Enzymatic modification of vegetable protein: Immobilization of Penicillium duponti enzyme on reconstituted collagen and the use of the immobilized-enzyme complex for solubilizing vegetable protein in a recycle reactor

Penicillium duponti enzyme was immobilized on reconstituted collagen by macromolecular complication, impregnation, and covalent crosslinking techniques. The immobilization of the enzyme on collagen has a twofold purpose: (1) providing a protein microenvironment for the proteolytic enzyme; and (2) extending the useful life the enzyme once immobilized on the collagen matrix. Two types of collagen were used, one produced by the United States Department of Agriculture and the other produced by FMC. The USDA collagen contained unhydrolyzed telepeptide linkages and required pretreatment to reduce collagenaselike activity of the enzyme. Activity analysis of the immobilized enzyme complex showed that membranes with enzyme loading less than 10 mg enzyme/g of wet membrane in the reactor were dimensionally stable. The degree of crosslinking was an important parameter. Membranes with structural opening up to three times the initial dry thickness were found to be the maximum limit for controlled release of enzyme from the collagen membrane during enzymatic reaction. Higher activities and better stability of the enzyme in collagen membrane were found for covalent crosslinking of the enzyme to treated collagen films. The hydrolysis of soybean vegetable protein with the immobilized enzyme in a recycle reactor at enzyme loading of mg/g of wet membrane at 40°C, pH 3.4, produced 56.5% of soluble protein in 10h. The production is equivalent to 1.84 h total contact time between the substrate and the immobilized enzyme. The average productivity based on a stable enzyme activity and 20g of dry membrane was 329 mg of protein/g/mg of active enzyme immobilized. The productivity of the free enzyme in a batch reactor was 62.5 mg protein/h/mg enzyme.     

Interaction of human leukocyte elastase with soluble and insoluble protein substrates. A practical kinetic approach

A progress-curve kinetic method was developed to investigate the interaction between human leukocyte elastase and macromolecular substrates, such as insoluble elastin and soluble plasma proteins. A fluorogenic, synthetic peptide (reporter substrate) was incubated in the presence of finely powdered elastin and enzyme under continuous stirring. The progress curves, which corresponded to the release of product from the reporter substrate, were very sensitive to the presence of various amounts of the macromolecular substrate. The kinetic parameters for the interaction between elastase and elastin were calculated using a pre-steady-state approach characteristic of slow-binding inhibitors. The interaction of elastase with the soluble protein substrates was studied with similar techniques, but formally treating the substrates as classical, fully competitive inhibitors. The adsorption of elastase on insoluble elastin was a time-dependent process consisting of at least three observable phases: The first step was a rapid formation of an encounter complex followed by a very slow step lasting several minutes, and the third step consisted of a steady-state release of products. On the contrary, elastase very rapidly formed productive complexes with bovine serum albumin and a human monoclonal immunoglobulin G. The progress-curve method was also suitable for analyzing the behavior of inhibitors in the presence of protein substrates. The kinetic parameters which characterize the interaction between elastase and protein substrates represent a practical tool to formulate hypotheses on the efficiency of inhibitors in vivo.     

Comparative Arrhenius plots of enzyme activity from penicillium

Comparison of the Arrhenius plots of three enzymes, formyltetrahydrofolate synthetase, glutathione reductase (GSSGR) and chorismate mutase (CM) from a thermophilic (Penicillium duponti) and a mesophilic (Penicillium chrysogenum) fungus reveals a fairly consistent pattern. In general, those enzymes extracted from mesophiles had lower activation energies than similar enzymes extracted from thermophiles. One enzyme studied, mesophilic glutathione reductase, exhibited a break in its Arrhenius plot. The allosteric enzyme studied showed slightly different sensitivities in the thermophilic versus the mesophilic extracts.     

