Effect of culturing processes and copper addition on laccase production by the white-rot fungus Fomes fomentarius MUCL 35117

Aim:  To produce high laccase activities from the white-rot fungus Fomes fomentarius .
Methods and Results:  Different culturing methods, viz, cell immobilization on stainless steel sponges and plastic material and solid-state fermentation (SSF) using wheat bran as substrate were used for laccase production by the white-rot fungus F. fomentarius . The SSF study expresses the highest laccase activities, nearly to 6400 U l⁻¹ after 13 days of laboratory flasks cultivation. When the wheat bran medium was supplemented with 2 mmol l⁻¹ copper sulfate, laccase activity increased by threefold in comparison to control cultures, reaching 27 864 U l⁻¹. With the medium thus optimized, further experiments were performed in a 3 l fixed-bed bioreactor (working volume 1·5 l) leading to a laccase activity of about 6230 U l⁻¹ on day 13.
Conclusions:  The results obtained clearly showed the superiority of wheat bran for laccase production over stainless steel sponges and plastic material. Supplementing the wheat bran solid medium with 2 mmol l⁻¹ copper sulfate allowed obtaining high activities at flask scale. The system was scaled to fixed-bed laboratory reactor.
Significance and Impact of the Study:  The high enzyme production along with the low-cost of the substrate, showed the suitability of the system F. fomentarius – SSF for industrial purposes.     

Influence of cultivating conditions on the alpha-galactosidase biosynthesis from a novel strain of Penicillium sp. in solid-state fermentation

AIMS: The work is intended to achieve optimum culture conditions of alpha-galactosidase production by a mutant strain Penicillium sp. in solid-state fermentation (SSF). METHODS AND RESULTS: Certain fermentation parameters involving incubation temperature, moisture content, initial pH value, inoculum and load size of medium, and incubation time were investigated separately. The optimal temperature and moisture level for alpha-galactosidase biosynthesis was found to be 30 degrees C and 50%, respectively. The range of pH 5.5-6.5 was favourable. About 40-50 g of medium in 250-ml flask and inoculum over 1.0 x 10(6) spores were suitable for enzyme production. Seventy-five hours of incubation was enough for maximum alpha-galactosidase production. Substrate as wheat bran supplemented with soyabean meal and beet pulp markedly improved the enzyme yield in trays. CONCLUSIONS: Under optimum culture conditions, the alpha-galactosidase activity from Penicillium sp. MAFIC-6 indicated 185.2 U g(-1) in tray of SSF. SIGNIFICANT AND IMPACT OF THE STUDY: The process on alpha-galactosidase production in laboratory scale may have a potentiality of scaling-up.     

l-leucine aminopeptidase production by filamentous Aspergillus fungi

AIMS: To screen various filamentous fungi belonging to Aspergillus spp. producing leucine and methionine aminopeptidases. METHODS AND RESULTS: Twenty-eight Aspergillus strains representing 14 species within the genus were screened for L-leucine aminopeptidase (LAP) production in two media in shake flask fermentation. Two Aspergillus sojae (NRRL 1988 and NRRL 6271) and one Aspergillus oryzae (NRRL 6270) strains were selected as the best producers for further studies. The peak LAP activities were 2.61, 2.59 and 1.30 IU ml(-1) for the three fungi on days 2, 5 and 4 respectively. In addition to LAP, L-methionine aminopeptidase (MAP) activity was also detected. Apart from submerged fermentation, the highest LAP yields by solid-state fermentation were 11.39, 17.40 and 13.02 IU g(-1) dry matter for the above fungi. The temperature and pH optimum of the enzyme was found to be in the range of 65-75 degrees C at pH 8.0-9.0 for all three fungi. Metal ions, Co(2+) and Fe(2+) in 2 mmol l(-1) concentration apparently enhanced the relative enzyme activity and heat stability. CONCLUSIONS: Two A. sojae (NRRL 1988 and NRRL 6271) and one A. oryzae (NRRL 6270) strains were found to be the best producers of LAP and MAP. The preliminary characterization studies revealed that the enzyme is considerably thermostable and belongs to the class metalloenzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: A good number of aspergilli were screened and the ability of the fungal aminopeptidase to release a particular N-terminal amino acid along with its high thermal stability, makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.     

Lipid bilayer tethered inside a nanoporous support: a solid-state nuclear magnetic resonance investigation

(31)P and (1)H solid-state nuclear magnetic resonance (NMR) experiments have been designed with the aim of studying directly the formation of supported bilayers tethered inside nanoporous aluminum oxide supports as a model of biomimetic membranes. The static and magic angle spinning (31)P NMR spectra of the supported bilayers have been compared with the experimental and simulated spectra of a simpler model with cylindrical geometry, namely a phospholipid bilayer adsorbed on an oriented polymer sheet. The broadening observed for the nanoporous model is most likely due to the presence of paramagnetic ions in the aluminum oxide. A phospholipid lateral diffusion coefficient of (2.8 +/- 0.4) x 10(-8) cm(2)/s has been measured for the tethered bilayer on a spherical support, indicating a good fluidity as compared with adsorbed membrane models.     

