Bing-Lan Liu

The Induction and Characterization of Phytase and Beyond

Abstract The hydrolysis of phytic acid, the principal storage form of phosphorus in seeds and pollen, to myo-inositol and phosphoric acid is a very important metabolic process in many biological systems. This dephosphorylation of free or bound inositol phosphate is believed to be mainly affected by phytase. Generally, phytase behaves like a monomeric protein of a molecular mass of approximately 40–200 kDa and shows a broad substrate specificity with optimal degradation of phytate occurring around pH 4.5–6.0 and a temperature at 45–60°C. Furthermore, it is found that the Aspergillus ficuum phytase consists of 594 amino acid residues and the secondary structure contains 17.3% α-helixes, 29% β-sheet, 32.6% turns, and 24.7% coils. The N-linked mannose and galactose of intact phytase from A. ficuum account for 27.3% of the molecular weight. This implies that the enzyme is a glycoprotein. Recently, the crystal structure of this phytase has been determined at 2.5 A resolution. In this review, the properties of various phytases are summarized and the digestion of phytate by phytase and its products are also discussed.

Optimization of growth medium for the production of spores from Bacillus thuringiensis using response surface methodology

Abstract The effects of cultivation medium compositions including tapioca, fishmeal, CaCO3 and (NH4)2SO4 for the growth of Bacillus thuringiensis YMB 96-1988 were accessed by using response surface methodology (RSM). The two-level (24–1) fractional factorial designs (FFD) which involve two concentrations of each nutrient, and the paths of steepest ascent were effective in searching for the major factors of the bacteria growth. This allows the fitting of a first order linear model to the data. In this study, supplementary CaCO3 showed a negative effect on the spore production based on the first order regression coefficients derived from SAS programme. Subsequently, a 23 central composite design (CCD) was used for allocation of treatment combinations. Preliminary studies showed that tapioca and fishmeal is believed to be the major factors for the growth of B. thuringiensis. Estimated optimum compositions for the production of spores by B. thuringiensis are as follows: tapioca, 5.01%; fishmeal 5.86%; (NH4)2SO4 0.06% and resulted in a maximum spore count of 8.56 × 108/ml was obtained. This value is close to the 8.35 × 108/ml spore density as counted from actual experimental observations.

Development and applications of destruxins: a review.

The insecticidal and phytotoxic activities of destruxins (dtxs) have been well studied. The cyclodepsipeptides, which are dtxs mainly isolated from the fungus Metarhizium anisopliae and other fungi, have been well characterized in vitro and in vivo. A succession of important function, such as antitumoral, antiviral, insecticidal, cytotoxic, immunosuppressant, phytotoxic, and antiproliferative effects have been observed. To date, 39 dtxs derivatives have been identified. Dtxs possess a variety of biological activities, including acting as virulence factors for specific insects, a V-ATPase inhibitor that provides a basis for the development of new drug to against osteoporosis, cancer, or biological control agents, etc. Here, we focus on some of the research progress made on understanding dtxs during the last decade, introduce some of the newly identified dtx members, especially from M. anisopliae, and give an overview of the applications of dtxs. Using the dtxs to learn about and moderate biological events has advanced significantly during the past year. We believe that several ongoing dtx application fields may benefit from the reviewed information herein.

Effect of immobilization on pH and thermal stability of Aspergillus ficuum phytase

Abstract The phytase from Aspergillus ficuum catalyzes the hydrolysis of phytic acid into phosphoric acid and myo -inositol. The activity of this enzyme was determined by monitoring the rate of inositol production using high-performance liquid chromography methodology. The maximum activity of the enzyme was found to be approximately pH 5 and had a temperature optimum of 50°C. Under this condition, the k cat of 96 s −1 was obtained, and the corresponding K m for the catalysis of phytic acid was 2.34 mM. The optimum pH for the immobilized phytase was not much different from the intact enzyme. However, the optimum temperature was increased to 58°C, which is 8°C higher than that of free enzyme. Apparent K m for the immobilized enzyme was 3.28 mM, and only 34.6% of the free enzyme activity ( k cat ) was retained.

Characterization study of the sporulation kinetics of Bacillus thuringiensis

A wild-type and an rDNA strain of Bacillus thuringiensis were cultured in a net-draft-tube modified 20-L airlift bioreactor. A comparison of the sporulation patterns suggests that the early sporulation strain has a lower final spore count. Results from off-gas analysis suggests that the CO(2) profile could be an alternative indication to spore counts for the examination of fermentation performance or even the mortality in bioassay of the cultivation product. The difference in mortality tests exhibited by the microorganism was attributed to different patterns of sporulation as well as different levels of gene control inside the cell itself. The sporulation kinetics of B. thuringiensis was simulated by a simple modified Hill equation, where the initial glucose concentration could affect the timing of the onset of sporulation. The equation matches well with the experimental sporulation data for B. thuringiensis in both wild-type and rDNA strains.

Purification and quantification of destruxins A and B from Metarhizium anisopliae

Abstract Purification of insecticidal cyclodepsipeptides, destruxins A (DA) and B (DB), from Metarhizium anisopliae fermentation broth was performed. The isolation scheme of these destruxins using ion-exchange chromatography, silica gel chromatography, and semi-preparative HPLC chromatography is presented. The quality of the semi-preparative products was unique. Over 90% purity (based on HPLC chromatograms) was achieved for both DA and DB. Purified destruxins were further identified employing the 1H NMR and fast atom bombardment MS methodology. The HPLC quantification methods for DA and DB in the fermentation broth have been established. The use of the purified destruxins as analytical standards demonstrated good correlation.