Water insoluble enzyme columns: Kinetic study on steady state and transient conditions

The kinetic behavior of water insoluble enzyme columns was studied using the hydrolysis of GPNA (N-glutaryl-L -phenylalanine-p-nitroanilide) by α-chymotrypsin as a model. Thermal denaturation of soluble and insoluble enzyme was compared in a batch reactor and in a tubular reactor. Convection of metabolites, enzyme reaction, diffusion of metabolites across porous particles and the kinetic behavior of a water insoluble enzyme column were tested. The respective and mutual effects of the first two phenomena were comprehensively analyzed for both stationary state and transient conditions. When the ingoing substrate concentration was varying with time, convection-reaction equations were solved by digital computation. The possible effects of diffusion limitations and unstirred layers are discussed.     

Kinetic studies on soluble and insoluble urokinases.

A water-insoluble urokinase (ins-UK) was prepared by covalent coupling to an electrostatically neutral polyacrylamide derivative. The esteratic activity retained by the bound enzyme is about 70 percent of that of the soluble urokinase (UK). Comparative kinetic studies of these two forms of the enzyme were undertaken on lysine esters: N-alpha-acetyl-L-lysine-methyl ester (ALEe) and N-alpha acetylglycyl-L-lysine methyl ester (AGLMe). It was first observed that these substrates both exhibit a marked inhibitory effect toward soluble UK, whereas this phenomenon was less manifest with the insoluble form of the enzyme. Michaelis constants and maximal velocities measured at 33 degrees C, for UK and ins-UK, were identical when ALMe was used, but slightly different with AGLMe. Determination of initial velocities, at a series of pH values shows only minimal differences in the behavior of the soluble enzyme with respect to that of the insoluble form. However, over a range of temperatures, differing Km values for these two enzyme forms were obtained using AGLMe as the substrate. These last results suggest possible interactions between the substrate and the insoluble carrier of the enzyme.     

Thermostable Acid Protease Produced by Penicillium duponti K1014, a True Thermophilic Fungus Newly Isolated from Compost

A thermophilic fungus, K1014, newly derived from a compost was selected on the basis of protease productivity as the only one of 81 isolates to produce high levels of acid protease. The fungus was named Penicillium duponti K1014 based on taxonomical studies. It grew in the temperature range of 28 to 58 C, and the optimum was 45 to 50 C. These temperature characteristics showed that the fungus was the most strongly thermophilic of all the fungi next to Humicola lanuginosa. When P. duponti K1014 was grown on moistened wheat bran, maximal accumulation of acid protease occurred after 2 days at 45 to 50 C. The addition of ammonium salts, but not nitrate, was effective for the production of the acid protease. The acid protease of P. duponti K1014 was stable at 60 C for 1 hr and retained more than 65% of original activity after the treatment for 1 hr at 70 C at pH 4.7. This thermal property was different from those of the ordinary acid proteases, indicating that the enzyme is a thermostable protein.     

Expression and purification of a soluble form of penicillin-binding protein 2 from both penicillin-susceptible and penicillin-resistant Neisseria gonorrhoeae

Resistance to penicillin in non-β-lactamase-producing strains of Neisseria gonorrhoeae (CMRNG strains) is mediated in part by the production of altered forms of penicillin-binding protein 2 (PBP 2) that have a decreased affinity for penicillin. The reduction in the affinity of PBP 2 is largely due to the insertion of an aspartic acid residue (Asp-345a) into the amino acid sequence of PBP 2. Truncated forms of N. gonorrhoeae PBP 2, which differed only by the insertion of Asp-345a, were constructed by placing the region of the penA genes encoding the periplasmic domain of PBP 2 (amino acids 42–581) into an ATG expression vector. When the recombinant PBP 2 molecules were over-expressed in Escherichia coli, insoluble PBP 2 inclusion bodies, which could be isolated by low-speed centrifugation of cell lysates, were formed. These insoluble aggregates were solubilized and the truncated PBP 2 polypeptides were partially purified by cation-exchange chromatography and gel filtration in the presence of denaturant prior to the refolding of the enzyme in vitro. After renaturation, gel filtration was used to separate monomeric soluble PBP 2 from improperly folded protein aggregates and other protein contaminants. A 4-liter culture of induced E. coli cells yielded 1.4 mg of soluble PBP 2 or PBP 2′ (PBP 2 containing the Asp-345a insertion), both of which were estimated to be 99% pure. The affinity of soluble PBP 2′ for [³H]penicillin G was decreased fourfold relative to that of soluble PBP 2, and their affinities were found to be identical to the affinities of the full-length PBP 2 enzymes that were previously determined in N. gonorrhoeae membranes. Furthermore, soluble PBP 2 displayed a rank order of affinity for several other β-lactam antibiotics that was consistent with the rank order of affinities previously reported for the native molecules. On the basis of these results, both of these soluble PBPs should be suitable for crystallization and X-ray crystallographic analysis.     