13C nuclear magnetic resonance studies on selectively labeled bacterial biofilms

Bacterial biofilms of Pseudomonas aeruginosa selectively labeled by introduction of 2-¹³C-glycerol was studied by solid-state and high-resolution nuclear magnetic resonance. The ¹³C nuclei were mainly integrated into mannuronate and guluronate, the two monomer units forming the bacterial alginate. The signal for the C5 position of the mannuronate, which was easily identified and well separated from other peaks, was analyzed for molecular mobility. The result indicated a high degree of motional freedom within the molecular network of the alginate. Despite the fact that the alginate was part of a solid aqueous gel phase, the reorientation mechanism of the monomer units came close to isotropic tumbling. Solid-state spectra of biofilms labeled in the described manner may serve as a valuable tool for noninvasive analyses of molecular mobility of the alginate component under various influences, thereby revealing important structural information. In addition, the effect of a monovalent electrolyte (LiCl) on the molecular mobility of alginate fragments in an aqueous solution was studied by determining the spin–lattice relaxation times, line widths and line shapes under variations of the ion concentration. The presence of ions accelerated overall motions but left rapid local motions virtually unaffected. Journal of Industrial Microbiology & Biotechnology (2001) 26, 62–69. Received 26 January 2000/ Accepted in revised form 30 August 2000     

Review: Model Peptides and the Physicochemical Approach to β-Amyloids

β-Amyloid peptides are the main protein components of neuritic plaques and may be important in the pathogenesis of Alzheimer's Disease. The determination of the structure of β-amyloid fibrils poses a challenge because of the limited solubility of β-amyloid peptides and the noncrystalline nature of fibrils formed from these peptides. In this paper, we describe several physicochemical approaches which have been used to examine fibrils and the fibrillogenesis of peptide models of β-amyloid. Recent advances in solid state NMR, such as the DRAWS pulse sequence, have made this approach a particularly attractive one for peptides such as β-amyloid, which are not yet amenable to high-resolution solution phase NMR and crystallography. The application of solid state NMR techniques has yielded information on a model peptide comprising residues 10–35 of human β-amyloid and indicates that in fibrils, this peptide assumes a parallel β-strand conformation, with all residues in exact register. In addition, we discuss the use of block copolymers of Aβ peptides and polyethylene glycol as probes for the pathways of fibrillogenesis. These methods can be combined with other new methods, such as high-resolution synchrotron X-ray diffraction and small angle neutron and X-ray scattering, to yield structural data of relevance not only to disease, but to the broader question of protein folding and self-assembly.     

Protein Motional Details Revealed by Complementary Structural Biology Techniques

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高产Monacolin K红曲菌株的复合诱变选育

利用紫外线和氯化锂对一株紫红曲Monascus purpureus菌株进行复合诱变试验。在最佳紫外照射时间为45s和最佳氯化锂浓度为1.0‰的情况下,获得了一株MonacolinK高产突变菌株Monascus purpureus ZT32且连续传接5代产量稳定,用紫外分光光度法检测其MonacolinK的含量为219.9μg/mL,较原始菌株高出2倍多,然后将其用于固态发酵,结果表明：红曲米中的MonacolinK的含量达到8.33mg/g,是原始菌株的3.3倍。     

The relation of the X-ray B-factor to protein dynamics: insights from recent dynamic solid-state NMR data

In addressing the potential use of B-factors derived from X-ray scattering data of proteins for the understanding the (functional) dynamics of proteins, we present a comparison of B-factors of five different proteins (SH3 domain, Crh, GB1, ubiquitin and thioredoxin) with data from recent solid-state nuclear magnetic resonance experiments reflecting true (rotational) dynamics on well-defined timescales. Apart from trivial correlations involving mobile loop regions and chain termini, we find no significant correlation of B-factors with the dynamic data on any of the investigated timescales, concluding that there is no unique and general correlation of B-factors with the internal reorientational dynamics of proteins.     

Domain swapping and amyloid fibril conformation

For several different proteins an apparent correlation has been observed between the propensity for dimerization by domain-swapping and the ability to aggregate into amyloid-like fibrils. Examples include the disease-related proteins β₂-microglobulin and transthyretin. This has led to proposals that the amyloid-formation pathway may feature extensive domain swapping. One possible consequence of such an aggregation pathway is that the resulting fibrils would incorporate structural elements that resemble the domain-swapped forms of the protein and, thus, reflect certain native-like structures or domain-interactions. In magic angle spinning solid-state NMR-based and other structural studies of such amyloid fibrils, it appears that many of these proteins form fibrils that are not native-like. Several fibrils, instead, have an in-register, parallel conformation, which is a common amyloid structural motif and is seen, for instance, in various prion fibrils. Such a lack of native structure in the fibrils suggests that the apparent connection between domain-swapping ability and amyloid-formation may be more subtle or complex than may be presumed at first glance.