The effects of bioprocess parameters on extracellular proteases in a recombinant <Emphasis Type="Italic">Aspergillus niger</Emphasis> B1-D

Although host proteases are often considered to have a negative impact upon heterologous protein production by filamentous fungi, relatively little is known about the pattern of their appearance in recombinant fungal bioprocesses. In the present study, we investigated extracellular proteases from a filamentous fungus, Aspergillus niger B1-D, genetically modified to secrete hen egg white lysozyme (HEWL). Our findings indicate that extracellular protease activity is only detected after the carbon source is completely utilised in batch cultures. The proteases are predominantly acid proteases and have optimal temperature for activity at around 45°C. Their activity could be partially inhibited by protease inhibitors, indicating the existence of at least four kinds of proteases in these culture fluids, aspartic-, serine-, cysteine-, and metallo-proteases. Oxygen enrichment does not have any noticeable effects on extracellular protease activity except that the onset of protease activity appears earlier in oxygen enrichment runs. Oxygen enrichment stimulates HEWL production substantially, and we propose that it is related to fungal morphology. Thermal stress imposed by raising process temperature (from 25 to 30 and 35°C) in early exponential phase, led to appearance of protease activity in the medium following the heat shock. Continued cultivation at high temperatures significantly reduced HEWL production, which was associated with increased activity of the extracellular proteases in these cultures.     

Acidic range titration of HEWL using a constant-pH molecular dynamics method

In this work, we present the first application to a protein of the stochastic constant-pH molecular dynamics (MD) method with the inclusion of proton tautomerism. The acidic titration of HEWL was performed under different conditions. Both generalized reaction field (GRF) and particle mesh Ewald (PME) methods were used in the treatment of the long range electrostatics and, even though the PME simulations revealed to be more stable, the better results were obtained using GRF (pK(a) RMSD of 0.82 for GRF and 1.13 for PME). The results using PME at different dielectric constants (2, 4, and 8) also revealed that there was no significant improvement in pK(a)'s prediction upon increasing the dielectric constant. The secondary structure analysis of HEWL revealed a remarkably stable protein in the acidic pH range. The beta-sheet strands (unlike the alpha-helices) seem to be destabilized upon pH decrease, suggesting that the beta-domain is less stable than the alpha-domain. The four principal alpha-helices were also ordered according to their stability in the acidic pH range and the results (4 < 1 < 2 approximately = 3) were consistent with the ones obtained in thermal denaturation studies.     

Sodium louroyl sarcosinate (sarkosyl) modulate amyloid fibril formation in hen egg white lysozyme (HEWL) at alkaline pH: a molecular insight study

Amyloid fibril formation is responsible for several neurodegenerative diseases and are formed when native proteins misfold and stick together with different interactive forces. In the present study, we have determined the mode of interaction of the anionic surfactant sarkosyl with hen egg white lysozyme (HEWL) [EC No. 3.2.1.17] at two pHs (9.0 and 13.0) and investigated its impact on fibrillogenesis. Our data suggested that sarkosyl is promoting amyloid fibril formation in HEWL at the concentration range between 0.9 and 3.0 mM and no amyloid fibril formation was observed in the concentration range of 3.0–20.0 mM at pH 9.0. The results were confirmed by several biophysical and computational techniques, such as turbidity measurement, dynamic light scattering, Raleigh scattering, ThT fluorescence, intrinsic fluorescence, far-UV CD and atomic force microscopy. Sarkosyl was unable to induce aggregation in HEWL at pH 13.0 as confirmed by turbidity and RLS measurements. HEWL forms larger amyloid fibrils in the presence of 1.6 mM of sarkosyl. The spectroscopic, microscopic and molecular docking data suggest that the negatively charged carboxylate group and 12-carbon hydrophobic tail of sarkosyl stimulate amyloid fibril formation in HEWL via electrostatic and hydrophobic interaction. This study leads to new insight into the process of suppression of fibrillogenesis in HEWL which can be prevented by designing ligands that can retard the electrostatic and hydrophobic interaction between sarkosyl and HEWL.     

