Process for isolation of cardanol from technical cashew (Anacardium occidentale L.) nut shell liquid.

Commercially available technical cashew (Anacardium occidentale L.) nut shell liquid (CNSL) contains mainly cardanol (decarboxylated anacardic acid) and cardol. Cardanol, the monophenolic component of technical CNSL, is widely used as a synthon for the preparation of a number of polymers and agricultural products. This paper describes the separation of cardanol from toxic cardol. Technical CNSL was dissolved in a mixture of methanol and ammonium hydroxide (8:5) and extracted with hexane to obtain cardanol. The resultant methanolic ammonia layer was extracted with a mixture of ethyl acetate and hexane to yield cardol. This is the first industrially feasible process based on solvent extractions for the isolation of cardanol from technical CNSL.

Novel method for isolation of major phenolic constituents from cashew (Anacardium occidentale L.) nut shell liquid.

Commercially available cashew (Anacardium occidentale L.) nut shell liquid (CNSL) mainly contains the phenolic constituents anacardic acid, cardol, and cardanol. These phenolic constituents are themselves heterogeneous, and each of them contains saturated, monoene, diene, and trienes in the fifteen-carbon side chain. This communication describes the separation of anacardic acid, cardol, and cardanol for industrial application. Anacardic acid was selectively isolated as calcium anacardate. The acid-free CNSL was treated with liquor ammonia and extracted with hexane/ethyl acetate (98:2) to separate the mono phenolic component, cardanol. Subsequently, ammonia solution was extracted with ethyl acetate/hexane (80:20) to obtain cardol.

The tertiary structure at 1.59 A resolution and the proposed amino acid sequence of a family-11 xylanase from the thermophilic fungus Paecilomyces varioti bainier.

We report the crystal structure at 1.59 A and the proposed amino acid sequence of an endo-1,4-beta-xylanase (PVX) from the thermophilic fungus Paecilomyces varioti Bainier (PvB), stable up to 75 degrees C. This fungus is attracting clinical attention as a pathogen causing post-surgical infections. Its xylanase, known as a skin-contact allergen, is the first protein from this fungus whose three-dimensional structure has been elucidated. The crystals of PVX conform to the space group P2(1)2(1)2(1 )with a=38.76 A, b=54.06 A and c=90.06 A. The structure was solved by molecular replacement techniques using polyalanine coordinates of the Thermomyces lanuginosus xylanase (PDB code 1YNA) and a careful model building based on the amino acid sequence known for two trypsin-digested peptide fragments (17 residues), the sequence and structural alignment of family-11 xylanases and electron density maps. The final refined model has 194 amino acid residues and 128 water molecules, with a crystallographic R-factor of 19.07 % and a free R-factor of 21.94 %. The structure belongs to an all-beta fold, with two curved beta-sheets, forming the cylindrical active-site cleft, and a lone alpha-helix, as present in other family-11 xylanases. We have carried out a quantitative comparison of the structure and sequence of the present thermophilic xylanase (PVX) with other available native structures of mesophiles and thermophiles, the first such detailed analysis to be carried out on family-11 xylanases. The analysis provides a basis for the rationalisation of the idea that the "hinge" region is made more compact in thermophiles by the addition of a disulphide bridge between Cys110 and Cys154 and a N-H.O hydrogen bond between Trp159 near the extremity of the lone alpha-helix and Trp138 on beta-strand B8. This work brings out explicitly the presence of the C-H.O and the C-H.pi type interactions in these enzymes. A complete description of structural stability of these enzymes needs to take account of these weaker interactions.

Crystal structure at 1.8 A resolution and proposed amino acid sequence of a thermostable xylanase from Thermoascus aurantiacus.

Thermoascus aurantiacus xylanase is a thermostable enzyme which hydrolyses xylan, a major hemicellulose component in the biosphere. Crystals belonging to P21 space group with a=41.7 A, b=68.1 A, c=51. 4 A and beta=113.6 degrees, Z=2 were grown that could diffract to better than 1.8 A resolution. The structure was solved by molecular replacement method using the Streptomyces lividans xylanase model. The amino acid sequence was determined from the electron density map aided by multiple alignment of related xylanase sequences. The sequence thus obtained provides a correction to the sequence reported earlier based on biochemical methods. The final refined protein model at 1.8 A resolution with 301 amino acid residues and 266 water molecules has an R-factor of 16.0 % and free R of 21.1 % with good stereochemistry. The single polypeptide chain assumes (alpha/beta)8 TIM-barrel fold and belongs to F/10 family of glycoside hydrolases. The active site consists of two glutamate residues located at the C terminus end of the beta-barrel, conforming to the double displacement mechanism for the enzyme action. A disulphide bond and more than ten salt bridges have been identified. In particular, the salt bridge Arg124-Glu232 which is almost buried, bridges the beta-strands beta4 and beta7 where the catalytic glutamate residues reside, and it may play a key role in the stability and activity at elevated temperature. To our knowledge, for the first time in the F/10 family xylanases, we observe a proline residue in the middle of the alpha-helix alpha6 which may be contributing to better packing. Earlier studies show that the enzyme retains its activity even at 70 degrees C. The refined protein model has allowed a detailed comparison with the other known structures in the F/10 family of enzymes. The possible causative factors for thermostability are discussed.

