Acid protease from Trichoderma reesei: limited proteolysis of fungal carbohydrases

Mechanisms regulating post-secretory limited proteolysis, carried out by the acid protease from Trichoderma reesei, were studied by following the release of α-galactosidase and multiple forms of cellobiohydrolase from this species. Both the rate of the proteolysis and the mode of action of the protease were affected by the pH of the culture medium, and only weakly depended on the amount of the enzyme. At pH between 2.7 and 3.5 the proteolytic reaction was limited, while at lower pH proteins were completely digested. Proteolysis depended on the degree of glycosylation of secreted enzymes. Inhibition of post-secretory deglycosylation decreased the rate of limited proteolysis in the culture medium in the course of fungal growth. Glucose and cellobiose, the main products of cellulose degradation carried out by the fungal cellulolytic complex, inhibited the proteolysis of the cellobiohydrolase in a concentration-dependent manner. A 32-kDa aspartic protease (EC 3.4.23.18) secreted by T. reesei was purified to homogeneity. The acid protease cleaved α-galactosidase and cellobiohydrolase into the same proteolytic fragments that had been isolated from the culture medium. Received: 4 December 1998 / Received revision: 22 February 1999 / Accepted: 5 March 1999     

Degradation of myofibrillar proteins by a calpain-like proteinase in the arm muscle of Octopus vulgaris

The effects of a calpain-like proteinase (CaDP) isolated from the arm muscle of Octopus vulgaris on the myofibrils and myofibrillar proteins isolated from the same tissue were examined. Our studies clearly showed that treatment of intact myofibrils with CaDP in the presence of 5 mM Ca²⁺ results in the degradation of the major myofibrillar proteins myosin, paramyosin, and actin. From the isolated α- and β-paramyosins only β-paramyosin is degraded by CaDP in the presence of 5 mM Ca²⁺ producing three groups of polypeptides of 80, 75, and 60 kDa, respectively. The degradation rate depends on the proteinase to substrate ratio, temperature, and time of proteolysis and is inhibited by the endogenous CaDP inhibitory factor (CIF), as well as by various known cysteine proteinase inhibitors (E-64, leupeptin, and antipain). From the other myofibrillar proteins examined myosin, but not actin, is degraded by CaDP; myosin heavy chain (MHC, 200 kDa) is degraded by CaDP producing four groups of polypeptides of lower molecular masses (155, 125, 115, and 102 kDa, respectively); the degradation rate depends on the incubation time and the proteinase to substrate ratio. Furthermore, CaDP undergoes limited autolysis in the presence of both the exogenous casein and the endogenous β-paramyosin producing two large active fragments of 52 and 50.6 kDa, respectively; CIF reversibly inhibits this CaDP autolysis. Accepted: 26 May 2000     

SDS-dependent proteases induced by ABA and its relation to Rubisco and Rubisco activase contents in rice leaves

Protease activities and its relation to the contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase were investigated in detached leaves of rice (Oryza sativa L.) floated on the solutions containing abscisic acid (ABA) or benzyladenine (BA). Rubisco and Rubisco activase contents were decreased during the time course and the decreases were enhanced by ABA and suppressed by BA. The decrease in Rubisco activase was faster than that in Rubisco. SDS-dependent protease activities at 50–70 kDa (rice SDS-dependent protease: RSP) analyzed by the gelatin containing PAGE were significantly enhanced by ABA. RSPs were also increased in attached leaves during senescence. RSPs had the pH optimum of 5.5, suggesting that RSPs are vacuolar protease. Both decrease in Rubisco and Rubisco activase contents and increase in RSPs activities were suppressed by cycloheximide. These findings indicate that the activities of RSPs are well correlated with the decrease in these protein contents. Immunoblotting analysis showed that Rubisco in the leaf extracts was completely degraded by 5 h at pH 5.5 with SDS where it was optimal condition for RSPs. However, the degradation of Rubisco did not proceed at pH 7.5 without SDS where it is near physiological condition for stromal proteins. Rubisco activase was degraded at similar rate under both conditions. These results suggest that RSPs can functions in a senescence related degradation system of chloroplast protein in rice leaves. Rubisco activase would be more susceptible to proteolysis than Rubisco under physiological condition and this could affect the contents of these proteins in leaves.     

