High yield preparation of ganglioside GM1 using recombinant sialidase from Cellulosimicrobium cellulans

Cellulosimicrobium cellulans employs extracellular sialidase to selectively convert polysialogangliosides to ganglioside GM1. We cloned this novel sialidase gene (ccsia) from C. cellulans sp. 21, and overexpressed recombinant sialidase (CcSia) protein in E. coli BL21 (DE3) by high cell density fermentation. The presence of an N-terminal hexa-His tag allowed for purification using nickel affinity chromatography (2.3-fold, specific activity 41.5 U/mg). As determined by gel electrophoresis and gel filtration chromatography, the molecular weight of CcSia was found to be about 75 kDa, consistent with sequence analysis (75,271 Da). CcSia transformed polysialogangliosides GD1a, GD1b and GT1b into GM1. For this reaction, the response surface approach showed that optimal conditions in a 1-L system were 2 h incubation at 32.5 °C and pH 5.2, with substrate concentrations of 10 g/L and crude enzyme concentration 1 g/L, respectively. Under above conditions, 10 g/L of ganglioside was completely converted to the product GM1 with a yield of 52%. Our studies demonstrate CcSia could be used for industrial preparation of ganglioside GM1 by the pharmaceutical industry.     

Identification of dipeptidyl peptidase 3 as the Angiotensin-(1–7) degrading peptidase in human HK-2 renal epithelial cells

Angiotensin-(1–7) (Ang-(1–7)) is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic and pro-oxidant effects of the Ang II-AT₁ receptor axis. We previously identified a peptidase activity from sheep brain, proximal tubules and human HK-2 proximal tubule cells that metabolized Ang-(1–7); thus, the present study isolated and identified the Ang-(1–7) peptidase. Utilizing ion exchange and hydrophobic interaction chromatography, a single 80 kDa protein band on SDS-PAGE was purified from HK-2 cells. The 80 kDa band was excised, the tryptic digest peptides analyzed by LC–MS and a protein was identified as the enzyme dipeptidyl peptidase 3 (DPP 3, EC: 3.4.14.4). A human DPP 3 antibody identified a single 80 kDa band in the purified enzyme preparation identical to recombinant human DPP 3. Both the purified Ang-(1–7) peptidase and DPP 3 exhibited an identical hydrolysis profile of Ang-(1–7) and both activities were abolished by the metallopeptidase inhibitor JMV-390. DPP 3 sequentially hydrolyzed Ang-(1–7) to Ang-(3–7) and rapidly converted Ang-(3–7) to Ang-(5–7). Kinetic analysis revealed that Ang-(3–7) was hydrolyzed at a greater rate than Ang-(1–7) [17.9 vs. 5.5 nmol/min/μg protein], and the Km for Ang-(3–7) was lower than Ang-(1–7) [3 vs. 12 μM]. Finally, chronic treatment of the HK-2 cells with 20 nM JMV-390 reduced intracellular DPP 3 activity and tended to augment the cellular levels of Ang-(1–7). We conclude that DPP 3 may influence the cellular expression of Ang-(1–7) and potentially reflect a therapeutic target to augment the actions of the peptide.     

Bioprocess development of the production of the mutant P-219-L human d-amino acid oxidase for high soluble fraction expression in recombinant Escherichia coli