Use of hen lysozyme for protection against bacterial contamination ofin vitro embryo cultures

Summary Curative treatments with antibiotics and hen egg white lysozyme (HEWL) were used to salvage embryo cultures contaminated withBaccillus subtilis. The use of HEWL gave good control ofBaccillus subtilis, but no control ofErwinia. HEWL was better than antibiotics, being much less phytotoxic. The antibiotics piperacillin, ampicillin and imipenem were also found to be ineffective againstErwinia. HEWL, at a final concentration of 1 mg per mL, was used as a preventive and curative agent for routine use in embryo culture ofTriticum aestivum and other Triticeae, as it cured from 30% to 50% of bacterial contamination problems over a one year period. Standardin vitro culture precautions remained essential, as certain bacteria were not controlled by HEWL.     

Effect of oxygen enrichment on morphology, growth, and heterologous protein production in chemostat cultures of Aspergillus niger B1-D.

The response of steady state chemostat cultures of a recombinant Aspergillus niger (B1-D), secreting both a heterologous enzyme (Hen Egg White Lysozyme [HEWL]) and a native enzyme (Glucoamylase), to varying levels of O2 enrichment of the process gas was evaluated. Formation of both the native and the foreign enzyme increased with increasing O2 supply. Conversely, biomass levels and total extracellular protein levels were generally not increased under O2 enriched conditions. Two distinct micromorphologies were apparent in these cultures, one, typically seen under O2 limiting conditions (i. e. at 0 and 10% enrichment levels), tended to be represented by long, sparsely branched hyphal elements, with low percentages of "active" length (i. e. how much of the hypha is cytoplasm filled); whilst, a second micromorphology, typical of O2 enriched cultures at 30 and 50% O2 enrichment, was represented by shorter hyphal elements, with more branching and a higher % "active" length. At these higher O2 levels, formation of a yellow pigment occurred, and signs of culture autolysis were noted. At 50% enrichment, a "stranded" aggregate morphology was apparent, possibly as a response to a hyperoxidant state. Production of both the native enzyme and HEWL correlated well with a simple morphological measure (tip number) or, with % "active" length. It is proposed the morphological changes noted in the cultures were associated with the increased production of both HEWL and glucoamylase.     

Comparative studies on extracellular protease secretion and glucoamylase production by free and immobilized <Emphasis Type="Italic">Aspergillus niger</Emphasis> cultures

The effects of cell immobilization on the secretion of extracellular proteases and glucoamylase production by Aspergillus niger were investigated under a variety of immobilization techniques and culture conditions. Immobilization was achieved by means of cell attachment on metal surfaces or spore entrapment and subsequent growth on porous Celite beads. Free-suspension cultures were compared with immobilized mycelium under culture conditions that included growth in shake flasks and an airlift bioreactor. Cell attachment on metal surfaces minimized the secretion of proteases while enhancing glucoamylase production by the fungus. Growth on Celite beads in shake-flask cultures reduced the specific activity of the secreted proteases from 128 to 61 U g⁻¹, while glucoamylase specific activity increased from 205 to 350 U g⁻¹. The effect was more pronounced in bioreactor cultures. A reduction of six orders of magnitude in protease specific activities was observed when the fungus grew immobilized on a rolled metal screen, which served as the draft tube of an airlift bioreactor. Received 29 October 2001/ Accepted in revised form 14 June 2002     

Comparative study of production of dextransucrase and dextran by cells of Leuconostoc mesenteroides immobilized on Celite and in calcium alginate beads

In fed-batch fermentation, cells of L. mesenteroides immobilized on three types of Celite were used to produce dextransucrase (DS) followed by production of dextran. A layer of calcium alginate on the porous Celite R630 particles improved their mechanical stability, increased the amount of soluble DS produced and decreased the cell leakage from the highly porous support. Enzyme production with the immobilized cell cultures was significantly affected by both pore and particle size. Immobilized cultures using Celite R648 (average particle radius of 200 mum and pore size of 0.14 mum) produced the highest total enzymatic activity, followed by Celite R633, alginate-coated Celite R630, Celite R630, and then calcium alginate beads. Culture of free cells produced about 18% more total enzymatic activity than immobilized cells in calcium alginate beads, but about 64% less than immobilized cells on Celite R630. It is expected that larger amounts of enzymatic activity than measured are immobilized inside the alginate-coated Celite R630 and calcium alginate beads due to the mass transfer limitation conferred by the dextran product formed therein. The dextran yield from conversion of sucrose to dextran and fructose with all such enzyme-enriched, immobilized-cell cultures was higher than that obtained from free-cell culture under similar conditions.     