Light-independent degradation of stromal proteins in intact chloroplasts isolated from Pisum sativum L. leaves: requirement for divalent cations

Intact chloroplasts were isolated from mature pea (Pisum sativum L.) leaves in order to study the degradation of several stromal proteins in organello. Changes in the abundances of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), glutamine synthetase (EC 6.3.1.2) and ferredoxin-dependent glutamine:α-ketoglutarate aminotransferase (glutamate synthase; EC 1.4.7.1) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Coomassie-staining of the gels or immunoblotting using specific antibodies for the different enzymes. Degradation of several stromal proteins was strongly stimulated when intact chloroplasts were incubated in the light in the presence of dithiothreitol. Since free radicals may artificially accumulate in the chloroplast under such conditions and interfere with the stability of stromal proteins, the general relevance of these processes remains questionable. In the absence of light, proteolysis proceeded slowly in isolated chloroplasts and was not stimulated by dithiothreitol. Inhibition by ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or excess zinc ions as well as the requirement for divalent cations suggested that a zinc-containing metalloprotease participated in this process. Furthermore, light-independent degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase was enhanced in chloroplasts isolated from leaves in which senescence was accelerated by nitrogen starvation. Our results indicate that light-independent stromal protein degradation in intact chloroplasts may be analogous to proteolysis that occurs in intact leaves during senescence. Received: 3 July 1997 / Accepted: 15 October 1997     

Degradation of naphthalene by cells of Pseudomonas sp. strain NGK 1 immobilized in alginate, agar and polyacrylamide

A Pseudomonas sp. strain NGK 1 (NCIM 5120) was immobilized in various matrices, namely, alginate, agar (1.8 × 10¹¹ cfu g⁻¹ beads) and polyacrylamide (1.6 × 10¹¹ cfu g⁻¹ beads). The degradation of naphthalene was studied, by freely suspended cells (4 × 10¹⁰ cfu ml⁻¹) and immobilized cells in batches, with shaken culture and continuous degradation in a packed-bed reactor. Free cells brought about the complete degradation of 25 mmol naphthalene after 3 days of incubation, whereas, a maximum of 30 mmol naphthalene was degraded by the bacteria after 3–4 days of incubation with 50 mmol and 75 mmol naphthalene, and no further degradation was observed even after 15 days of incubation. Alginate-entrapped cells had degraded 25 mmol naphthalene after 3.5 days of incubation, whereas agar- and polyacrylamide-entrapped cells took 2.5 days; 50 mmol naphthalene was completely degraded by the immobilized cells after 6–7 days of incubation. Maximum amounts of 55 mmol, 70 mmol and 67 mmol naphthalene were degraded, from an initial 75 mmol naphthalene, by the alginate-, agar- and polyacrylamide-entrapped cells after 15 days of incubation. When the cell concentrations were doubled, 25 mmol and 50 mmol naphthalene were degraded after 2 and 5.5 days of incubation by the immobilized cells. Complete degradation of 75 mmol naphthalene occurred after 10 days incubation with agar- and polyacrylamide-entrapped␣cells, whereas only 60 mmol naphthalene was degraded by alginate-entrapped cells after 15 days of␣incubation. Further, with 25 mmol naphthalene, alginate-, agar- and polyacrylamide-entrapped cells (1.8 × 10¹¹ cfu g⁻¹ beads) could be reused 18, 12 and 23 times respectively. During continuous degradation in a packed-bed reactor, 80 mmol naphthalene 100 ml⁻¹ h⁻¹ was degraded by alginate- and polyacrylamide-entrapped cells whereas 80 mmol naphthalene 125 ml⁻¹␣h⁻¹ was degraded by agar-entrapped cells. Received: 21 October 1997 / Received revision: 15 January 1998 / Accepted: 18 January 1998     

Purification and characterization of lanatoside 15′-O-acetylesterase from Digitalis lanata Ehrh.