High-level expression of a soluble snake venom enzyme,gloshedobin, in E. coli in the presence of metal ions

The mature gene of gloshedobin, a snake venom thrombin-like enzyme from the snake, Gloydius shedaoensis, was cloned and expressed in strain E. coli BL21(DE3). Having been induced by IPTG, the recombinant gloshedobin was in both soluble and insoluble forms. To avoid inclusion body formation, expression was optimized at 25 °C. Furthermore, a 50% increase in solubilization of the target protein was obtained by adding 0.1 mM Mg²⁺ to the medium. The purified recombinant gloshedobin gave a 44 kDa band on SDS-PAGE gel.     

Expression of Giardia duodenalis beta-tubulin as a soluble protein in Escherichia coli

The beta-tubulin gene of the parasitic protozoan Giardia duodenalis has been expressed for the first time using a novel and direct method. The protein was expressed in both soluble and insoluble forms in an Escherichia coli-based expression system. The level of expression was found to be affected by several variables including the incubation temperature, length of time for which expression was carried out, and the E. coli culture volume. The protein expression system contributed no additional amino acids to the final fusion protein and the polyhistidine fusion sequence was easily removed from the beta-tubulin protein using a specific enterokinase enzyme. The expression system also provided a means of preparing a soluble protein and purifying it by a relatively straightforward affinity chromatography method to give a very high level of protein purity. This makes the protein suitable for a number of applications for characterization including beta-tubulin antibody assays, alpha-/beta-tubulin-binding regions, and beta-tubulin folding intermediates.     

Cellulase production with Penicillium iriense (n. sp.)

Summary A new cellulase producing species of penicillium, named Penicillium iriense, has been isolated. Cultures of this fungus in liquid media containing cellulose as carbon source. excrete into the medium an enzyme complex able to degrade both soluble and insoluble forms of cellulose. This complex has been separated into five protein fractions. Three of them are endowed with CM-cellulase activity, one contains a cellobiase and one contains a C₁-like factor. These fractions show a moderate synergism in the attack of cotton fibres.     

Maize genotypes classified as null at theglu locus have β-glucosidase activity and immunoreactive protein

