Purification and characterization of a novel β-glucosidase from Aspergillus flavus and its application in saccharification of soybean meal

Abstract

Aspergillus flavus has been regarded as a potential candidate for its production of industrial enzymes, but the details of β-glucosidase from this strain is very limited. In herein, we first reported a novel β-glucosidase (AfBglA) with the molecular mass of 94.2?kDa from A. flavus. AfBglA was optimally active at pH 4.5 and 60?°C and is stable between pH 3.5 and 9.0 and at a temperature of up to 55?°C for 30?min remaining more than 90% of its initial activity. It showed an excellent tolerance to Trypsin, Pepsin, Compound Protease, and Flavourzyme and its activity was not inhibited by specific certain cations. AfBglA displayed broad substrate specificity, it acted on all tested pNP-glycosides and barley glucan, indicating this novel β-glucosidase exhibited a β-1, 3-1, 4-glucanase activity. Moreover, the AfBglA could effectively hydrolyze the soybean meal suspension into glucose and exhibit a strong tolerance to the inhibition of glucose at a concentration of 20.0?g/L during the saccharification. The maximum amount of the glucose obtained by AfBglA corresponded to 67.0?g/kg soybean meal. All of these properties mentioned above indicated that the AfBglA possibly attractive for food and feed industry and saccharification of cellulolytic materials.     

Partial Purification and Characterization of Chromate Reductase of a Novel Ochrobactrum sp. Strain Cr-B4

Hexavalent chromium contamination is a serious problem due to its high toxicity and carcinogenic effects on the biological systems. The enzymatic reduction of toxic Cr(VI) to the less toxic Cr(III) is an efficient technology for detoxification of Cr(VI)-contaminated industrial effluents. In this regard, a chromate reductase enzyme from a novel Ochrobactrum sp. strain Cr-B4, having the ability to detoxify Cr(VI) contaminated sites, has been partially purified and characterized. The molecular mass of this chromate reductase was found to be 31.53 kD, with a specific activity 14.26 U/mg without any addition of electron donors. The temperature and pH optima for chromate reductase activity were 40°C and 8.0, respectively. The activation energy (E_a) for the chromate reductase was found to be 34.7 kJ/mol up to 40°C and the activation energy for its deactivation (E_d) was found to be 79.6 kJ/mol over a temperature range of 50–80°C. The frequency factor for activation of chromate reductase was found to be 566.79 s^?1, and for deactivation of chromate reductase it was found to be 265.66 × 10³ s^?1. The reductase activity of this enzyme was affected by the presence of various heavy metals and complexing agents, some of which (ethylenediamine tetraacetic acid [EDTA], mercaptoethanol, NaN₃, Pb²⁺, Ni²⁺, Zn²⁺, and Cd²⁺) inhibited the enzyme activity, while metals like Cu²⁺ and Fe³⁺ significantly enhanced the reductase activity. The enzyme followed Michaelis–Menten kinetics with K_m of 104.29 µM and a V_max of 4.64 µM/min/mg.     

PURIFICATION AND CHARACTERIZATION OF PHOSPHODIESTERASE I FROM Walterinnesia aegyptia VENOM

A phosphodiesterase I (EC 3.1.4.1; PDE-I) was purified from Walterinnesia aegyptia venom by preparative native polyacrylamide gel electrophoresis (PAGE). A single protein band was observed in analytical native PAGE and sodium dodecyl sulfate (SDS)-PAGE. PDE-I was a single-chain glycoprotein with an estimated molecular mass of 158 kD (SDS-PAGE). The enzyme was free of 5′-nucleotidase and alkaline phosphatase activities. The optimum pH and temperature were 9.0 and 60°C, respectively. The energy of activation (Ea) was 96.4, the V_max and K_m were 1.14 µM/min/mg and 1.9 × 10^?3 M, respectively, and the K_cat and K_sp were 7 s^?1 and 60 M ^?1 min^?1 respectively. Cysteine was a noncompetitive inhibitor, with K_i = 6.2 × 10^?3 M and an IC₅₀ of 2.6 mM, whereas adenosine diphosphate was a competitive inhibitor, with K_i = 0.8 × 10^?3 M and an IC₅₀ of 8.3 mM. Glutathione, o-phenanthroline, zinc, and ethylenediamine tetraacetic acid (EDTA) inhibited PDE-I activity whereas Mg²⁺ slightly potentiated the activity. PDE-I hydrolyzed thymidine-5′-monophosphate p-nitrophenyl ester most readily, whereas cyclic 3′-5′-AMP was least susceptible to hydrolysis. PDE-I was not lethal to mice at a dose of 4.0 mg/kg, ip, but had an anticoagulant effect on human plasma. These findings indicate that W. aegyptia PDE-I shares various characteristics with this enzyme from other snake venoms.     