In order to examine the structure–activity relationship and the substrate specificity of human d-amino acid oxidase (h.DAO), a single amino acid mutation had been established as proline-219-luecine (P-219-L). The gene encoding mutant h.DAO has been cloned and expressed in Escherichia coli BL21 (DE3). It was observed that the host cell was negatively affected by the expressed mutant h.DAO, resulting in a remarkable decrease in the cell growth and consequently the amount of the produced enzyme. To overcome this problem, we investigated several factors that may affect the cell growth rate and the mutant h.DAO production such as optimization of the glucose concentration as a main carbon source and the yeast extract concentration as a main nitrogen source, optimization of dissolved oxygen (DO%) concentration and the addition of benzyl alcohol (BA, which can artificially induce a strong heat shock response at low temperature), to enhance the production of natively folded soluble fraction of the recombinant protein. These parameters were tested on both shake flask level and fed-batch bioreactor level. The Western blot analysis and the enzyme activity assay indicated the higher level of the mutant expression towards enhancement of the conditions by using our designed approach.The specific activity (which was used as an indicator for the level of the desired protein produced = U/mg protein) and the OD_600 nm of the host cells (which was used as an indicator for the cell growth), reached to be 0.061 U/mg protein and 3.44, respectively upon using fed-batch culture system containing the optimized medium composition (15 g/l glucose and 5 g/l yeast extract). While upon using the shake flask level, these values were 0.032 and 1.1, respectively. Enhancement of the cell growth and the enzyme production was noticed after DO% optimization upon using 500 rpm agitation speed and 1.8 v.v.m. (volume volume minute) aeration. The specific activity for the mutant enzyme and the OD_600 nm of the host cells reached to be 0.14 U/mg protein and 7.1, respectively. Finally upon using the optimized culture composition (15 g/l glucose and 5 g/l yeast extract), optimized DO% (using 500 rpm agitation speed and 1.8 v.v.m.) and 0.1 mM BA at the fed-batch bioreactor level, the specific activity and the OD_600 nm of the host cells increased significantly to be 0.21 U/mg protein and 11.3, respectively at 24 h culture. These results indicate the importance of our approaches to overproducing mutant h.DAO in soluble form in E. coli.     

Bio-reduction of hexachloroplatinic acid to platinum nanoparticles employing Acinetobacter calcoaceticus

Acinetobacter calcoaceticus PUCM 1011 efficiently synthesized platinum nanoparticles (PtNP) of size 2–3 nm intracellularly when challenged with hexachloroplatinic acid. Salt concentration (1 mM), temperature (30 °C), pH (7) and incubation period (72 h) influenced the efficiency of monodisperse cuboidal PtNP synthesis. Resolution of ordered lattice fringes with “d” value of 0.23 nm corresponding to (1 1 1) plane and EDAX confirmed presence of metallic platinum. AFM, TEM and HR-TEM confirmed synthesis of PtNP and its effect on cell viability. Total cell protein profile for 120 h with an interval of 24 h after PtNP synthesis revealed prominent four protein bands (97, 66, 43 and 29 kDa) when compared to control. Combinations of three proteins initiated PtNP synthesis within 4 h in range of 1–4 nm and few in picometers under HR-TEM. This is the first report of PtNP synthesis employing whole cell and total cell protein of A. calcoaceticus.     

Iron containing keratinolytic metallo-protease produced by Chryseobacterium gleum

Chryseobacterium gleum exhibited complete dissolution of whole chicken-feathers (10 g l^?1, pH 8) after 72 h at 30 °C through synthesis of keratinolytic protease when inoculated at 1% (v/v). This enzyme was purified to 67-fold with yield of 2.25% having a specific activity of 1670 U mg^?1 and ～36 kDa Mw. MALDI-TOF MS of this keratinase showed some similarity with the keratinase peptides of Bacillus subtilis (BOFXJ2). The keratinase action was inhibited by EDTA, iodoacetamide and metal ions like mercury, copper and zinc (1 mM each), while it was enhanced by iron and calcium. Keratinase showed presence of 3 mM of Fe M^?1 as tested by atomic absorption spectroscopy and addition of Fe in its apoenzyme retained about 79% of original residual feather degradation activity which portrayed it to be metalloprotease. Purified keratinase revealed significant degradation (85%) of feather concentrate (20 g l^?1) to 3.9 μM ml^?1 of free amino groups in 24 h at an initial pH of 8.0, 30 °C and 120 rpm shaking. This keratinase activity can be controlled precisely by presence of chemical or metal ions which could be of use in biotechnology industry while the culture can be used in poultry waste management.     