Maize β-glucosidase (β-d-glucoside glucohydrolase; EC 3.2.1.21) was extracted from coleoptiles of 15 maize genotypes (3 normals, 10 nulls, and 2 hybrids) in two fractions, the soluble and the insoluble. The enzyme activity was measured spectrophotometrically in the soluble fraction and also studied on zymograms after native gel electrophoresis and isoelectric focusing. The enzyme was purified from a normal genotype by anion-exchange chromatography and preparative electrophoresis. Antisera were raised in four rabbits, and the soluble and the insoluble extracts of each genotype were analyzed for a cross-reacting material by ELISA and immunoblotting. The results showed that extracts from both the normal and the null genotypes had β-glucosidase activity, and the activity measured spectrophotometrically was 2- to 10-fold higher in normals than in nulls. Zymograms of the null genotypes were devoid of distinct bands that were present in those of normals and hybrids from crosses between normals and nulls. Zymograms of both the normal and the null genotypes had a diffuse, smeared zone of activity at the cathodic end of native gels. A cross-reacting antigen was present in extracts of both genotypes when assayed by ELISA and a 60-kD polypeptide (β-glucosidase monomer) was detected by four different monospecific β-glucosidase antisera on Western blots by immunostaining. Moreover, six of seven null genotypes had a larger amount of their 60-kD polypeptide in the insoluble fraction than in the soluble fraction. These data show that both the null and the normal genotypes have similar amounts of the enzyme protein, but the enzyme occurs mostly as insoluble or poorly soluble polymers in nulls, and the monogenic inheritance reported for the null alleles of theglu locus is likely to be for a factor encoded by another locus which affects directly or indirectly the solubility of the enzyme by increasing its polymerization into large quaternary structures.     

Physiological consequence of expression of soluble and active hen egg white lysozyme in Escherichia coli

Hen egg white lysozyme was expressed as a protein fusion with the OmpA signal sequence and an octapeptide linker in Escherichia coli. The expression yielded soluble and enzymatically active lysozyme. Lysozyme activity was detected in the periplasmic space, in the cytosol and in the insoluble cytosolic fraction of E. coli. The results indicate that the environmental conditions in both the cytosol and the periplasmic space of E. coli were sufficient for correct protein folding and disulphide bond formation of eukaryotic recombinant lysozyme. However, the expression of active enzyme in E. coli consequently led to bacterial cell lysis due to hydrolysis of the peptidoglucan. Correspondence to: B. Fischer     

High level of expression of the Toxoplasma gondii-recombinant Rop2 protein in Escherichia coli as a soluble form for optimal use in diagnosis

The rhoptry 2 protein (Rop2) is an interesting protein of Toxoplasma gondii that is involved in the parasite invasion of host cell, it has three T-cell epitopes and high antigenic value. However, the expression of Rop2 as a recombinant protein in Escherichia coli is not an easy task, showing low levels of expression or degradation and solubility problems. Using a recombinant Rop2_196–561 fused to 6 histidine residues, we showed high levels of expression in bacteria growing in terrific broth. rRop2_196–561 was purified mainly as a soluble product and in high concentrations (approx 1 mg/mL) under native conditions (40 mM imidazol in phosphate buffer). However, after a cycle of freezing-thawing rRop2_196–561 became insoluble. When glycerol was added to 26%, immediately after purification, the protein stayed soluble after cycles of freezing-thawing. Finally, it was demonstrated that under these conditions soluble rRop2_196–561 keeps its diagnostic value in contrast with the insoluble protein.     

Requirement for a major soluble protein in the conversion of lanosterol to cholesterol by membrane-bound enzymes

The conversion of the 30-carbon atom sterol, lanosterol, to cholesterol by a series of membrane-bound rat liver enzymes requires one major soluble protein called squalene and sterol carrier protein (SCP). This homogenous low-molecular-weight liver protein was previously known to function with membrane-bound enzymes catalyzing cholesterol synthesis from 27-carbon atom precursor sterols. To define characteristics of the multienzyme system catalyzing lanosterol metabolism and the role of SCP in this process, a rapid spectroscopic assay was developed, i.e., formation of Δ^5,7-cholestadienol from lanosterol. In addition to SCP, the cofactor requirements for synthesis of cholesterol from lanosterol are NAD, NADPH, and oxygen. Metal ions, reducing agents, heme, or heme-containing proteins are not required. Another homogeneous, low-molecular-weight protein, which accompanies SCP during purification steps, does not support sterol metabolism by membrane-bound enzymes. The broad functions of SCP in cholesterol synthesis and metabolism coupled with its remarkable abundance (~8% of the liver-soluble proteins), ubiquitous occurrence, and recently discovered functions in fatty acid metabolism suggest SCP plays an important regulatory role in lipid metabolism.     

