Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus

The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from Aspergillus clavatus (AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production.     

Isolation and some properties of a nonspecific extracellular ribonuclease of Aspergillus clavatus]

RNase has been isolated from the homogenate of the Aspergillus clavatus mycelium by gel filtration through Sephadex G-75, chromatography on CM-cellulose and DEAE-cellulose. By gel filtration and electrophoresis in polyacrylamide gel the preparation has been shown to be homogeneous. The enzyme is acid protein with the isoelectric point at pH 4.4 and molecular weight of 27,000. RNase has pH optimum at 6.0--6.2 and temperature optimum 60 degrees for RNA action. The enzyme splits RNA completely in the absence of metal ions. Ions Zn2+, Cu+2, Ag+1 and Ni+2 at a concentration of 10(-4) M are strong inhibitors of RNase activity.     

Biosynthesis of glyantrypine by Aspergillus clavatus

The biosynthesis of glyantrypine from radiolabelled amino acid precursors has been shown experimentally to involve anthranilic acid, tryptophan and glycine. Low values for percentage incorporation of radiolabel into glyantrypine were partly influenced by a complex array of other novel alkaloids shown by the radiolabelling experiments to be related to glyantrypine. Interpretation of radiolabel incorporation from [14C-carboxyl]-anthranilic acid into microbial metabolites seen to contain an anthranilyl moiety in various biosynthetic arrangements is discussed. The possibility of diversion of anthranilic acid from the kynurenine pathway to glyantrypine biosynthesis is recognised.     

棒曲霉Asp-195v产蛋白酶的酶学性质研究

对液体发酵的棒曲霉Asp-195v菌株所产蛋白酶的活力进行了研究,并通过分离纯化获得了电泳纯的酶蛋白。研究结果表明,该蛋白酶的最适反应温度为40℃,在30-50℃温度范围内相对活力可保持在70%以上;最适pH为7,pH稳定范围在4-8;Mn2+对该蛋白酶活力有明显的激活作用,K+、Ag+、Cu2+、Fe2+、Mg2+、Zn2+、Ca2+、Al3+和Fe3+离子则有明显的抑制作用,尤其是Hg2+和Pb2+对酶活的抑制作用更加强烈;其他试剂如葡萄糖、EDTA对酶活的抑制作用不明显,而蔗糖、SDS和Tween-20对酶活的抑制明显;以酪氨酸为底物采用双倒数作图法测得Vmax为30.40mmol/min,Km为97.53mmol/L。该酶的表观分子量为30.1kDa。     

Large scale synthesis of dinucleoside monophosphates catalyzed by ribonuclease from Aspergillus clavatus

A gram scale enzymatic synthesis of eight, dinucleoside monophosphates (ApC, ApU, CpC, CpU, GpC, GpU, UpC, and UpU) is described. The synthesis involves a reaction between the appropriate ribonucleoside-2′,3′-cyclie phosphates and cytidine or uridine in the presence of ribonuelease from Aspergillus clavatus at 30°C. The enzyme is removed from the reaction mixture by chromatography on Bio-Gel P–4, and the dinucleoside monophosphate is further purified by chromatography on a DEAE-Sephadex A–25, column. A procedure for the large scale preparation of the ribonuclease from Aspergillus clavatus is also described.     

Interaction of intracellular RNAase from Aspergillus clavatus with derivatives of its substrates]

Using spectrophotometric and kinetic methods and also the methods of protection of Aspergillus clavatus RNAse (EC 3.1.4.23) by adenine nucleotides and their components against inactivation by means of acylation or heating, it was found that RNAse-nucleotide complex was formed by association of one enzyme molecule with one nucleotide molecule. It was also shown that all components of nucleotides (base, ribose and phosphate) take part in the formation of such complex and the removal of one of them (base or phosphate) lead to loosening of bindings of remaining fragments (ribose-5'-monophosphate, adenine) with the active site of RNAse, and to disappearance of bends within the pH range of 3.0-4.0 on the plot of pKi (5'-MP) versus pH, within the pH range of 5.5-7.0 on the plot of oKi (Ado) versus pH. The possibility of participation of associative pair RNAse imidasole groups - nucleotide phosphate groups and RNAse carboxylic group - nucleotide base in the mechanism of formation of enzyme-nucleotide (enzyme-substrate) complexes is postulated.     

Substrate specificity of Aspergillus clavatus intracellular acid RNAase]

Substrate specificity of intracellular acid RNAse from Aspergillus clavatus, has been studied using different RNAs, synthetic polynucleotides and diribonucleoside monophosphates as substrates. The enzyme was shown to be a RNAse, non-specific to the chemical nature of bases adjacent to the disrupted phosphodiesther bonds in the molecules of RNA. It has been demonstrated that the order of nucleotide release from RNA coincides with the order of weakening of the enzyme binding to substrates XpY, depending on the base X. Purine bases increase substrates XpY binding with the enzyme and hamper their splitting. The effect of pyrimidine bases on adsorption and catalytic functions of the enzyme is contrary to that of purine bases cited above.     

Identification and engineering of the cytochalasin gene cluster from Aspergillus clavatus NRRL 1

Cytochalasins are a group of fungal secondary metabolites with diverse structures and bioactivities, including cytochalasin E produced by Aspergillus clavatus, which is a potent anti-angiogenic agent. Here, we report the identification and characterization of the cytochalasin gene cluster from A. clavatus NRRL 1. As a producer of cytochalasin E and K, the genome of A. clavatus was analyzed and the ∼30 kb ccs gene cluster was identified based on the presence of a polyketide synthase–nonribosomal peptide synthetases (PKS–NRPS) and a putative Baeyer–Villiger monooxygenase (BVMO). Deletion of the central PKS–NRPS gene, ccsA, abolished the production of cytochalasin E and K, confirming the association between the natural products and the gene cluster. Based on bioinformatic analysis, a putative biosynthetic pathway is proposed. Furthermore, overexpression of the pathway specific regulator ccsR elevated the titer of cytochalasin E from 25 mg/L to 175 mg/L. Our results not only shed light on the biosynthesis of cytochalasins, but also provided genetic tools for increasing and engineering the production.