Importance of tyrosine residues of Bacillus stearothermophilus serine hydroxymethyltransferase in cofactor binding and L-allo-Thr cleavage

Serine hydroxymethyltransferase (SHMT) from Bacillus stearothermophilus (bsSHMT) is a pyridoxal 5′-phosphate-dependent enzyme that catalyses the conversion of L-serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. In addition, the enzyme catalyses the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids and transamination. In this article, we have examined the mechanism of the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids by SHMT. The three-dimensional structure and biochemical properties of Y51F and Y61A bsSHMTs and their complexes with substrates, especially L-allo-Thr, show that the cleavage of 3-hydroxy amino acids could proceed via Cα proton abstraction rather than hydroxyl proton removal. Both mutations result in a complete loss of tetrahydrofolate-dependent and tetrahydrofolate-independent activities. The mutation of Y51 to F strongly affects the binding of pyridoxal 5′-phosphate, possibly as a consequence of a change in the orientation of the phenyl ring in Y51F bsSHMT. The mutant enzyme could be completely reconstituted with pyridoxal 5′-phosphate. However, there was an alteration in the λmax value of the internal aldimine (396 nm), a decrease in the rate of reduction with NaCNBH3 and a loss of the intermediate in the interaction with methoxyamine (MA). The mutation of Y61 to A results in the loss of interaction with Cα and Cβ of the substrates. X-Ray structure and visible CD studies show that the mutant is capable of forming an external aldimine. However, the formation of the quinonoid intermediate is hindered. It is suggested that Y61 is involved in the abstraction of the Cα proton from 3-hydroxy amino acids. A new mechanism for the cleavage of 3-hydroxy amino acids via Cα proton abstraction by SHMT is proposed

Purification and characterization of a new endoglucanase from Aspergillus aculeatus

Endoglucanase has been isolated from Aspergillus aculeatus. The purified enzyme showed a single band and had a molecular weight of 45000 Da as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a specific activity of 1.4 units/mg. The purified enzyme was identified as endoglucanase, showing a high specific activity toward CM-cellulose and low specific activity toward Avicel. The activity of the isolated enzyme was optimum at a pH of 5.0 and temperature of 40°C, respectively. The isoelectric point of the enzyme was 4.3. Tm was found to be 57°C. The treatment of the endoglucanase with diethylpyrocarbonate resulted in the modification of the histidine residues present in the enzyme, with a concomitant loss of 70% of the original enzymatic activity. However, carbodiimide completely inactivated the endoglucanase. The results show that the enzyme is able to sustain 50% of its activity even when heated at 90°C for a period of 5 h. Endoglucanase can be used in the controlled hydrolysis of cellulose and other cellulose-rich foods. It can be used in the development of targeted functional foods from agrimaterials for value addition in the food chain

Infuence of metal ions on structure and catalytic activity of papain

18-27Papain is an endoprotease belonging to cysteine protease family. The catalytic activity of papain in presence of two different metal ions namely zinc and cadmium has been investigated. Both the metal ions are potent inhibitors of the enzyme activity in a concentration dependent manner. The enzyme loses 50% of its activity at 2x10-4 M of CdCI2 and 4x10-4 M of ZnCI2. It is completely inactivated above 1x10-3 M concentration of either ZnCI2 or CdCI2. Of the two metal ions zinc with a ki value of 5x10-5 M is a more potent inhibitor than cadmium which has a ki value of 8x10-5 M. Both the metal ions have higher affinity for active site than the substrate. At concentrations above 1x10-2 M of metal ions the inhibition is not reversible. Calorimetric studies showed decreased thermal stability of papain upon binding of these metal ions. Far UV circular dichroic spectral data showed only small changes in the β-structure content upon binding of these metal ions. These data are also supported by decrease in the apparent thermal transition temperature of papain by 5°C upon binding of metal ions indicating destabilization of the papain molecule. The mechanism of both partial and complete inactivation of papain in presence of these two metal ions both at lower and higher <span style="font-size:14.0pt;font-family:HiddenHorzOCR; mso-bidi-font-family:HiddenHorzOCR">concentration has been explained. </span

3D modelled trimeric structure of cupincin.

<p>View of the trimeric structure of cupincin as modelled in Swiss-Model (The Swiss Institute of Bioinformatics) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152819#pone.0152819.ref029" target="_blank">29</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152819#pone.0152819.ref031" target="_blank">31</a>]. Each subunit of the trimer is coloured differently (<a href="http://swissmodel.expasy.org/workspace/" target="_blank">http://swissmodel.expasy.org/workspace/</a>).</p

Whey protein hydrolysate: Functional properties, nutritional quality and utilization in beverage formulation

