Enzymatic degradation of liposome-grafted poly(hydroxyethyl L-glutamine)

Liposomes coated with poly(hydroxyethyl L-glutamine) (PHEG) show prolonged circulation times and biodistribution patterns comparable to PEG-coated liposomes. While PEG is a nondegradable polymer, PHEG is expected to be hydrolyzed by proteases. In this study the enzymatic degradability of PHEG both in its free form and grafted onto liposomes was investigated, using the proteases papain, pronase E, and cathepsin B. Enzymatic action was monitored with a ninhydrin assay, which quantifies amine groups formed due to hydrolysis of amide bonds, and the degradation products were characterized by MALDI-ToF mass spectrometry. PHEG, both in its free form and when grafted onto liposomes, showed degradation into low molecular weight peptides by the enzymes. Thus, we present a polymer-coated long-circulating liposome with an enzymatically degradable coating polymer, avoiding the risk of cellular accumulation.     

Enzymatic degradation of lignocellulosic biomass by continuous process using laccase and cellulases with the aid of scaffoldin for ethanol production

Biological processes for the degradation of intractable materials are still not considered to be practical due to the slow rates of enzymatic degradation. Cellulosomes are complexed enzyme systems with great degradative potential and one of the strategies for overcoming this problem. In this study, the laccase CueO from Escherichia coli was fused to the dockerin domain of a cellulosome system and further assembled with the scaffoldin miniCbpA, forming a laccase–miniCbpA complex. Compared to the individual subunits, laccase–miniCbpA complex caused a noticeable 2.1-fold increase in enzyme activity levels and enhanced degradation of various synthetic dyes, showing an increase of approximately 1.6-fold. Also, pretreated barley straw by laccase complexes was efficiently converted to bioethanol using a cellulase producing Saccharomyces cerevisiae strain. The laccase complexes caused a 2.6-fold increase in the amount of reduced sugar with an insoluble substrate in conditions with an identical amount of enzymes. The cellulolytic yeast with the aid of laccase complexes produced 2.34 g/L ethanol after 72 h, indicating an increase of approximately 2.1-fold compared to fermentation without the laccase complexes. This demonstrates the feasibility of developing an efficient laccase complex based on the cellulosome and this strategy may be used to degrade recalcitrant materials.     

Enzymatic hydrolysis of steam-pretreated lignocellulosic materials with Trichoderma atroviride enzymes produced in-house

Background  

Improvement of the process of cellulase production and development of more efficient lignocellulose-degrading enzymes are necessary in order to reduce the cost of enzymes required in the biomass-to-bioethanol process.     

Recycle of cellulases and the use of lignocellulosic residue for enzyme production after hydrolysis of steam-pretreated aspenwood

Summary Various modes of substrate and enzyme addition were used to hydrolyze a 10% concentration (w/v) of steam-exploded, water-and-alkali extracted aspenwood withTrichoderma harzianum E58 cellulases. Although cellulose conversion was high (94–100%), enzyme recovery was low in all cases. Low enzyme recovery was due to a combination of thermal inactivation and adsorption of the cellulases onto the lignocellulosic residue. Enzyme recycle was not feasible as the activity of the recovered cellulases towards crystalline cellulose was low. However, the residual material from enzyme hydrolysis was a suitable carbon source for cellulase enzyme production byT. harzianum based on enzyme yield and hydrolytic potential. These residues could only be used up to a 1% substrate concentration, since at higher substrate loadings cellulase production was reduced, likely because of lignin inhibitors.     

Enzymatic degradation of alginate by marine fungi

A total of 72 pre-selected strains of 19 species of marine fungi were tested for their ability to decompose sodium alginate, calcium alginate or freshly prepared calcium alginate gel. Active alginate decomposition was evident in 18 strains (25% of total tested). These belong to only three different species: Asteromyces cruciatus, Corollospora intermedia, and Dendryphiella salina. In broth culture, decomposition of sodium alginate by the two deuteromycetes was followed by gravimetric, electrometric, viscometric, photometric and chromatographic methods in order to characterize the alginase enzyme system and its degradation products. The alginase enzyme complex consisted of at least two different enzyme components: the already known alginate lyase (eliminase) and a new endo-alginate hydrolase. In summary, a model is presented on the alginase-mediated structural and molecular decomposition of sodium alginate by marine fungi.     

Enzymatic degradation of nitriles by Klebsiella oxytoca

Klebsiella oxytoca, isolated from cyanide-containing wastewater, was able to utilize many nitriles as sole source of nitrogen. The major objective of this study was to explore the ability of K. oxytoca to utilize some nitriles and then further evaluate the pathways of transformation of cyanide compounds by K. oxytoca. Results from this study indicate that succinonitrile and valeronitrile were the most optimal sources of nitrogen for the growth of K. oxytoca. The biodegradation of acetonitrile proceeded with the formation of acetamide followed by acetic acid. The production of ammonia was also detected in this biodegradation experiment. Similar results were observed in the propionitrile biodegradation experiments. Collectively, this study suggests that the breakdown of acetonitrile or propionitrile by this bacterium was via a two-step enzymatic hydrolysis with amides as the intermediates and organic acids plus with ammonia as the end products.     