A truncated glucoamylase gene fusion for heterologous protein secretion from Aspergillus niger

Abstract The secreted yield of hen egg-white lysozyme (HEWL) from the filamentous fungus Aspergillus niger was increased 10–20-fold by constructing a novel gene fusion. The cDNA sequence encoding mature HEWL was fused in frame to part of the native A. niger gene encoding glucoamylase ( gla A), separated by a proteolytic cleavage site for in vivo processing. Using this construct, peak secreted HEWL yields of 1 g/l were obtained in A. niger shake flask cultures compared to about 50 mg/l when using an expression cassette lacking any gla A coding sequence. The portion of gla A used in the gene fusion encoded the first 498 amino acids of glucoamylase (G498) and comprised its secretion signal, the catalytic domain and most of the O-glycosylated linker region which, in the entire glucoamylase molecule, spatially separates and links the catalytic and starch-binding domains.     

Inhibition of extracellular protease secretion by Aspergillus niger using cell immobilization

A wild-type A. niger strain was employed as a model to investigate the effect of cell immobilization on extracellular protease secretion during fermentation. A metal-coated pad of polyester latex felt was used to immobilize the cells in shake flasks. Compared with free suspension culture, the maximum specific activity of the extracellular protease from immobilized cells was reduced from 129 units/g to 28 units/g. © Rapid Science Ltd. 1998     

Changes in protein and protease activity during the growth and senescence of Paul's Scarlet rose

A study was performed to determine the relationship between the protein content and protease activity in suspension cultures of rose (Rosa cv Paul's Scarlet) grown over a 30 day period. Protein levels and protease activity were calculated on a per culture and per cell basis. Older nongrowing 14 day-old cultures possessed the largest total protease activity, but the highest concentration of protease activity per cell was in young 4 day-old rapidly dividing cells.     

Inactivation of polycaprolactone depolymerase (cutinase) in Fusarium cultures by an extracellular protease

Cultures of Fusarium moniliforme grown on polycaprolactone (PCL) or on cutin as a sole source of carbon and energy had low levels of detectable PCL depolymerase (cutinase) activity in the supernatant medium. A small peak of depolymerase activity was observed after hyphal accumulation had ceased, but this activity soon declined. The low level of the peak of activity and its decline were attributable to proteolytic inactivation of the depolymerase. A decrease in the pH of cultures coincided with the appearance of protease activity in the supernatant at about the same time as the appearance of the transient peak of depolymerase activity. Addition of protease substrates (bovine serum albumin, casein) to the culture at this time caused a dramatic although temporary increase in PCL depolymerase activity. The same effect was seen for cultures of F. solani pisi. Use of a different buffer system for the medium prevented a drop in pH and resulted in higher and stable levels of PCL depolymerase activity. Received 1 July 1998/ Accepted in revised form 6 December 1998     

Activation of protease by sol-gel entrapment into organically modified hybrid silicates

To prepare an immobilized protease with a high activity for transesterification of vinyl n-butyrate with 3-methyl-1-butanol (isoamyl alcohol) in organic media, a protease was entrapped into organic–inorganic hybrid silica gel on Celite 545 by the sol-gel method. When propyltrimethoxysilane was used as the organic silane precursor mixed with tetramethoxysilane at a molar ratio of 16:1, the hybrid gel-entrapped protease on Celite 545 had 8 times the activity of the protease deposited on Celite 545 from 35 to 85°C.     

Acid protease activity during germination of microcysts of the cellular slime mold Polysphondylium pallidum.