Production of cyclodepsipeptides destruxin A and B from Metarhizium anisopliae.

Maltose and peptone were the best carbon and nitrogen sources for the production of destruxins from Metarhizium anisopliae. With the addition of 0.1% (w/v) β‐alanine to the basal medium, the yields of cyclodepsipeptides DA and DB were 7.2 and 279 mg/L, respectively, which was 2‐fold higher than that of control experiment. Response surface methodology (RSM) was applied to optimize the compositions of maltose, peptone, β‐alanine, and glucose used in a shaker‐flask cultivation of M. anisopliae for the production of DA and DB. Estimated optimal compositions for the DA production were maltose 2.58%, peptone 0.72%, β‐alanine 0.02%, and glucose 0.55%. The predicted DA yield was 18.5 mg/L. On the other hand, the optimal compositions for DB production were maltose 2.51%, peptone 0.75%, β‐alanine 0.02%, and glucose 0.43%. A maximum DB yield of 232 mg/L was predicted. These were confirmed by cultivation experiments conducted at the optimized conditions for maximum destruxins production in a shaker‐flask. Furthermore, a modest high level of DA (49 mg/L) and DB (268 mg/L) yields were obtained by employing the response surface methodology optimized DB production medium in a no‐baffle, stirred‐tank fermentor.

Effect of rice--glycerol complex medium on the production of Lovastatin by Monascus ruber.

Response surface methodology (RSM) was employed to study the effect of the composition of the rice-glycerol complex medium on the production of lovastatin (Lvs) by the ascomyceteMonascus ruber in mixed solid-liquid (or submerged) cultures at 25°C. Four components (rice powder, peptone, glycerol, glucose) were studied to evaluate, the approximate polynomial for all dependent variables, explaining their effects on the production of Lvs. The best composition derived from RSM regression was (in g/L) rice powder 34.4, peptone 10.8, , glucose 129, KNO3 8.0, MgSO4·7H2O 4.0 and glycerol 36.4 mL/L. With this composition, the Lvs production was 157 mg/L after 10 d of cultivation. In comparison with glycerol and glucose, the rice powder becomes a more suitable carbon source and represents a great potential for the production of Lvs.

Destruxin B Isolated from Entomopathogenic Fungus Metarhizium anisopliae Induces Apoptosis via a Bcl-2 Family-Dependent Mitochondrial Pathway in Human Nonsmall Cell Lung Cancer Cells

Destruxin B, isolated from entomopathogenic fungus Metarhizium anisopliae, is one of the cyclodepsipeptides with insecticidal and anticancer activities. In this study, destruxin B was extracted and purified by ion-exchange chromatography, silica gel chromatography, and semipreparative high-performance liquid chromatography. The potential anticancer effects and molecular mechanisms of destruxin B in human nonsmall cell lung cancer cell lines were characterized. Our results showed that destruxin B induced apoptotic cell death in A549 cells. This event was accompanied by the activation of caspase-2, -3, and -9. Moreover, destruxin B increased the expression level of proapoptotic molecule, PUMA, while decreased antiapoptotic molecule Mcl-1. Additionally, the translocation of Bax from cytosol to mitochondrial membrane was observed upon destruxin B treatment. Knockdown of Bax by shRNA effectively attenuated destruxin-B-triggered apoptosis in A549 cells. Interestingly, similar toxic effects and underlying mechanisms including caspase activation, upregulation of PUMA, and downregulation of Mcl-1 were also observed in a p53-null lung cancer H1299 cell line upon destruxin B treatment. Taken together, our findings suggest that destruxin-B-induced apoptosis in human nonsmall cell lung cancer cells is via a Bcl-2 family-dependent mitochondrial pathway.

Pulmonary toxicity of thuringiensin administered intratracheally in Sprague-Dawley rats.

The purpose of this work is to evaluate the pulmonary toxicity of purified thuringiensin in Sprague-Dawley rats. Rats were intratracheally instillated with 0, 0.4, 0.8, 1.6, 3.2, 6.4 and 9.6 mg/kg body weight of thuringiensin. The results indicated that the acute pulmonary LD(50) of thuringiensin for rats was 4.4 mg/kg. The total number of inflammatory cells and lactate dehydrogenase (LDH) activity in the bronchoalveolar lavage (BAL) fluid increased in a dose-dependent manner after thuringiensin instillation. Furthermore, an effective dose of 1.6 mg/kg was selected for the time course study of pulmonary toxicity. The treated animals showed a significant increase in the weights of the lungs, hydroxyproline levels in the lungs and total number of cells in BAL fluid 2, 4, 7, 14, 28 and 56 days after treatment. In comparison with the control, the total protein concentrations in BAL fluid were increased by 361, 615, 116, 41, 34 and 41%, after 2, 4, 7, 14, 28 and 56 days, respectively. The LDH activity in BAL fluid showed a significant increase after 1, 2, 4, 7, 14, 28 and 56 days. The increases in fibronectin levels were 164, 552, 490, 769, 335, 257 and 61% at the corresponding times, but neither tumor necrosis factor nor interleukin-1 increased. The treated rats presented abnormal histology including distributed inflammation in the bronchioles and alveoli, bronchial cellular necrosis on days 1 and 2, and areas of septal thickening with cellular infiltration and collagen deposit in the intestinal and alveolar spaces on days 4-56. Based on these biochemical and pathological parameters, intratracheal instillation of purified thuringiensin might cause significant pulmonary toxicity in rats.