Efficiency of new fungal cellulase systems in boosting enzymatic degradation of barley straw lignocellulose

This study examined the cellulytic effects on steam-pretreated barley straw of cellulose-degrading enzyme systems from the five thermophilic fungi Chaetomium thermophilum, Thielavia terrestris, Thermoascus aurantiacus, Corynascus thermophilus, and Myceliophthora thermophila and from the mesophile Penicillum funiculosum. The catalytic glucose release was compared after treatments with each of the crude enzyme systems when added to a benchmark blend of a commercial cellulase product, Celluclast, derived from Trichoderma reesei and a beta-glucosidase, Novozym 188, from Aspergillus niger. The enzymatic treatments were evaluated in an experimental design template comprising a span of pH (3.5-6.5) and temperature (35-65 degrees C) reaction combinations. The addition to Celluclast + Novozym 188 of low dosages of the crude enzyme systems, corresponding to 10 wt % of the total enzyme protein load, increased the catalytic glucose yields significantly as compared to those obtained with the benchmark Celluclast + Novozyme 188 blend. A comparison of glucose yields obtained on steam-pretreated barley straw and microcrystalline cellulose, Avicel, indicated that the yield improvements were mainly due to the presence of highly active endoglucanase activity/activities in the experimental enzyme preparations. The data demonstrated the feasibility of boosting the widely studied T. reeseicellulase enzyme system with additional enzymatic activity to achieve faster lignocellulose degradation. We conclude that this supplementation strategy appears feasible as a first step in identifying truly promising fungal enzyme sources for fast development of improved, commercially viable, enzyme preparations for lignocellulose degradation.     

Rotational diffusion of the ADP/ATP translocator in the inner membrane of mitochondria and in proteoliposomes

The ADP/ATP translocator was selectively labeled with the triplet probe eosin-5-maleimide (EMA) after pretreatment with N-ethylmaleimide in beef heart mitochondria, as reported previously for submitochondrial particles (Müller, M., Krebs, J. J. R., Cherry, R. J., and Kawato, S. (1982) J. Biol. Chem. 257, 1117-1120). The EMA binding was completely inhibited by carboxyatractylate. 0.7-1.1 molecules of EMA conjugated with 1 molecule of the dimeric translocator with Mr approximately 65,000. The EMA binding decreased [14C]ADP uptake by about approximately 25%. The EMA-labeled translocator bongkrekate complex was purified and reconstituted in liposomes by removing Triton X-100 with Amberlite XAD-2. The liposomes were composed of phosphatidylcholine/phosphatidylethanolamine/cardiolipin and the lipid to protein ratio by weight was (L/P) = 60. Rotational diffusion of the ADP/ATP translocator around the membrane normal was measured in reconstituted proteoliposomes and in the mitochondrial inner membranes by observing the flash-induced absorption anisotropy, r(t), of EMA. In proteoliposomes with L/P = 60, the translocator was rotating with an approximate average rotational relaxation time of phi congruent to 246 microseconds and a normalized time-independent anisotrophy [r3/rr(0)]min congruent to 0.55. In intact mitochondria, values of phi congruent to 405 microseconds and r3/rr(0) congruent to 0.79 were obtained. The higher value of r3/rr(0) in mitochondria compared with proteoliposomes indicates the co-existence of rotating and immobile translocator (phi greater than 20 ms) in the inner mitochondrial membrane. Based on the assumption that all the translocator is rotating in the lipid-rich proteoliposomes, the population of the mobile translocator at 20 degrees C was calculated to be approximately 47%. By removing the outer membrane, the mobile population was increased to approximately 70% in mitoplasts, while approximately 53% of the translocator was rotating in submitochondrial particles. The above results indicate a significant difference in protein-protein interactions of the ADP/ATP translocator in the different types of inner membranes of mitochondria. The immobile population of the translocator could be due to nonspecific protein aggregates caused by the very high concentration of proteins in the inner membrane of mitochondria (L/P approximately 0.4).     