Mass spectrometry in the analysis of grape and wine proteins

Wine proteins play an important role in the quality of wine, because they affect taste, clarity and stability of product. The majority of wine proteins are in the range of 20–30 kDa. Different mass spectrometry (MS) techniques have been successfully applied to study the grape and wine proteins. By liquid chromatography (LC) electrospray ionization (ESI) MS and nano-LC/MS, nine dipeptides and 80 peptides were unambiguously identified in Champagne and Sauvignon Blanc wines, respectively. Using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and surface-enhanced laser desorption/ionization TOF, the protein and peptide fingerprints in Chardonnay, Sauvignon Blanc and Muscat of Alexandria wines were determined. MALDI-TOF identified the mesocarp proteome of six Vitis grape varieties. Proteins in different grape tissue extracts were also studied. The major grape pathogenic-related proteins are chitinases and thaumatin-like proteins, which both persist through the vinification process and cause hazes and sediments in bottled wines. ESI-MS, LC/ESI-MS and MALDI-TOF analysis of these proteins in grape and wine were also used to characterize different grape varieties.     

POTENTIAL USE OF LEAF BIOMASS,ARAUCARIA HETEROPHYLLA FOR REMOVAL OF Pb+2

The present investigation attempt to analyze the biosorption behavior of novel biosorbent, Araucaria heterophylla (green plant) biomass, for removal of Pb⁺² from solution as the function of initial metal ion concentration, pH, temperature, sorbent dosage and biomass particle size. The maximum biosorption was found to be 95.12% at pH 5 and biosorption capacity (q_e) of Cd⁺² is 9.643 mg/g. The Langmuir and Freundlich equilibrium adsorption isotherms were studied and observed that Freundlich model is best fit than the Langmuir model with correlation coefficient of 0.9927. Kinetic studies indicated that the biosorption process of Cd⁺² followed well pseudo second order model with R² 0.999. The process is exothermic and, spontaneous. The chemical functional groups –OH, CH₂ stretching vibrations, C?O of alcohol, C?O of amide, P?O stretching vibrations, –CH, were involved in the process. The XRD pattern of the A. heterophylla was found to be mostly amorphous in nature. The SEM studies showed Pb⁺² biosorption on selective grains of the biosorbent. It was concluded that A. heterophylla leaf powder can be used as an effective, low cost, and environmentally friendly biosorbent for the removal of Pb⁺² from aqueous solution.     

Synthetic Studies on Formamidopyrimidines Related to Clofarabine

Degradation of clofarabine (3) in 0.9% saline solution at 100°C afforded three degradation products which were determined to be formamidopyrimidines 4–6.Compounds 4 and 5 were assigned as C_1′ anomers on the basis of one-dimensional and two-dimensional NMR experiments, whereas 6 was found to be the formamidopyrimidine lacking the sugar moiety. An improved procedure for the synthesis of formamidopyrimidines was developed, wherein benzoylated clofarabine (11) was treated with allyl chloroformate, followed by deprotection of the alloc group with catalytic Pd(PPh₃)₄ and dimedone. A synthesis of compound 6 from 4 is also described.     

Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: Expression and biochemical characterization

A thermo-alkaliphilic lipase from Bacillus subtilis DR8806 was functionally expressed as an N-terminal 6xHis-tagged recombinant enzyme in Escherichia coli BL21 using pET-28a(+) expression vector. Sequence analysis revealed an open reading frame of 639 bp encoding a 212-amino acid protein containing the well-conserved Ala-His-Ser-Met-Gly motif. One-step purification of the His-tagged recombinant lipase was achieved using Ni-NTA affinity chromatography with a specific activity of 1364 U/mg. The purified enzyme with an apparent molecular mass of 26.8 kDa demonstrated the maximum activity at 70 °C and pH 8.0 for hydrolysis of p-nitrophenylbutyrate as substrate. The enzyme activity was strongly inhibited by divalent ions of heavy metals such as Hg²⁺ and Cu²⁺, while retained over 90% of the original activity in the presence of several reagents including DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)), SDS (sodium dodecyl sulfate), urea, DMF (dimethylformamide), DTT (dithiothreitol), glycerol and Triton X-100. While being considerably stable in organic solvents, imidazolium-based ionic liquids (ILs) had stimulatory effects on the activity of purified lipase. Remarkable stabilization of enzyme at alkaline pH and in ionic liquids as well as its thermostability/thermoactivity are among the most fundamental characteristics which offer great potential for various biotechnological applications including detergent formulation, bioremediation processes and biotransformation in non-aqueous media.     