Enzymatic saccharification of cornstalk by onsite cellulases produced by Trichoderma viride for enhanced biohydrogen production

Lignocellulosic biomass, if properly saccharified, could be an ideal feedstock for biohydrogen production. However, the high cellulases cost is the key obstacle to its development. In this work, cost‐effective enzyme produced by Trichoderma viride was used to saccharify cornstalk. To obtain high sugar yield, a central composite design of response surface method was used to optimize enzymatic saccharification process. Experimental results showed that the enzymatic saccharification rate reached the highest of 81.2% when pH, temperature, cellulases and substrate concentration were 5, 49.7 °C, 35.7 IU g^?1, and 38.5 g L^?1, respectively. The cornstalk hydrolysate was subsequently introduced to fermentation by Thermoanaerobacterium thermosaccharolyticum W16, the yield of hydrogen reached the highest level of 90.6 ml H₂ g^?1 pretreated cornstalk. The present results indicate the potential of using T. thermosaccharolyticum W16 for high yield conversion of cornstalk hydrolysate, which was saccharified by onsite enzyme produced by T. viride.     

Biostoning of denims by Penicillium occitanis (Pol6) cellulases

The Pol6 mutant of Penicillium occitanis, secreting a large quantity of cellulases, was cultivated in fermentor using a local paper pulp as an inducer substrate. A high titer of extracellular cellulase activity was reached after a fed batch process: 23 IU ml⁻¹ filter paper activity, 21 IU ml⁻¹ CMCases activity (endoglucanase units) and 25 mg ml⁻¹ of proteins. Various tests were done to compare the action of the P. occitanis cellulases with those commercially available and with the traditional stonewashing process. This cellulase preparation was successfully applied in a biostoning process at an industrial scale. The abrasive effect of the P. occitanis cellulases was very uniform and with an efficiency comparable to that obtained by the commercial ones.     

Enzymatic degradation of endomorphins

Centrally acting plant opiates, such as morphine, are the most frequently used analgesics for the relief of severe pain, even though their undesired side effects are serious limitation to their usefulness. The search for new therapeutics that could replace morphine has been mainly focused on the development of peptide analogs or peptidomimetics with high selectivity for one receptor type and high bioavailability, that is good blood-brain barrier permeability and enzymatic stability. Drugs, in order to be effective, must be able to reach the target tissue and to remain metabolically stable to produce the desired effects. The study of naturally occurring peptides provides a rational and powerful approach in the design of peptide therapeutics. Endogenous opioid peptides, endomorphin-1 and endomorphin-2, are two potent and highly selective mu-opioid receptor agonists, discovered only a decade ago, which display potent analgesic activity. However, extensive studies on the possible use of endomorphins as analgesics instead of morphine met with failure due to their instability. This review deals with the recent investigations that allowed determine degradation pathways of endomorphins in vitro and in vivo and propose modifications that will lead to more stable analogs.     

Enzymatic degradation of chlorodiamino-s-triazine

2-Chloro-4,6-diamino-s-triazine (CAAT) is a metabolite of atrazine biodegradation in soils. Atrazine chlorohydrolase (AtzA) catalyzes the dechlorination of atrazine but is unreactive with CAAT. In this study, melamine deaminase (TriA), which is 98% identical to AtzA, catalyzed deamination of CAAT to produce 2-chloro-4-amino-6-hydroxy-s-triazine (CAOT). CAOT underwent dechlorination via hydroxyatrazine ethylaminohydrolase (AtzB) to yield ammelide. This represents a newly discovered dechlorination reaction for AtzB. Ammelide was subsequently hydrolyzed by N-isopropylammelide isopropylaminohydrolase to produce cyanuric acid, a compound metabolized by a variety of soil bacteria.     

Modulation of chromatin superstructure induced by poly(ADP-ribose) synthesis and degradation

It has been demonstrated recently by Poirier et al. (Poirier, G. G., de Murcia, G., Jongstra-Bilen, J., Niedergang, C., and Mandel, P. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3423-3427) that poly(ADP-ribosyl)ation of pancreatic nucleosomes causes relaxation of the chromatin superstructure through H1 modification. The in vitro effect of poly(ADP-ribose) synthesis and degradation on calf thymus chromatin was investigated by the time course incorporation of ADP-ribose, electron microscopy, analytical ultracentrifugation, and autoradiography of the protein acceptors. Purified calf thymus poly(ADP-ribose) polymerase and partially purified bull testis poly(ADP-ribose) glycohydrolase were used. Degradation of ADP-ribose units on hyper(ADP-ribosyl)ated H1 by poly(ADP-ribose) glycohydrolase restores the native condensed chromatin superstructure. This reversible conformational change induced by poly(ADP-ribosyl)ation on nucleosomal arrangement could be one of the mechanisms by which the accessibility of DNA polymerases and/or excision-repair enzymes is favored, the native structure being fully restorable.     