Crystallization and preliminary X-ray crystallographic studies of thermostable xylanase crystals isolated from Paecilomyces varioti.

A highly thermostable xylanase isolated from the thermophilic fungus Paecilomyces varioti has been crystallized by the vapour diffusion method. The isolation of this enzyme by crystallization directly from the culture filtrate projects this fungus as an important source for large-scale production of pure xylanase. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with the unit cell dimensions a = 38.48 A, b = 53.87 A and c = 90.23 A. Four molecules occupy a volume of 187,039.4 A3 along with 34% of solvent. The data collected with an area detector to the resolution of 2.7 A were used to calculate the unit cell parameters and Matthews' constant. The optical behaviour of the crystal was studied at different temperatures to understand its thermal stability.

Crystallization and preliminary X-ray diffraction analysis of crystals of Thermoascus aurantiacus xylanase.

Crystals suitable for high resolution X-ray diffraction analysis have been grown of the 29,774-Da protein, xylanase (1,-4-beta-xylan xylanohydrolase EC 3.2.1.8) from the thermophilic fungus Thermoascus aurantiacus. This protein, an endoxylanase demonstrates the hydrolysis of beta-(1-4)-D-xylose linkage in xylans and crystallizes as monoclinic pinacoids in the presence of ammonium sulphate buffered at pH 6.5, and also with neutral polyethylene glycol 6000. The crystals belong to space group P2(1) and have cell dimensions, a = 41.2 A, b = 67.76 A, c = 51.8 A; beta = 113.2 degrees.

The primary structure of xylanase from Thermoascus aurantiacus.

The amino acid sequence of xylanase isolated from the culture medium of Thermoascus aurantiacus was determined. It had 269 amino acid residues with an alpha-N-acetyl group at the amino terminus. The structure of blocked N-terminal 11 amino acid tryptic peptide except for acetylalanine was determined by sequence analysis of peptides derived from partial acid hydrolysis and from thermolysin digestion. The blocked N-terminal amino acid was determined as N-acetylalanine by electron ionization mass spectrometry. The sequence comparison of xylanase from T. aurantiacus with the xylanases of alkalophilic Bacillus sp C-125 and Cryptococcus albidus showed 40% similarity. Xylanase from T. aurantiacus had up to 15% similarity with the other two xylanases known. All the five xylanases showed a higher degree of similarity at the level of secondary structure.

Role of a disulfide cross-link in the conformational stability of a thermostable xylanase.

The role of a S-S cross-link in the conformational stability of xylanase from Humicola lanuginosa has been investigated using CD, UV absorption spectroscopy, and RIA displacement studies. Our studies show that reduction and carboxymethylation of the S-S cross-link in xylanase results in a gross conformational perturbation of the protein. The secondary structure analysis of the CD spectra indicates that the xylanase with an intact S-S contains 66% beta-sheet structure and remaining random coil. Cleavage of the S-S bond results in a loss of 25% beta-sheet structure. Thermal denaturation studies using CD spectroscopy and pH-dependent tyrosine ionization studies using UV spectroscopy show that the presence of disulfide cross-link offers resistance against unfolding by extremes of temperature and pH. Further, we demonstrate that the heat-induced changes in xylanase with intact S-S bond are almost totally reversible, while those in the S-S cleaved enzyme fail to show any significant reversal. Our studies support the present theory that S-S cross-links exert their stabilizing effect in proteins by destabilizing the unfolded state of the protein and forcing it back to a more folded state.

Purification and properties of xylanase from the thermophilic fungus, Humicola lanuginosa (Griffon and Maublanc) Bunce.