Extracellular expression of a thermostable phytase (phyA) in Kluyveromyces lactis

Functional expression of a thermostable phytase from A. niger was achieved in Kluyveromyces lactis GG799 cells. Effective secretion of recombinant enzyme (198 U ml⁻¹) in the fermentation broth at 72 h incubation at 22 °C was obtained. Purified enzyme showed a specific activity of 72 U mg⁻¹) and was detected on SDS-PAGE as a heavily glycosylated protein with a molecular weight of ≥140 kDa. Optimum temperature of the enzyme was at 55 °C and it showed a characteristic bi-hump pH profile with two pH optima (at pH 2.5 and 5.5). Enzyme showed considerable pepsin resistance with 60% activity retention after incubation with pepsin at the ratio of 1:1000. Enzyme was thermostable retaining 69 and 37% activity at 90 and 100 °C for 10 min respectively and remained active at these temperatures till 1 h. Deglycosylation studies demonstrated negligible effect of N-linked glycans on thermal properties. Multiple sequence alignment data revealed a conserved Asn at position 345 of this phytase which might contribute to its thermal properties. This thermostable phytase coupled with its noticeable protease resistance could be a better alternative to current commercial phytases.     

Effect of different levels of concentrate allowances on rumen fluid pH,nutrient digestion,nitrogen retention and growth performance of weaner lambs

This experiment was conducted for 90 d to assess the effect of feeding graded levels of concentrate allowance on rumen fermentation characteristics, performance and nutrient utilisation of weaner lambs on restricted or high concentrate allowance using 60 weaner lambs of initial average live weight of 13.90 kg BW in a randomized design. The experimental treatments were 15 or 25 g kg⁻¹ BW or ad libitum concentrate allowance. Roughage source which contained Khejri (Prosopis cineratia) and Siris (Albizia lebback) leaves in 50:50 ratio was offered ad libitum to all the animals. Lambs supplemented with 15 g or ad libitum concentrate had similar dry matter intake (4.2 kg/100 kg BW) but significantly (p < 0.01) lower than 25 g concentrate supplemented group (4.9 kg/100 kg BW). Organic matter and CP intakes increased with increasing concentrate supplementation. Apparent digestibilities of dry matter, organic matter, CP, NDF, ADF and cellulose were significantly (p < 0.01) higher in ad libitum concentrate supplemented than 15 and 25 g concentrate supplemented lambs. Daily ME intake was significantly (p < 0.05) higher in 25 g and ad libitum concentrate supplemented lambs while ME intake kg⁻¹ gain was lower in ad libitum concentrate supplemented lambs (57 MJ kg⁻¹ gain) than those supplemented with 15 or 25 g concentrate (91 MJ kg⁻¹ gain). Generally, average daily gain increased with increasing levels of concentrate supplementation. Ad libitum concentrate supplemented lambs had significantly (p < 0.01) higher daily gains (151 g) than 15 and 25 g concentrate supplemented lambs (77 and 98 g, respectively). Feed efficiency was similar for 15 and 25 g concentrate supplemented lambs but significantly (p < 0.01) lower than the ad libitum concentrate supplemented lambs. All animals were in positive N-balance and the N-balance increased with increasing concentrate supplementation. Mean rumen fluid pH was significantly (6.6, p < 0.01) lower in ad libitum concentrate supplemented lambs compared to 15 or 25 g concentrate fed lambs (6.9). Rumen NH₃-N and total-N-concentrations peaked at 3 h post-feeding. Optimum rumen fluid pH, better nutrient digestibilities, higher N-retention improved growth by 49% of ad libitum concentrate fed lambs.     

Efficient cloning and expression of a thermostable nitrile hydratase in Escherichia coli using an auto-induction fed-batch strategy

A nitrile hydratase (NHase) gene from Aurantimonas manganoxydans was cloned and expressed in Escherichia coli BL21 (DE3). A downstream gene adjacent to the β-subunit was necessary for the functional expression of the recombinant NHase. The structural gene order of the Co-type NHase was α-subunit beyond β-subunit, different from the order typically reported for Co-type NHase genes. The NHase exhibited adequate thermal stability, with a half-life of 1.5 h at 50 °C. The NHase efficiently hydrated 3-cyanopyridine to produce nicotinamide. In a 1-L reaction mixture, 3.6 mol of 3-cyanopyridine was completely converted to nicotinamide in four feedings, exhibiting a productivity of 187 g nicotinamide/g dry cell weight/h. An industrial auto-induction medium was applied to produce the recombinant NHase in 10-L fermenter. A glycerol-limited feeding method was performed, and a final activity of 2170 U/mL culture was achieved. These results suggested that the recombinant NHase was efficiently cloned and produced in E. coli.     