New Taxa of Lespedeza (Leguminosae) from China

Lespedeza fasciculiflora Franch. var. hengduanshanensis C. J. Chen and L. davurica (Laxm.) Schindl. Subsp. huangheensis C. J. Chen are described from the Hengduan Mountains of SW China and from the Yellow Rivervalley respectively.     

Enzymatic chlorophyll degradation in wheat near-isogenic lines elicited by cereal aphid (Homoptera: Aphididae) feeding

Chlorophyll degradation enzyme (i.e., chlorophyllase, Mg-dechelatase, and chlorophyll oxidase) activities of aphid-infested and uninfested 'Tugela' and Tugela near-isogenic wheat lines (i.e., Tugela-Dn1, Tugela-Dn2, and Tugela-Dn5) were assayed. Chlorophyllase activity was higher in bird cherry-oat aphid, Rhopalosiphum padi (L.) (Homoptera: Aphididae),-infested wheat lines compared with Russian wheat aphid, Diuraphis noxia (Mordvilko) (Homoptera: Aphididae)]-infested and uninfested plants. Mg-dechelatase activity was higher in D. noxia-infested wheat lines than in R. padi-infested and uninfested plants. Also, Mg-dechelatase activity was lower in Tugela wheat infested with D. noxia than in Tugela near-isogenic lines with Dn genes. Based on the in vitro assays of chlorophyll degradation enzyme (i.e., chlorophyllase and Mg-dechelatase) activities, we proposed that the chlorotic symptoms observed on D. noxia-infested Tugela wheat were most likely to be elicited by unbalanced chlorophyll biosynthesis and degradation.     

Enzymatic activity and degradation of different kinds of organic wastes by Saccobolus saccoboloides (Fungi,Ascomycotina)

The ability to degrade organic solid wastes by the fungus Saccobolus saccoboloides was studied. The organism, unusual in such studies, was cultivated in synthetic liquid media with agitation, and on day 8 of growth the mycelium was passed to flasks with trimming. On day 16 of growth, the trimming degradation was assesed by carboxymethylcellulase, xylanase, and amylase activities evaluation, and NaOH 1% hydrolysis. Later on, the type of waste was modified (trimming, filter paper, newspaper, cardboard, sawdust and wood shaving were used) as well as the mass (300-1800 mg/flask). In these cases the enzymatic activities increased between 300 and 600 mg/flask. The total separation of the cellular components in all types of paper and cardboard was observed, together with a high loss of weight. S. saccoboloides was not able to degrade the wood wastes     

Enzymatic degradation of succinyl-coenzyme A by rat liver homogenates

When a dilute suspension of the mitochondrial fraction of rat liver homogenates was incubated with chemically synthesized succinyl-CoA, a product was rapidly formed which was retained at pH 3.9 on Dowex 50 (H⁺). Although its acid-base properties were indistinguishable from those of δ-aminolevulinic acid, the product did not form a pyrrole with acetylacetone, nor was its enzymatic formation dependent on added glycine. The enzyme which cleaved succinyl-CoA to the δ-aminolevulinic acid-like product was inhibited by phenylmethyl sulfonylfluoride. The first substance formed by the peptidase was the unstable thioester of succinic acid and cysteamine which underwent rearrangement to the more stable N-succinyl cysteamine above pH 4.0.It is apparent that the assay of δ-aminolevulinic acid synthetase (EC 2.3.1.37) by the ion-exchange method of Ebert et al. (Ebert, P.S., Tschudy, D.P., Choudhry, J.N. and Chirigos, M.A. (1970) Biochim. Biophys. Acta 208, 236–250) can yield erroneous results with succinyl-coenzyme A as substrate, especially when incubations are carried out for less than 25 min.     

Enzymatic degradation of block copolymers prepared from epsilon-caprolactone and poly(ethylene glycol)

Block copolymers were prepared by ring-opening polymerization of epsilon-caprolactone in the presence of monohydroxyl or dihydroxyl poly(ethylene glycol) (PEG), using Zn powder as catalyst. The resulting poly(epsilon-caprolactone) (PCL)-PEG diblock and PCL-PEG-PCL triblock copolymers were characterized by various analytical techniques such as NMR, size-exclusion chromatography, differential scanning calorimetry, and X-ray diffraction. Both copolymers were semicrystalline polymers, the crystalline structure being of the PCL type. Films were prepared by casting dichloromethane solutions of the polymers on a glass plate. Square samples with dimensions of 10 x 10 mm were allowed to degrade in a pH = 7.0 phosphate buffer solution containing Pseudomonas lipase. Data showed that the introduction of PEG blocks did not decrease the degradation rate of poly(epsilon-caprolactone).