Rotation of cytochrome P-450. II. Specific interactions of cytochrome P-450 with NADPH-cytochrome P-450 reductase in phospholipid vesicles

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles using a cholate dialysis technique. The co-reconstitution of the enzymes was demonstrated in proteoliposomes fractionated by centrifugation in a glycerol gradient. The proteoliposomes catalyzed the N-demethylation of a variety of substrates. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. The rotational mobility of cytochrome P-450, when reconstituted alone, was found to be dependent on the lipid to protein ratio by weight (L/P450) (Kawato, S., Gut, J., Cherry, R. J., Winterhalter, K. H., and Richter, C. (1982) J. Biol. Chem. 257, 7023-7029). About 35% of cytochrome P-450 was immobilized and the rest was rotating with a mean rotational relaxation time phi 1 of about 95 mus in L/P450 = 1 vesicle. In L/P450 = 10 vesicles, about 10% of P-450 was immobile and the rest was rotating with phi 1 congruent to 55 mus. Co-reconstitution of equimolar amounts of NADPH-cytochrome P-450 reductase into the above vesicles results in completely mobile cytochrome P-450 with a phi 1 congruent to 40 mus. Only a small decrease in the immobile fraction of cytochrome P-450 is observed when the molar ratio of cytochrome P-450 to the reductase is 5. The results suggest the formation of a monomolecular 1:1 complex between cytochrome P-450 and NADPH-cytochrome P-450 reductase in the liposomes.     

Divergence of the hyperthermophilic archaea Pyrococcus furiosus and P. horikoshii inferred from complete genomic sequences.

Divergence of the hyperthermophilic Archaea, Pyrococcus furiosus and Pyrococcus horikoshii, was assessed by analysis of complete genomic sequences of both species. The average nucleotide identity between the genomic sequences is 70-75% within ORFs. The P. furiosus genome (1.908 mbp) is 170 kbp larger than the P. horikoshii genome (1.738 mbp) and the latter displays significant deletions in coding regions, including the trp, his, aro, leu-ile-val, arg, pro, cys, thr, and mal operons. P. horikoshii is auxotrophic for tryptophan and histidine and is unable to utilize maltose, unlike P. furiosus. In addition, the genomes differ considerably in gene order, displaying displacements and inversions. Six allelic intein sites are common to both Pyrococcus genomes, and two intein insertions occur in each species and not the other. The bacteria-like methylated chemotaxis proteins form a functional group in P. horikoshii, but are absent in P. furiosus. Two paralogous families of ferredoxin oxidoreductases provide evidence of gene duplication preceding the divergence of the Pyrococcus species.     

A technique for simultaneous antikinetochore immunofluorescence staining and Q-banding in chromosomes from human lymphocytes

Antikinetochore immunofluorescence staining has been used in several studies to determine whether a second kinetochore is present, active, or both, in multicentric chromosomes. All of these studies have used tissue culture cells, and contended with the problem of obtaining well spread, banded metaphase chromosomes without affecting the kinetochore staining. We have adapted hypotonic, centrifugation and chromosome staining techniques to obtain simultaneous Q-banding and bright kinetochore staining of chromosomes from human lymphocytes.     

Cytogenetic characterization of 20 lymphoblastoid lines derived from human individuals differing in bleomycin sensitivity

Summary Forty lymphoblastoid (lymphoid) lines were established from 42 volunteer blood donors, including healthy individuals and patients with head and neck carcinomas. Each peripheral blood sample was split into two portions, one for the establishment of a lymphoid line and the other for short-term culture, which was used to estimate bleomycin sensitivity by cytogenetic procedures. Twenty lymphoid lines were selected at random to compare bleomycin sensitivity with data obtained from short-term lymphocyte cultures. In each set, bleomycin sensitivity of lymphoid cells was similar to that of the lymphocytes. The lymphoid lines, which can be propagated for an unlimited supply of relatively homogeneous cellular material, will be useful for a variety of future investigations. This investigation was supported by grants from the John S. Dunn Foundation, Houston, TX, the Esther Knispel Fund administered by The University of Texas M. D. Anderson Cancer Center, Houston, TX, and Department of Health and Human Services PHS grant DE 07007.     

Polarised absorption spectroscopy of chlorophyll-lipid membranes

A technique has been developed for measuring visible absorption spectra of chlorophyll in lipid membranes. An expression is derived which enables the directions of the transition moments of the different absorption bands to be determined from polarisation data. It is found that the transition moments of the principal blue and red absorption bands of chlorophyll a make angles of 26° and 36.5° respectively with the plane of the membrane. On the assumption that these two transitions lie in the plane of the porphyrin ring and are mutually perpendicular, it may be deduced that the plane of the porphyrin ring is tilted at approx. 48° to the membrane surface. For chlorophyll b the transition moments of the blue and red bands are found to make angles of 29.5° and 36.5° with the plane of the bilayer, giving an angle of tilt of the porphyrin ring of approx. 51°.

These results are compared with measurements of dichroism in vivo.