Lanatoside 15′-O-acetylesterase (LAE) from in-vitro-cultivated cells of Digitalis lanata Ehrh. was isolated and partially sequenced. The enzyme was extracted with citrate buffer from acetone dry powder. It was purified in a two-step chromatographical procedure including Phenyl Sepharose hydrophobic interaction chromatography followed by CM Sepharose cation-exchange chromatography to more than 330 μmol · s⁻¹ · (g protein)⁻¹. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified protein showed a major band at 39 kDa. The protein was identified by correlation of band intensity on SDS-PAGE and enzyme activity of CM Sepharose column fractions. Size-exclusion chromatography on Sephacryl 200 revealed a single activity peak with an apparent molecular mass of about 85 kDa. Electrophoresis under nondenaturating conditions of purified LAE showed only one band with esterase activity. The intensity of this band was correlated with that of the 39-kDa band after SDS-PAGE. About 30% of the protein, including the N-terminus and several fragments obtained by Lys-C protease digestion, was sequenced. A fragment obtained by Lys-C digestion showed partial homology to other hydrolases and apoplasmic proteins. It included the probable location of an active-site histidine. The activity of LAE was high in non-morphogenic D. lanata cell strains selected for high activities in the chemical transformation of cardenolides, but rather low in the proembryogenic masses of the embryogenic cell strain VIII. It increased during the development of somatic embryos. The LAE activity in leaves of D. lanata plants was in the range 4–24 nmol · s⁻¹ · (g protein)⁻¹. Received: 25 March 1997 / Accepted: 3 July 1997     

Characterization of ATP-dependent proteolysis in embryos of the brine shrimp, Artemia franciscana

Under anoxia, embryos of Artemia franciscana enter a state of quiescence. During this time protein synthesis is depressed, and continued degradation of proteins could jeopardize the ability to recover from quiescence upon return to favorable conditions. In this study, we developed an assay for monitoring ATP/ubiquitin-dependent proteolysis in order to establish the presence of this degradation mechanism in A. franciscana embryos, and to describe some characteristics that may regulate its function during anoxia-induced quiescence. For lysates experimentally depleted of adenylates, supplementation with ATP and ubiquitin stimulated protein degradation rates by 92 ± 17% (mean ± SE) compared to control rates. The stimulation by ATP was maximal at concentrations ≥11 μmol · l⁻¹. In the presence of ATP and ubiquitin, ubiquitin-conjugated proteins were produced by lysates during the course of the 4-h assays, as detected by Western blotting. Acute acidification of lysates to values approximating the intracellular pH observed under anoxia completely inhibited ATP/ubiquitin-dependent proteolysis. Depressed degradation was also observed under conditions where net ATP hydrolysis occurred. These results suggest that ATP/ubiquitin-dependent proteolysis is markedly inhibited under cellular conditions promoted by anoxia. Inhibition of proteolysis during quiescence may be one critical factor that increases macromolecular stability, which may ultimately govern the duration of embryo survival under anoxia. Accepted: 2 November 1999     

Association of carbonic anhydrase with a Calvin cycle enzyme complex in Nicotiana tabacum

Chloroplast-localized carbonic anhydrase (CA; EC 4.2.1.1), an enzyme which catalyzes the reversible hydration of CO₂, appears to be associated with other enzymes of the Calvin cycle in a large multienzyme complex. Gel-filtration fast protein liquid chromatography (FPLC) of soluble proteins obtained by osmotic lysis of tobacco (Nicotiana tabacum L. cv. Carlson) chloroplasts results in the co-elution of a protein complex of greater than 600 kDa which includes CA, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoribulokinase (PRK), and ribose-5-phosphate isomerase. Anion-exchange FPLC of chloroplast extracts indicates that there is an association of CA with other proteins that modifies its elution profile in a NaCl gradient, and that Rubisco co-elutes with the fractions containing CA. Following a protocol described by Süss et al. (1993, Proc Natl Acad Sci USA 90: 5514–5518), limited protease treatment of chloroplast extracts was used to show that the association of PRK with other chloroplast proteins appears to protect a number of lysine and arginine residues which may be involved in specific protein-protein interactions. A similar treatment of CA indicates some protection of these residues when CA is associated with other chloroplast polypeptides but the level of protection is not as profound as that exhibited by PRK. In concert with previously published immunolocalization studies, these data indicate that CA may be associated with Rubisco at the stromal periphery of a Calvin cycle enzyme complex in which PRK is more centrally located and associated with thylakoid membranes. Received: 2 June 1997 / Accepted: 28 June 1997     

Treatment of cyanide-containing wastewater from the food industry in a laboratory-scale fixed-bed methanogenic reactor