Expression and purification of 15 kDa granulysin utilizing an insect cell secretion system

Granulysin is an antimicrobial and proinflammatory protein expressed in activated human T cells and natural killer cells. A single mRNA produces the 15 kDa isoform which is then cleaved at the amino and carboxy termini to produce the 9 kDa isoform. Recombinant 9 kDa granulysin has been studied in detail but little is known about the function of the 15 kDa isoform, and no protocol has been published describing expression and purification of this form. Two commercially available preparations of the recombinant 15 kDa granulysin contain tags that may affect function. Here we describe for the first time a method to produce 15 kDa granulysin as a secreted protein from insect cells. The 15 kDa granulysin is purified using a HiTrap Heparin column and a Resource S column. A typical a yield of purified 15 kDa granulysin is 0.6 mg/L of insect cell supernatant.     

Superoxide dismutases from larvae of the camel tick Hyalomma dromedarii

Three superoxide dismutases (EC 1.15.1.1) (TLSOD1, TLSOD2 and TLSOD3) were purified from larvae of the camel tick Hyalomma dromedarii by ammonium sulfate precipitation, ion exchange and gel filtration columns. SDS-PAGE revealed that the subunit molecular masses of the SODs are 40 ± 2 kDa, 67 ± 1.5 kDa and 45 ± 2.6 kDa for TLSOD1, TLSOD2 and TLSOD3, respectively. TLSOD1 and TLSOD2 are monomeric proteins, while TLSOD3 isoenzyme exhibits dimeric structure with native molecular mass of 90 kDa. The pI values are estimated at pH 8.0, pH 7.2 and pH 6.6 for the three SODs which displayed pH optima at 7.6, 8.0 and 7.8, respectively. CuCl₂ and ZnCl₂ increase the activity of TLSOD2 and TLSOD3, while MnCl₂ increases the activity of TLSOD1. KCN inhibits the activity of TLSOD2 and TLSOD3, while a remarkable resistance of TLSOD1 isoenzyme was detected. TLSOD1 is suggested to be a manganese containing isoenzyme while TLSOD2 and TLSOD3 are suggested to be copper/zinc-containing isoenzymes. These results indicate the presence of three different forms of SODs in the larval stage of camel tick. This finding will contribute to our understanding of the physiology of these ectoparasites and the development of non-traditional methods to control them.     

Antimicrobial activity against Shigella sonnei and probiotic properties of wild lactobacilli from fermented food

Four lactobacilli strains (Lactobacillus paracasei subp. paracasei M5-L, Lactobacillus rhamnosus J10-L, Lactobacillus casei Q8-L and L. rhamnosus GG (LGG), were systematically assessed for the production of antimicrobial substances active towards Shigella sonnei, Escherichia coli and Salmonella typhimurium. Agar-well assay showed that the four lactobacilli strains displayed strong antibacterial activity towards S. sonnei. The nature of antimicrobial substances was also investigated and shown to be dependent on the production of organic acids, in particular the lactic acid. Time-kill assay showed that the viability of the S. sonnei was decreased by 2.7–3.6 log CFU/ml after contact with CFCS (cell-free culture supernatants) of four lactobacilli for 2 h, which confirmed the result of the agar-well assay. Further analysis of the organic acid composition in the CFCS revealed that the content of lactic acid range from 227 to 293 mM. In addition, the aggregations properties, adherence properties and tolerance to simulated gastrointestinal conditions were also investigated in vitro tests. The result suggested that the M5-L, J10-L and Q8-L strains possess desirable antimicrobial activity towards S. sonnei and probiotic properties as LGG and could be potentially used as novel probiotic strains in the food industry.     