The objective of the study was to analyze the functional and nutritional properties of enzymatically hydrolyzed whey protein concentrate (WPC) and to formulate a beverage mix. WPC hydrolysates were produced using fungal protease and papain, at time intervals of 20, 40 and 60min and were analyzed for proximate composition and functional properties. A beverage was formulated with hydrolyzed WPC, skim milk powder, cocoa, liquid glucose, sugar and vegetable fat and analyzed for physicochemical properties, sensory attributes and keeping quality. Results revealed that the protein content of WPC was 75.6 and decreased slightly on enzyme treatment (69.6). The water absorption capacity of WPC was 10ml/100g and increased in enzyme treated samples from 16 to 34ml/100g with increase in the time of hydrolysis. Emulsion capacity (45ml of oil/g of control WPC) showed a decreasing trend with increasing time of hydrolysis. Enzyme treatment slightly increased the foam capacity in three samples but lowered foam stability in all. The gel filtration pattern of enzyme treated samples showed an increase in low molecular weight fractions. The amino acid profile showed higher content of methionine in samples treated with enzymes, compared to the control. The in vitro protein digestibility of untreated WPC was 25 and increased in all treated samples to varying degrees (69–70). Formulated beverage had 52 protein, 10 fat and 6.6 ash. There were no significant differences in the sensory attributes of formulated and commercial beverage. The formulated beverage could be stored well in a PET container for 30 days

Hydrolytic specificity of cupincin on neurotensin.

<p><b>(A).</b> RP-HPLC profile showing the blank neurotensin (a). Cleavage pattern of neurotensin hydrolyzed by cupincin at 2hours (b), 4hours (c) and 6 hours (d) of incubation. The peptides were resolved using a Grace Vydac C-18 column (4.6 × 250mm). The solvents used were 0.1% TFA and 70% acetonitrile containing 0.05% TFA. The peptides were detected at 230 nm. <b>(B).</b> Amino acid sequence of neurotensin, arrow indicating the cleavage position of cupincin.</p

Preferential extractability of γ-oryzanol from dried soapstock using different solvents

Background: γ-Oryzanol from rice bran has lately gained potential importance because of its proven health benefits. Thus the extractability of γ-oryzanol from the soapstock of crude rice bran oil is important from the perspective of future large-scale production, which would give value addition to this by-product obtained from the rice bran oil industry. The aim of the present study was to investigate the extraction of γ-oryzanol from the drum-dried soapstock of rice bran oil using various solvents. Results: It was found that γ-oryzanol could be extracted most effectively using ethyl acetate, followed by dichloromethane and ethyl methyl ketone. All components of γ-oryzanol have an alcohol group in the ferulate portion giving rise to relatively high polarity, thereby increasing the extraction in more polar solvents efficiently. Ethyl acetate showed maximum extractability of γ-oryzanol by the Soxhlet method. To quantify γ-oryzanol, reverse phase high-performance liquid chromatography (RP-HPLC) was used for fingerprinting the γ-oryzanol analogues with respect to standard γ-oryzanol. Conclusion: A new RP-HPLC method for determining the individual components of γ-oryzanol has been reported that can be used for performing an online characterisation of γ-oryzanol analogues by liquid chromatography/mass spectrometry

Hydrolytic specificity of cupincin on B-chain of insulin.

<p><b>(A).</b> RP-HPLC profile showing the blank Insulin B chain (a). Cleavage pattern of B chain of insulin hydrolyzed by cupincin at 1hour (b), 2hours (b) and 3 hours (d) of incubation. The peptides were resolved using a Grace Vydac C-18 column (4.6 × 250mm). The solvents used were 0.1% Trifluoroacetic acid (TFA) and 70% acetonitrile containing 0.05% TFA. The peptides were detected at 230 nm. <b>(B).</b> Amino acid sequence of B-chain of insulin, arrow indicating the cleavage position of cupincin.</p

Deduced amino acid sequence of cupincin.

<p>The putative signal sequence is underlined (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152819#pone.0152819.s006" target="_blank">S6 Fig</a>). Amino terminal sequence as determined by Edman degradation method is indicated in red. Peptide sequences obtained by in-gel trypsin digestion and MS/MS analysis are indicated in blue (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152819#pone.0152819.s008" target="_blank">S8A–S8G Figs</a>). OsI_13867 (NCBI) or LOC Os06g43830.1 (TIGR data base, <a href="http://rice.plantbiology.msu.edu/" target="_blank">http://rice.plantbiology.msu.edu/</a>) was identified as cupincin.</p

Elution profile of cupincin on analytical gel filtration column. Assessment of the molecular mass of cupincin.

<p>x-axis- retention time in minutes, y-axis- Absorbance unit (Au) @ 280ηm. The column was calibrated with 1) ϒ-Globulin, 2) Bovine serum albumin, 3) Carbonic anhydrase, 4) Cytochrome C and 5) Aprotinin. The column was equilibrated with 0.1 M sodium phosphate buffer pH 7.0 and the proteins were eluted at a flow rate of 1 mL /min. The samples were run for 25 minutes. Retention time of cupincin was determined to be 5.516 minutes corresponding to the mass of around 135 kDa as estimated based on the calibration curve constructed with known molecular mass proteins in the range of 6.5kDa-160 kDa (inset).</p