During the process of producing cassava starch from Manihot esculenta roots, large amounts of cyanoglycosides were released, which rapidly decayed to CN⁻ following enzymatic hydrolysis. Depending on the varying cyanoglycoside content of the cassava varieties, the cyanide concentration in the wastewater was as high as 200 mg/l. To simulate anaerobic stabilization, a wastewater with a chemical oxygen demand (COD) of about 20 g/l was prepared from cassava roots and was fermented in a fixed-bed methanogenic reactor. The start-up phase for a 99% degradation of low concentrations of cyanide (10 mg/l) required about 6 months. After establishment of the biofilm, a cyanide concentration of up to 150 mg CN⁻/l in the fresh wastewater was degraded during anaerobic treatment at a hydraulic retention time of 3 days. All nitrogen from the degraded cyanide was converted to organic nitrogen by the biomass of the effluent. The cyanide-degrading biocoenosis of the anaerobic reactor could tolerate shock concentrations of cyanide up to 240 mg CN⁻/l for a short time. Up to 5 mmol/l NH₄Cl (i.e. 70 mg N/l = 265 mg NH₄Cl/l) in the fresh wastewater did not affect cyanide degradation. The bleaching agent sulphite, however, had a negative effect on COD and cyanide removal. For anaerobic treatment, the maximum COD space loading was 12 g l⁻¹ day⁻¹, equivalent to a hydraulic retention time of 1.8 days. The COD removal efficiency was around 90%. The maximum permanent cyanide space loading was 50 mg CN⁻ l⁻¹ day⁻¹, with tolerable shock loadings up to 75 mg CN⁻ l⁻¹day⁻¹. Under steady-state conditions, the cyanide concentration of the effluent was lower than 0.5 mg/l. Received: 15 August 1997 / Received revision: 10 October 1997 / Accepted: 14 October 1997     

Thermophilic acidification of dairy wastewater

Acidification of simulated dairy wastewater was conducted in an upflow reactor at 55 °C. Results showed that the degree of acidification decreased with the increase in chemical oxygen demand (COD) loading rate, from 60.8% at 4 g l⁻¹ day⁻¹ to 27.1% at 24 g l⁻¹ day⁻¹. Carbohydrate was readily degraded at all loading rates, but degradation of protein and lipid decreased with the increase in loading rate. Most carbohydrate degradation occurred at the reactor bottom, whereas protein was degraded mainly after the carbohydrate became depleted. The predominant acidification products were acetate, propionate, butyrate and ethanol, whereas formate, i-butyrate, valerate, i-valerate, caproate, lactate, methanol, propanol and butanol were present in lesser quantities. The increase in loading rate resulted in the increase of propionate and the decrease of acetate, but had little effect on ethanol and butyrate productions. Only 2.5–8.8% of influent COD was converted to hydrogen and methane. The biomass yield was 0.30–0.43 mg VSS mg⁻¹ COD. Received: 8 December 1999 / Received revision: 14 February 2000 / Accepted: 25 February 2000     

Massive breakdown of the Photosystem II polypeptides in a mutant of the cyanobacterium Synechocystis sp. PCC 6803

Degradation of the D1 protein of the Photosystem II (PS II) complex was studied in the Fad6/desA::Km^r mutant of a cyanobacterium Synechocystis sp. PCC 6803. The D1 protein of the mutant was degraded during solubilization of thylakoid membranes with SDS at 0°C in darkness, giving rise to the 23 kDa amino-terminal and 10 kDa carboxy-terminal fragments. Moreover, the D2 and CP43 proteins were also degraded under such conditions of solubilization. Degradation of the D2 protein generated 24, 17 and 15.5 kDa fragments, and degradation of the CP43 protein gave rise to 28, 27.5, 26 and 16 kDa fragments. The presence of Ca²⁺ and urea protected the D1, D2 and CP43 proteins against degradation. Degradation of the D1 protein was also inhibited by the presence of a serine protease inhibitor suggesting that the putative protease involved belonged to the serine class of proteases. The protease had the optimum activity at pH 7.5; it was active at low temperature (0°C) but a brief heating (65°C) during solubilization destroyed the activity. Interestingly, the protease was active in isolated thylakoid membranes in complete darkness, suggesting that proteolysis may be a non-ATP-dependent process. Proteolytic activity present in thylakoid membranes seemed to reside outside of the PS II complex, as demonstrated by the 2-dimensional gel electrophoresis. These results represent the first (in vitro) demonstration of strong activity of a putative ATP-independent serine-type protease that causes degradation of the D1 protein in cyanobacterial thylakoid membranes without any induction by visible or UV light, by active oxygen species or by any chemical treatments.     