Expression,purification and partial characterization of a xanthine oxidase (XOD) in Arthrobacter sp.

A xanthine oxidase (XOD) was expressed, purified and partially characterized from Arthrobacter sp. with a negative immune protocol. To determine the optimal inducer for XOD, xanthine, hypoxanthine and uric acid were added into the medium of cultivation. The results revealed that with the inducement of about 14 mM xanthine, the highest XOD activity could be detected. To separate XOD from Arthrobacter sp., the cells were first cultured without any inducement; then the total proteins of the collected cells were extracted and immunized to rabbits for the polyclonal antibodies. These antibodies were then coupled with sepharose CL 6 B, and the medium was further employed to deplete most of the cells’ back ground proteins. Began with ～20 mg crude protein from disrupted cells was subjected to the antibody medium, and ～1.45 mg protein was detected in unbinding fractions with ～92.0% of activity. The extracted xanthine oxidase was ～85% pure with native-PAGE analysis, and ～90% pure with SDS-PAGE analysis, the yield of protein was ～7.4%. The specific activity of the enzyme was 36.0 U/mg. The native enzyme should be a dimer (～280 kDa) of a protein composed with two different peptides with the mass of approximately 55.5 and 85.5 kDa, respectively. The optimal pH and temperature of this enzyme were determined at about pH 7 and 50 °C. Furthermore, EDTA revealed almost no influences on the activity.     

Isolation and antiproliferative activity of Lotus corniculatus lectin towards human tumour cell lines

The objective of the study was to investigate the anti cancer activity of a lectin isolated from Lotus corniculatus seeds. A tetrameric 70 kDa galactose specific lectin was purified using two step simple purification protocol which involved affinity chromatography on AF-BlueHC650M and gel filtration on Sephadex G-100. The lectin was adsorbed on AF-BlueHC650M and desorbed using 1 M NaCl in the starting buffer. Gel filtration on Sephadex G-100 yielded a major peak absorbance that gave two bands of 15 kDa and 20 kDa in SDS PAGE. Hemagglutination activity was completely preserved, when the temperature was in the range of 20–60 °C. However, drastic reduction in activity occurred at temperatures above 60 °C. Full hemagglutination activity was retained at ambient pH 4–12. Thereafter no activity was observed above pH 13. Hemaglutination of the lectin was inhibited by d-galactose. The lectin showed a strong antiproliferative activity towards human leukemic (THP-1) cancer cells followed by lung cancer (HOP62) cells and HCT116 with an IC₅₀ of 39 μg/ml and 50 μg/ml and 60 μg/ml respectively. Flow cytometry analysis showed an increase in the percentage of cells in sub G0G1 phase confirming that Lotus corniculatus lectin induced apoptosis. Morphological observations showed that Lotus corniculatus lectin (LCL) treated THP-1 cells displayed apparent apoptosis characteristics such as nuclear fragmentation, appearance of membrane enclosed apoptotic bodies and DNA fragmentation. Lotus corniculatus lectin (LCL) effectively inhibits the cell migration in a dose dependent manner as indicated by the wound healing assay.     

A novel angiotensin I-converting enzyme (ACE) inhibitory peptide from a marine Chlorella ellipsoidea and its antihypertensive effect in spontaneously hypertensive rats

Marine Chlorella ellipsoidea protein was hydrolyzed using Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, trypsin, α-chymotrypsin, pepsin and papain. Alcalase-proteolytic hydrolysate exhibited the highest ACE inhibitory activity among them and was fractionated into three ranges of molecular weight (below 5 kDa, 5–10 kDa and above 10 kDa). The below 5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. During consecutive purification, a potent ACE inhibitory peptide from marine C. ellipsoidea, which was composed of 4 amino acids, Val–Glu–Gly–Tyr (MW: 467.2 Da, IC₅₀ value: 128.4 μM), was isolated. Lineweaver–Burk plots suggest that the peptide purified acts as a competitive inhibitor against ACE and stable against gastrointestinal enzymes of pepsin, trypsin and α-chymotrypsin. Furthermore, antihypertensive effect in spontaneously hypertensive rats (SHRs) also revealed that oral administration of purified peptide can decrease systolic blood pressure significantly. The results suggest that marine C. ellipsoidea would be an attractive raw material for the manufacture of antihypertensive nutraceutical ingredients.     