Characterization of polyhydroxyalkanoates accumulated by a moderately halophilic salt pan isolate Bacillus megaterium strain H16

Aim

Characterization of polyhydroxyalkanoates (PHA) accumulated by halophilic bacteria isolated from solar salterns.

Methods and Results

Twenty‐six halophilic isolates were obtained from solar salterns of Goa, India. They were screened for accumulation of PHA by Sudan black B, Nile blue A and Nile red stains. Strains H15, H16 and H26 were selected based on their intensity of Nile blue A/Nile red fluorescence. On the basis of phenotypic and genotypic characterization, the three isolates were identified as Bacillus megaterium. Growth kinetics and polymer accumulating capacity of strain H16 were studied in E2 mineral media with 2% glucose with/without NaCl. In the absence of NaCl, strain H16 accumulated PHA to 40·0% (w/w) of cell dry weight (CDW) at 42 h of growth, whereas in presence of 5% w/v NaCl, the culture showed longer lag phase of up to 24 h and accumulated a maximum PHA of 39% (w/w) CDW at 54 h of growth. The infrared spectra of both the polymers exhibited peaks at 1733·9 cm^?1 characteristic of C=O. Scans of ¹H nuclear magnetic resonance (NMR) showed a doublet at 2·5 ppm corresponding to methylene group (‐CH₂), the signal at 5·3 ppm corresponded to methine group (‐CH‐), and another signal at 1·3 ppm corresponded to the methyl group (‐CH₃). Scans of ¹³C NMR showed prominent peaks at 20, 40, 67–68 and 170 ppm, indicating the polymer to be homopolymer of 3‐hydroxybutyrates. The polymer is stable up to a temperature of 160°C.

Conclusion

Three moderately halophilic isolates (strain H15, H16 and H26) capable of accumulating PHA were isolated from solar salterns of Ribandar Goa, India, and identified as B. megaterium based on phenotypic and genotypic characterization. Strain H16 accumulated polyhydroxybutyrate in the presence and absence of NaCl up to 40% of its CDW.

Significance and Impact of the Study

This strain would be better suited for production of PHA at industrial level due to its tolerance to high concentration of NaCl.     

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

CTLA4-Ig is a highly glycosylated therapeutic fusion protein that contains multiple N- and O-glycosylation sites. Glycosylation plays a vital role in protein solubility, stability, serum half-life, activity, and immunogenicity. For a CTLA4-Ig biosimilar development program, comparative analytical data, especially the glycosylation data, can influence decisions about the type and amount of animal and clinical data needed to establish biosimilarity. Because of the limited clinical experience with biosimilars before approval, a comprehensive level of knowledge about the biosimilar candidates is needed to achieve subsequent development. Liquid chromatography-mass spectrometry (LC–MS) is a versatile technique for characterizing N- and O-glycosylation modification of recombinant therapeutic proteins, including 3 levels: intact protein analysis, peptide mapping analysis, and released glycans analysis. In this report, an in-depth characterization of glycosylation of a candidate biosimilar was carried out using a systematic approach: N- and O-linked glycans were identified and electron-transfer dissociation was then used to pinpoint the 4 occupied O-glycosylation sites for the first time. As the results show, the approach provides a set of routine tools that combine accurate intact mass measurement, peptide mapping, and released glycan profiling. This approach can be used to comprehensively research a candidate biosimilar Fc-fusion protein and provides a basis for future studies addressing the similarity of CTLA4-Ig biosimilars.     

类芽胞杆菌碱性果胶裂解酶的纯化与酶学性质表征

从类芽胞杆菌Paenibacillus sp.WZ008的发酵上清液中纯化得到一个高活力碱性果胶裂解酶,经SDS-PAGE电泳估算其亚基相对分子质量为4.5×104。通过对该酶进行酶学性质研究发现：该酶能催化裂解果胶酸、低酯果胶和高酯果胶;酶催化反应最适温度范围为55～60℃,最适pH为9.6,在最适条件下以低酯果胶为底物酶的比酶活达3 021.6 U/mg;Ca2＋能增强该酶的活力,而Mn2＋,Ba2＋和EDTA强烈抑制该酶活力;当没有Ca2＋存在时,高度酯化的果胶是该酶的最适底物,在4 mmol/L Ca2＋存在时,该酶以果胶酸为底物比酶活最高（25 467 U/mg）。该酶N端序列比对分析发现与类芽胞杆菌Paenibacillus amylolyticus strain 27c64果胶裂解酶高度同源。