Generation of a histidine-tagged antibotulinum toxin antibody fragment in E. coli: effects of post-induction temperature on yield and IMAC binding-affinity

Recombinant E. coli clones expressing a 50-kDa poly-histidine tail tagged antibody fragment against botulinum toxin (bt-Fab) were initially screened for yield and binding affinity. One clone was selected for bioprocess development. The selected bt-Fab vector was induced by addition of IPTG and the protein was targeted to the periplasm by inclusion of a pelB leader sequence. A histidine₆ affinity ligand at the heavy chain C-terminus facilitated single-step purification by immobilized metal-affinity chromatography (IMAC). Notably, the effects of post-induction temperature on bt-Fab expression and downstream purification were evaluated. Our results demonstrated that fermentation conditions interfered with purification on the IMAC column at 37°C. Protease analysis by gelatin polyacrylamide gel electrophoresis (GPAGE) indicated the presence of a membrane-bound ∼39 kDa protease activity shortly after induction. The appearance of the protease activity was inversely correlated with the bt-Fab yield. The protease was purified and was shown to degrade bt-Fab. A simple kinetic model was developed describing temporal regulation of protease and bt-Fab degradation. Partially degraded bt-Fab was unrecoverable by IMAC, presumably due to the loss of the His₆ affinity ligand. The amount of purified bt-Fab obtained per liter of fermentation broth was typically ∼1 mg. Received 18 August 1997/ Accepted in revised form 4 October 1998     

Extracellular production of an intact and biologically active human growth hormone by the Bacillus brevis system

The characteristic features of the Bacillus brevis system are very high productivity of heterologous proteins and very low extracellular protease activity. However, degradation of some heterologous proteins, especially mammalian proteins, can be observed and resulted in a lowering of protein productivity. By using a mutant expressing low levels of proteases and the addition of EDTA to the medium, intact human growth hormone (hGH) was successfully produced with the B. brevis system. Signal peptide modification with higher basicity in the amino terminal region and higher hydrophobicity in the middle region brought about a twelve-fold increase in hGH production. The hGH yield was further elevated to 240 mg L⁻¹ by optimization of culture conditions. Thus, biologically active and mature hGH can be efficiently produced directly in the medium with the B. brevis system. Received 06 March 1997/ Accepted in revised form 03 July 1997     

Production of recombinant herpes simplex virus protease in 10-L stirred vessels using a baculovirus–insect cell expression system

A gene expression system using recombinant Autographa californica nuclear polyhedrosis virus (baculovirus) and Sf-9 cells has been scaled up to the 10-L tank level and shown to be capable of producing herpes simplex virus (HSV) protease in serum-free media. High densities of Spodoptera frugiperda (Sf-9) cells were achieved by modifying two 10-L Biolafitte fermenters specifically for insect cell growth. The existing Rushton impellers were replaced by marine impellers to reduce shear and the aeration system was modified to allow external addition of air/O₂ mixtures at low flow rates through either the sparge line or into the head space of the fermenter. To inoculate the tanks, Sf-9 cells were adapted to grow to high cell densities (6–10 × 10⁶ cells ml⁻¹) in shake flasks in serum-free media. With these procedures, cell densities of 5 × 10⁶ cells ml⁻¹ were routinely achieved in the 10-L tanks. These cells were readily infected with recombinant baculovirus expressing the 247-amino acid catalytic domain of the HSV-1 strain 17 protease UL26 gene as a glutathione-S-transferase (GST) fusion protein (GST-247). Three days after infection at a multiplicity of infection (MOI) of 3 pfu cell⁻¹, the GST-247 fusion protein was purified from a cytoplasmic lysate by Glutathione Sepharose 4-B affinity chromatography with reproducible yields of 11–38 mg L⁻¹ of recombinant protein and ≥ 90% purity. Maximum production of this protein was observed at a cell density of 5.0 × 10⁶ cells ml⁻¹. Received 09 December 1996/ Accepted in revised form 13 April 1997     

Metabolism of EDTA and its metal chelates by whole cells and cell-free extracts of strain BNC1