Insulin-sensitizing activities of tanshinones,diterpene compounds of the root of Salvia miltiorrhiza Bunge

In this study, the effects of the extract and four tanshinone compounds from the dried root of Salvia miltiorrhiza Bunge (Labiatae) on the tyrosine phosphorylation of the insulin receptor (IR) β-subunit and the downstream signaling were examined in Chinese-hamster ovary cells expressing human insulin receptors (CHO/IR cells) as well as in 3T3-L1 adipocytes. In addition the translocation of the glucose transporter 4 was investigated in 3T3-L1 adipocytes. Total extract of Danshen (1–10 μg/ml) and the four tanshinones (10 μM) did not show any activity, but the total extract and the tanshinone I, IIA and 15, 16-dihydrotanshinone I except cryptotanshinone enhanced the activity of insulin (1 nM) on the tyrosine phosphorylation of the IR as well as the activation of the downstream kinases Akt, ERK1/2, and GSK3β. In the adipocytes the same IR-downstream signaling and the translocation of glucose transporter 4 were demonstrated by the three tanshinones in the presence of insulin. These insulin-sensitizing activities of tanshinones may be useful for developing a new class of specific IR activators as anti-diabetic agents.     

Characterization of a highly efficient heterodimeric xylosidase from Humicola insolens

A heterodimeric xylosidase (E.C. 3.2.1.37) with robust activity is secreted among the plant cell wall degrading enzymes produced by the saprophytic fungus Humicola insolens. The xylosidase has been purified to homogeneity by gel filtration and cation exchange chromatography, and demonstrated to be composed of two protein subunits of 68 and 17 kDa with a molecular mass in solution of approximately 85 kDa based on a combination of SDS-PAGE, size exclusion chromatography and analytical ultracentrifugation. Peptide sequence identities from the subunits indicate the 68 kDa subunit contains a catalytic protein domain and the 17 kDa subunit a carbohydrate binding module. The xylosidase has wide biotechnological potential with maximum activity exhibited at 70 °C and kinetic constants with p-nitrophenol xylopyranoside substrate that suggest it has the highest catalytic efficiency recorded to date (Vmax 22.17 μmoles/min/mg, Km 1.74 mM and Kcat 6787/s).     

Succinic acid production from sucrose and molasses by metabolically engineered E. coli using a cell surface display system

To achieve sucrose-metabolizing capability, different sucrose utilization operons have been introduced into E. coli that cannot utilize sucrose. However, these engineered strains still suffer from low growth rates and low sucrose uptake rates. In this study, cell surface display system was adopted in engineered E. coli AFP111 for succinic acid production from sucrose and molasses directly. Invertase (CscA) from E. coli W was successfully anchored to outer membrane by fusion with OmpC anchoring motif, and the displayed CscA showed high extracellular activity. Compared with the sucrose permease system, the cell surface display system consumed less ATP during sucrose metabolism. When less ATP was consumed by AFP111/pTrcC-cscA, the succinic acid productivity from sucrose was 23% higher than that by AFP111/pCR2.1-cscBKA that having the sucrose permease system. As a result, 41 g L⁻¹ and 36.3 g L⁻¹ succinic acid were produced by AFP111/pTrcC-cscA from sucrose and sugarcane molasses respectively at 34 h in 3-L fermentor during dual-phase fermentation. In addition, 79 g L⁻¹ succinic acid was accumulated with recovered AFP111/pTrcC-cscA cells at the end of dual-phase fermentation in 3-L fermentor, and the overall yield was 1.19 mol mol⁻¹ hexose.