Extracts of dormant microcysts of Polysphondylium pallidum demonstrate pH optima for the hydrolysis of casein at 3.5 and 6.0. During germination the intracellular pH 6.0 caseinolytic specific activity does not change significantly. The pH 6.0 protease is also active on azo-albumin, revealing the same developmental pattern with this substrate. Both acid protease activities are excreted during the germination process. Addition of purified nonspecific protease to cultures speeds up germination, suggesting that the excreted protease may play a role in removal of the microcyst wall. When cycloheximide is added to cultures, complete germination (emergence) is stopped whereas the pH 6.0 protease activity still accumulates to between 50 and 60% of the maximum control activity. Although this suggests that post-translational controls might mediate the accumulation of a portion of the pH 6.0 protease increase, mixing and dilution experiments with cell extracts do not reveal the differential presence of soluble activators or inhibitors of this activity at different developmental stages. The presence of tightly bound enzyme-inhibitor complexes for protease B in dormant microcysts has not been ruled out and is currently under study.     

Synthesis of antioxidant propyl gallate using tannase from Aspergillus niger van Teighem in nonaqueous media

Tannase from Aspergillus niger van Teighem has been used for synthesis of food additive antioxidant propyl gallate by direct transesterification of tannic acid. The optimized yield of 86% was obtained by using simultaneously pH tuned enzyme, immobilized on Celite and using the right amount of water in the non aqueous media.     

Antiviral effects of bacteria isolated from manure

The objectives of this study were to determine the role of microbial activity in inactivation of hepatitis A virus (HAV) and to learn how the virus is inactivated. Of 31 bacterial strains isolated from animal manure, 10 efficiently inactivated HAV in fluid thioglycollate medium, with D₁₀ values (time, in days, required for a 90% reduction of virus titer) of 10 at 30°C. The D₁₀ value of the control suspension without bacteria was 35.1. Most of the 10 strains raised the pH of the medium during growth; comparisons suggested that alkalinity was not a principal antiviral property of these cultures. Cell-free filtrates of nine of these strains caused net 90% inactivation of HAV within 6 days at 37°C; the other did not. The inactivation capacity of four of the nine culture filtrates was significantly reduced by incubation with selected protease inhibitors before the virus was added. These protease inhibitors did not affect the activities of the other five culture filtrates. Fractions prepared by ultrafiltration (nominal molecular weights <1,000) from two of these cultures inactivated HAV suggesting that their mode of action was not enzymatic. Correspondence to: D.O. Cliver. mis|Department of Animal Health and Biomedical Sciences     

Inhibition of Clostridium botulinum 52A toxicity and protease activity by sodium acid pyrophosphate in media systems

The effects of two pH levels (5.55 or 5.85) in combination with 0.4% sodium acid pyrophosphate (SAPP), NaH2PO4 X H2O, Na2HPO4 X 7H2O, or NaCl on the growth and toxicity of Clostridium botulinum 52A were studied. Absorbancy measurements at 630 nm, microscopic observations, and the mouse bioassay procedure were used to observe the effects. At pH 5.55 and 5.85 most control cultures exhibited toxicity when cell lysis began. Vegetative cell development was normal (4 micron long; 1 micron wide). SAPP-containing (0.4%) treatment cultures displayed similar growth and lysis but no or delayed (48 h) toxicity. Cells grown in the SAPP treatment culture were longer and wider (6 micron long; 1.5 micron wide) than in most other treatment cultures. Trypsinization of nontoxic supernatants from 0.4% SAPP resulted in toxicity. Addition of 0.4% SAPP to toxic C. botulinum supernatant delayed but did not prevent death of mice. The addition of various levels of SAPP to toxic supernatants resulted in a decrease in zone size with an increase in the level of SAPP (9 mm with 0.4% SAPP to 7 mm with 1.0% SAPP), using a dual substrate protease assay. A decrease in the zone size also occurred with the supernatant from cultures grown in the presence of SAPP and with Bacillus polymyxa protease dilutions containing 0.4% SAPP. Results suggest that the actual production or function of the protease responsible for toxin activation may have been inhibited by the presence of SAPP.