and the high product yield on energy source were driven by the high enzyme activity. Therefore, it was concluded that oxygenase activity of whole-cell biocatalysts is one of the critical factors to determine their catalytic performance.     

Co-autodisplay of Z-domains and bovine caseins on the outer membrane of E. coli

In this work, two proteins, Z-domains and bovine casein, were autodisplayed on the outer membrane of the same Escherichia coli cells by co-transformation of two different autodisplay vectors. On the basis of SDS-PAGE densitometry, Z-domains and bovine casein were expressed at 3.12 × 10⁵ and 1.55 × 10⁵ proteins/E. coli cell, respectively. The co-autodisplayed Z-domains had antibody-binding activity and the bovine casein had adhesive properties. E. coli with co-autodisplayed proteins were analyzed by fluorescence assisted cell sorting (FACS). E. coli with co-autodisplayed Z-domains and bovine casein aggregated due to hydrophobic interaction. For application to immunoassays, the Z-domain activity was estimated after (1) immobilizing the E. coli and (2) forming an OM layer. E. coli with co-autodisplayed two proteins that were immobilized on a polystyrene microplate had the same antibody-binding activity as did E. coli with autodisplayed Z-domains only. The OM layer from the co-transformed E. coli had Z-domains and bovine casein expressed at a 1:2 ratio from antibody-binding activity measurements.     

Comparison of two matrices for selective recovery of C595 diabody fragment (dbFv) from Escherichia coli lysates

A comparative study of the performance of two types of adsorbent (Streamline Quartz Base and Upfront Matrices), derivatized with the same affinity ligand (RPAP) to recover C595 diabody fragment (dbFv) from Escherichia coli lysates has been undertaken. Both streamline and Upfront Matrices are characterized by a particle size range of 100–300 μm. Streamline has a density of 1.20 g cm⁻³ and ligand concentration of 0.85 μmol ml⁻¹. Upfront has a density of 1.35 g cm⁻³ and ligand concentration of 0.83 μmol ml⁻¹. The release of C595 dbFv from E. coli cells was achieved by a chemical lysis method. The recovery performance of both adsorbents was evaluated in terms of operational productivity and elution yield of C595 dbFv in packed bed (clarified feedstock) and expanded bed (unclarified and clarified feedstock) chromatography systems. Streamline and Upfront adsorbents exhibited diabody operational productivities of 131 and 202 mg l⁻¹, respectively, with an elution yield of 92 and 94%, respectively, in packed bed operation. Streamline and Upfront adsorbents exhibited diabody operational productivities of 54.5 and 123.7 mg l⁻¹, respectively, with an elution yield of 89 and 92%, respectively, in expanded bed operation.     

In vivo effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression in striped catfish (Pangasianodon hypophthalmus)

Lipolysaccharide (LPS), a component of outer membrane protein of gram-negative bacteria, reportedly stimulates fish immune system. However, mechanisms driving this immunomodulatory effect are yet unknown. To determine effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression of striped catfish (Pangasianodon hypophthalmus), juvenile fish (20–25 g) were injected with 3, 15 or 45 mg E.coli LPS/kg and challenged with Edwardsiella ictaluri. Plasma cortisol and glucose were rather low and did not differ (p < 0.05) among treatments. All LPS treatments differed regarding blood cell count and immune variables such as plasma and spleen lysozyme, complement activity and antibody titer, 3 mg LPS/kg yielding best results; red blood cell count was not affected by LPS treatment. Accumulated mortalities after bacterial challenge were 23.4, 32.8, 37.7 and 52.5% for treatment 3, 15, 45 mg LPS/kg fish and control respectively. Proteomic analysis of peripheral blood mononuclear cells (PBMC) confirmed that LPS induced differentially over-expressed immune proteins such as complement component C3 and lysozyme C2 precursor. Regulation of other proteins such as Wap65, alpha-2 macroglobulin-3 and transferrin precursor was also demonstrated. Striped catfish injected with E.coli LPS enhanced innate immune responses.     

Simultaneous saccharification and fermentation of kraft pulp by recombinant Escherichia coli for phenyllactic acid production

Simultaneous saccharification and fermentation (SSF) of renewable cellulose for the production of 3-phenyllactic acid (PhLA) by recombinant Escherichia coli was investigated. Kraft pulp recovered from biomass fractionation processes was used as a model cellulosic feedstock and was hydrolyzed using 10–50 filter paper unit (FPU) g⁻¹ kraft pulp of a commercial cellulase mixture, which increased the glucose yield from 21% to 72% in an enzyme dose-dependent manner. PhLA fermentation of the hydrolyzed kraft pulp by a recombinant E. coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens TK1 produced 1.9 mM PhLA. The PhLA yield obtained using separate hydrolysis and fermentation was enhanced from 5.8% to 42% by process integration into SSF of kraft pulp (20 g L⁻¹) in a complex medium (pH 7.0) at 37 °C. The PhLA yield was negatively correlated with the initial glucose concentration, with a five-fold higher PhLA yield observed in culture medium containing 10 g L⁻¹ glucose compared to 100 g L⁻¹. Taken together, these results suggest that the PhLA yield from cellulose in kraft pulp can be improved by SSF under glucose-limited conditions.