An extracellular xylanase was purified to homogeneity from the culture filtrate of the thermophilic fungus, Humicola lanuginosa (Griffon and Maublanc) Bunce and its properties were studied. A fourfold purification and a yield of 8% were achieved. The molecular weight of the protein was found to be 22,500 based on electrophoretic mobility and 29,000 by gel filtration behavior. The protein is rich in acidic amino acids, glycine and tyrosine, and poor in sulfur-containing amino acids. The kinetic properties of the enzyme are similar to those of other fungal xylanases. The enzyme shows high affinity toward larchwood xylan (Km = 0.91 mg/ml) and hydrolyzes only xylan. The enzyme becomes inactivated when stored for more than 2 months at -20 degrees C in the dry state. Such an inactivation has not been reported so far for any xylanase. Using chromatographic techniques, one species of protein differing from the native protein in charge but enzymatically active was isolated in low yields. However, a large molecular-weight species of the protein devoid of enzyme activity was isolated in substantial quantities and further characterized. Based on ultracentrifugation and gel electrophoretic studies, it was concluded that this species may be an aggregate of the native protein and that such an aggregation might be taking place on storage in the dry state at -20 degrees C, leading to loss in activity.

Purification and characterization of two cellobiohydrolases from Chaetomium thermophile var. coprophile.

Cellobiohydrolases I and II were purified to homogeneity from culture filtrates of a thermophilic fungus, Chaetomium thermophile var. coprophile, by using a combination of ion-exchange and gel filtration chromatographic procedures. The molecular weights of cellobiohydrolase I and II were estimated to be 60,000 and 40,000 and the enzymes were found to be glycoproteins containing 17 and 22.8% carbohydrate, respectively. The two forms differed in their amino-acid composition mainly with respect to threonine, alanine, methionine and arginine. Antibodies produced against either form of cellobiohydrolases failed to cross-react with the other. The tryptic maps of the two enzymes were found to be different. The temperature optima for cellobiohydrolase I and II were 75 and 70 degrees C, and they were optimally active at pH 5.8 and 6.4, respectively. Both enzymes were stable at higher temperatures and were able to degrade crystalline cellulosic materials.

Purification of xylanase, beta-glucosidase, endocellulase, and exocellulase from a thermophilic fungus, Thermoascus aurantiacus.

A strain of thermophilic fungus, Thermoascus aurantiacus, was isolated from local soil. From the culture filtrates of the organism grown on blotting paper, a xylanase, beta-glucosidase, exocellulase, and endocellulase were obtained in large amounts in highly purified form by employing ion-exchange and gel-permeation chromatography. The xylanase was crystallized. The xylanase and endocellulase were stable at 70 degrees C for 8 h, whereas the beta-glucosidase and exocellulase were less stable at 70 degrees C.

Degradation of larchwood xylan by enzymes of a thermophilic fungus, Thermoascus aurantiacus.

Proteins from the culture filtrates of Thermoascus aurantiacus grown on paper were found to hydrolyze larchwood xylan completely to form xylose and 4-O-methyl-alpha-D-glucuronic acid. Partial hydrolysis of xylan by a xylanase purified from the culture filtrates resulted in the formation of neutral xylooligosaccharides of dp from 2 to 6 and acidic xylooligosaccharides of dp from 5 to 8. Each of these acidic sugars contained a single molecule of 4-O-methyl-alpha-D-glucuronic acid as a branch. Extensive hydrolysis of these oligosaccharides or xylan by xylanase led to the isolation of xylose, xylobiose, and an aldotetrauronic acid as terminal products. The structure of the aldotetrauronic acid was established by NMR as (2(2)-O-alpha-D,4-O-methyl-alpha-D-glucurono)-xylotriose. A beta-glucosidase, also purified from the culture filtrates, hydrolyzed xylan and the neutral or the acidic xylooligosaccharides from the nonreducing end to release only xylose. Neither xylanase nor beta-glucosidase hydrolyzed the beta-(1----4) linkage between the xylose carrying the branch and the adjacent xylose residue on each side.

Purification and characterization of an alpha-D-glucuronidase from a thermophilic fungus, Thermoascus aurantiacus.

An alpha-D-glucuronidase was purified from the culture filtrates of Thermoascus aurantiacus. A simple colorimetric method for its assay is reported. The enzyme is a single polypeptide chain with a molecular weight of 118,000. It acts optimally at pH 4.5. It shows maximum activity at 65 degrees C. The t 1/2 at 70 degrees C was 40 min. It specifically cleaved the alpha-(1----2) linkage between 4-O-methyl-alpha-D-glucuronic acid and the xylose residue in xylan and several glucurono-xylooligosaccharides.

Influence of deamidation(s) in the 67-74 region of ribonuclease on its refolding.