The influence of metal ions on the metabolism of ethylenediaminetetraacetate (EDTA) by whole cells and cell-free extracts of strain BNC1 was investigated. Metal-EDTA chelates with thermodynamic stability constants below 10¹² were readily mineralized by whole cells with maximum specific turnover rates of 15 (MnEDTA) to 20 (Ca-, Mg-, and BaEDTA) μmol g protein⁻¹ min⁻¹. With the exception of ZnEDTA, chelates with stability constants greater than 10¹² were not oxidized at a significant rate. However, it was shown for Fe(III)EDTA that even strong complexes can be degraded after pretreatment by addition of calcium and magnesium salts in the pH range 9–11. The range of EDTA chelates converted by cell-free extracts of strain BNC1 did not depend on their thermodynamic stabilities. The EDTA chelates of Ba²⁺, Co²⁺, Mg²⁺, Mn²⁺, and Zn²⁺ were oxidized whereas Ca-, Cd-, Cu-, Fe-, Pb-, and SnEDTA were not. The first catabolic enzyme appears to be an EDTA monooxygenase since it requires O₂, NADH, and FMN for its activity and yields glyoxylate and ethylenediaminetriacetate as products. The latter is further degraded via N,N′-ethylenediaminediacetate. The maximum specific turnover rate with MgEDTA, the favoured EDTA species, was 50–130 μmol g protein⁻¹ min⁻¹, and the K _m value was 120 μmol/l (K _s for whole cells = 8 μmol/l). Whole cells as well as cell-free extracts of strain BNC1 also converted several structural analogues of EDTA. Received: 4 July 1997 / Received revision: 25 September 1997 / Accepted: 29 September 1997     

Biodegradation of cyanides, cyanates and thiocyanates to ammonia and carbon dioxide by immobilized cells of Pseudomonas putida

Pseudomonas putida utilizes cyanide as the sole source of carbon and nitrogen. Agar, alginate, and carrageenan were screened as the encapsulating matrices for P. putida. Alginate-immobilized cells of P. putida degraded sodium cyanide (NaCN) more efficiently than non-immobilized cells or cells immobilized in agar or carrageenan. The end products of biodegradation of cyanide were identified as ammonia (NH₃) and carbon dioxide (CO₂). These products changed the medium pH. In bioreactors, the rate of cyanide degradation increased with an increase in the rate of aeration. Maximum utilization of cyanide was observed at 200 ml min⁻¹ of aeration. Immobilized cells of P. putida degraded cyanides, cyanates and thiocyanates to NH₃ and CO₂. Use of Na[¹⁴C]-CN showed that 70% of carbon of Na[¹⁴C]-CN was converted into ¹⁴CO₂ and only 10% was associated with the cell biomass. The substrate-dependent kinetics indicated that the K _m and V _max values of P. putida for the substrate, NaCN were 14 mM and 29 nmol of oxygen consumed mg protein⁻¹ min⁻¹ respectively. Received 29 January 1996/ Accepted in revised form 19 September 1997     

Mesophilic and thermophilic anaerobic digestion of source-sorted organic wastes: effect of ammonia on glucose degradation and methane production

The wet organic fraction of household wastes was digested anaerobically at 37 °C and 55 °C. At both temperatures the volatile solids loading was increased from 1 g l⁻¹ day⁻¹ to 9.65 g l⁻¹ day⁻¹, by reducing the nominal hydraulic retention time from 93 days to 19 days. The volatile solids removal in the reactors at both temperatures for the same loading rates was in a similar range and was still 65% at 19 days hydraulic retention time. Although more biogas was produced in the thermophilic reactor, the energy conservation in methane was slightly lower, because of a lower methane content, compared to the biogas of the mesophilic reactor. The slightly lower amount of energy conserved in the methane of the thermophilic digester was presumably balanced by the hydrogen that escaped into the gas phase and thus was no longer available for methanogenesis. In the thermophilic process, 1.4 g/l ammonia was released, whereas in the mesophilic process only 1 g/l ammonia was generated, presumably from protein degradation. Inhibition studies of methane production and glucose fermentation revealed a K _i (50%) of 3 g/l and 3.7 g/l ammonia (equivalent to 0.22 g/l and 0.28 g/l free NH₃) at 37 °C and a K _i (50%) of 3.5 g/l and 3.4 g/l ammonia (equivalent to 0.69 g/l and 0.68 g/l free NH₃) at 55 °C. This indicated that the thermophilic flora tolerated at least twice as much of free NH₃ than the mesophilic flora and, furthermore, that the thermophilic flora was able to degrade more protein. The apparent ammonia concentrations in the mesophilic and in the thermophilic biowaste reactor were low enough not to inhibit glucose fermentation and methane production of either process significantly, but may have been high enough to inhibit protein degradation. The data indicated either that the mesophilic and thermophilic protein degraders revealed a different sensitivity towards free ammonia or that the mesophilic population contained less versatile protein degraders, leaving more protein undegraded. Received: 26 March 1997 / Received revision: 13 May 1997 / Accepted: 19 May 1997     

Effect of Mg2+ on the Structure and Function of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Mg²⁺ in various concentrations was added to purified Rubisco in vitro to gain insight into the mechanism of molecular interactions between Mg²⁺ and Rubisco. The enzyme activity assays showed that the reaction between Rubisco and Mg²⁺ was two order, which means that the enhancement of Rubisco activity was accelerated by low concentration of Mg²⁺ and slowed by high concentration of Mg²⁺. The kinetics constant (K _m) and V _max was 1.91 μM and 1.13 μmol CO₂ mg⁻¹ protein∙min⁻¹, respectively, at a low concentration of Mg²⁺, and 3.45 μM and 0.32 μmol CO₂∙mg⁻¹ protein∙min⁻¹, respectively, at a high concentration of Mg²⁺. By UV absorption and fluorescence spectroscopy assays, the Mg²⁺ was determined to be directly bound to Rubisco; the binding site of Mg²⁺ to Rubisco was 0.275, the binding constants (K _A) of the binding site were 6.33 × 10⁴ and 5.5 × 10⁴ l·mol⁻¹. Based on the analysis of the circular dichroism (CD) spectra, it was concluded that the binding of Mg²⁺ did not alter the secondary structure of Rubisco, suggesting that the observed enhancement of Rubisco carboxylase activity was caused by a subtle structural change in the active site through the formation of the complex with Mg²⁺.     

Cardenolide 16′-O-glucohydrolase from Digitalis lanata. Purification and characterization

A three-step chromatographic procedure was developed for purification of cardenolide 16′-O- glucohydrolase (CGH) from Digitalis lanata Ehrh. leaves, including Phenyl-Sepharose hydrophobic interaction chromatography followed by SP-Sepharose cation exchange and Q-Sepharose anion-exchange chromatography. Starting with acetone dry powder the purification resulted in an 760-fold enrichment of CGH. Molecular weight, substrate specificity, pH optimum and temperature stability of CGH were determined. Antibodies against CGH were prepared in rabbits. The SDS gel electrophoresis of protein extracts from leaves of D. lanata and other D. species showed bands at 70␣kDa and 36 kDa reacting with the antibodies. The 70-kDa protein is the main protein stained with CGH antibodies in freshly prepared extracts of D. lanata. It may represent undegraded CGH. The 36-kDa protein is enriched in aged CGH preparations. It is probably a degradation product. Proteins related to 70-kDa and 36-kDa bands also occur in crude protein preparations from leaves of D. heywoodii P. et M. Silva, D. mariana Boiss., D. purpurea L., and D. thapsi L. indicating that CGH is also present in these species. Purified CGH was digested with proteases V8 and Lys-C and the resulting fragments obtained were sequenced. One fragment had the typical amino-acid sequence of the catalytic center of family-1 glycosyl hydrolases (EC 3.2.1.x). Cardenolide 16′-O-glucohydrolase, like the other members of this enzyme family, appeared to have a glutamic acid residue directly involved in glycosidic bond cleavage as a nucleophile. Received: 26 April 1997 / Accepted: 20 December 1997     

Maize glutathione-dependent formaldehyde dehydrogenase: protein sequence and catalytic properties

Glutathione-dependent formaldehyde dehydrogenase (FDH; EC 1.2.1.1) has been purified 3900-fold from maize cell-suspension cultures to a specific activity of 4.68 μmol (mg protein)⁻¹ min⁻¹. The homogeneous enzyme consisted of two identical subunits with a molecular mass of 42 kDa, and an isoelectric point of 5.8. Eight tryptic peptides were sequenced and gave a perfect fit to the protein sequence derived from maize Fdh cDNA (J. Fliegmann and H. Sandermann, 1997, Plant Mol Biol 34: 843–854). There was 62% identity with the eucaryotic FDH consensus sequence. Michaelis constants of approx. 20 μm (formaldehyde), approx. 50 μm (glutathione) and approx. 31 μm (NAD⁺) were determined for the maize enzyme as well as for FDH partially purified from dog lung. Besides S-hydroxymethylglutathione, pentanol-1, octanol-1, and ω-hydroxyfatty acids served as substrates for both FDH preparations. The unusual substrate specificity indicates that FDH may be involved in the detoxification of long-chain lipid peroxidation products. Received: 1 April 1998 / Accepted: 18 November 1998