The influence of chemical mutation featuring the selective conversion of asparagine or glutamine to aspartic or glutamic acid, respectively, on the kinetics of refolding of reduced RNase has been studied. The monodeamidated derivatives of RNase A, viz. RNase Aa1a, Aa1b, and Aa1c having their deamidations in the region 67-74, were found to regain nearly their original enzymatic activity. However, a marked difference in the kinetics of refolding is seen, the order of regain of enzymic activity being RNase A greater than Aa1c congruent to Aa1a greater than Aa1b. The similarities in the distinct elution positions on Amberlite XE-64, gel electrophoretic mobilities, and u.v. spectra of reoxidized and native derivatives indicated that the native structures are formed. The slower rate of reappearance of enzymic activity in the case of the monodeamidated derivatives appears to result from altered interactions in the early stages of refolding. The roles of some amino acid residues of the 67-74 region in the pathway of refolding of RNase A are discussed.

Isolation and characterization of monodeamidated derivatives of bovine pancreatic ribonuclease A.

The isolation and characterization of the initial intermediates formed during the irreversible acid denaturation of enzyme Ribonuclease A are described. The products obtained when RNase A is maintained in 0.5 M HCl at 30 degrees for periods up to 20 h have been analyzed by ion-exchange chromatography on Amberlite XE-64. Four distinct components were found to elute earlier to RNase A; these have been designated RNase Aa2, Aa1c, Aa1b, and Aa1a in order of their elution. With the exception of RNase Aa2, the other components are nearly as active as RNase A. Polyacrylamide gel electrophoresis at near-neutral pH indicated that RNase Aa1a, Aa1b, and Aa1c are monodeamidated derivatives of RNase A; RNase Aa1c contains, in addition, a small amount of a dideamidated component. RNase Aa2, which has 75% enzymic activity as compared to RNase A, consists of dideamidated and higher deamidated derivatives of RNase A. Except for differences in the proteolytic susceptibilities at an elevated temperature or acidic pH, the monodeamidated derivatives were found to have very nearly the same enzymic activity and the compact folded structure as the native enzyme. Fingerprint analyses of the tryptic peptides of monodeamidated derivatives have shown that the deamidations are restricted to an amide cluster in the region 67-74 of the polypeptide chain. The initial acid-catalyzed deamidation occurs in and around the 65-72 disulfide loop giving rise to at least three distinct monodeamidated derivatives of RNase A without an appreciable change in the catalytic activity and conformation of the ribonuclease molecule. Significance of this specific deamidation occurring in highly acidic conditions, and the biological implications of the physiological deamidation reactions of proteins are discussed.

Irreversible thermal denaturation of bovine pancreatic ribonuclease-A. Physico-chemical characterization of initial products.

The isolation and characterization of the products formed during the irreversible thermal denaturation of enzyme RNAase-A are described. RNAase-A, when maintained in aqueous solution at pH 7.0 and 70 degrees for 2 h, gives soluble products which have been fractionated by gel filtration on Sephadex G-75 into four components. These components are designated RNAase-At1, RNAase-At2, RNAase-At3 and RNAase-At4 according to the order of their elution from Sephadex G-75. RNAase-At4 shows the same specific activity towards yeast RNA as native RNAase-A and is virtually indistinguishable from it by the physical methods employed. However, chromatography on CM-cellulose separates it into three components that show the same u.v. spectra and specific activity towards yeast RNA as native RNAase-A. RNAase-At1, RNAase-At2 and RNAase-At3 are all structurally altered derivatives of RNAase-A and they exhibit low specific activity (5-10%) towards yeast RNA. In the presence of added S-protein, all these derivatives show greatly enhanced enzymic activity. RNAase-At1 and RNAase-At2 are polymers, covalently crosslinked by intermolecular disulfide bridges; whereas RNAase-At3 is a monomer. Physical studies such as 1H-n.m.r., sedimentation analysis, u.v. absorption spectra and CD spectra reveal that RNAase-At3 is a unfolded derivative of RNAase-A. However, it is seen to possess sufficient residual structure which gives rise to a low but easily detectable enzymic activity.

Chemical modification of methionines of ribonuclease A with o-benzoquinone.

The accessibility of methionines in RNAase A to reaction with OBQ has been studied at highly acidic pH. The differences between the rate constants of reactions of the methionine and methionines of RNAase A with OBQ is a reflection on the limited accessibility of methionines in the protein conformation. Nevertheless, at sufficiently high OBQ concentration, all the four methionines of the enzyme can be modified. At lower concentration of OBQ, a derivative may be prepared in which a specific methionine is modified. The introduced chromophore ionizes at around pH 3 in this derivative. The derivative has partial activity towards RNA which is enhanced on addition of S-protein.