Effect of molecular chaperones on the soluble expression of alginate lyase inE. coli

When the alginate lyase gene (aly) fromPseudoalteromonas elyakovii was expressed inE. coli, most of the gene product was organized as aggregated insoluble particles known as inclusion bodies. To examine the effects of chaperones on soluble and nonaggregated form of alginate lyase inE. coli, we constructed plasmids designed to permit the coexpression ofaly and the DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results indicate that coexpression ofaly with the Dnak/DnaJ/GrpE chaperone together had a marked effect on the yield alginate lyase as a soluble and active form of the enzyme. It is speculated this result occurs through facilitation of the correct folding of the protein. The optimal concentration ofl-arabinose required for the induction of the DnaK/DnaJ/GrpE chaperone was found to be 0.05 mg/mL. An analysis of the protein bands on SDS-PAGE gel indicated that at least 37% of total alginate lyase was produced in the soluble fraction when the DnaK/DnaJ/GrpE chaperone was coexpressed.     

Properties of soluble fusions between mammalian aspartic proteinases and bacterial maltose-binding protein

The mammalian aspartic proteinases procathepsin D and pepsinogen form insoluble inclusion bodies when expressed in bacteria. They become soluble but nonnative when synthesized as fusions to the carboxy terminus of E. coli maltose-binding protein (MBP). Since these nonnative states of the two aspartic proteinases showed no tendency to form insoluble aggregates, their biophysical properties were analyzed. The MBP portions were properly folded as shown by binding to amylose, but the aspartic proteinase moieties failed to bind pepstatin and lacked enzymatic activity, indicating that they were not correctly folded. When treated with proteinase K, only the MBP portion of the fusions was resistant to proteolysis. The fusion between MBP and cathepsin D had increased hydrophobic surface exposure compared to the two unfused partners, as determined by bis-ANS binding. Ultracentrifugal sedimentation analysis of MBP–procathepsin D and MBP–pepsinogen revealed species with very large and heterogeneous sedimentation values. Refolding of the fusions from 8 M urea generated proteins no larger than dimers. Refolded MBP–pepsinogen was proteolytically active, while only a few percent of renatured MBP–procathepsin D was obtained. The results suggest that MBP–aspartic proteinase fusions can provide a source of soluble but nonnative folding states of the mammalian polypeptides in the absence of aggregation.     

Active,soluble recombinant melittin purified by extracting insoluble lysate of Escherichia coli without denaturation

Cell lytic peptides are a class of drugs that can be used to selectively kill invading organisms or diseased cells. Several of these peptides have been identified as potential therapeutics. Herein, we report a novel process for purifying recombinant melittin, a cell lytic peptide that inserts into the membranes of cells causing cell lysis, from Escherichia coli. The process involves surfactant and low pH to solubilize melittin fusion proteins from the insoluble fraction of bacterial lysates. We are able to significantly improve purity of the final product and confirm the activity of the peptide. The process yields recombinant melittin that is effective when used to treat U‐87 MG glioma cells and inhibits growth of the gram‐positive pathogenic bacterium Streptococcus pyogenes. We demonstrate a method of repeated extraction of the insoluble protein fraction with mild detergent at a low pH that is able to generate a yield of pure, soluble melittin of ～0.5–1 mg/L of E. coli culture. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1150–1157, 2013     

Effect of protein malnutrition on the metabolism of uteral collagen in the rat

Protein malnutrition in the rat resulted in a reduction in the syntehsis of collagen as determined by changes in the specific and total radioactivity incorporated following a single injection of [³H]-proline. It was also accompanied by a retardation in the maturation of soluble to insoluble collagen. In addition, protein deficiency was accompanied by decreased rates of catabolism of both soluble and insoluble collagen. Presented at Symposium of the Society of Biological Chemists, India, held at Lucknow in October, 1979.     

Total soluble protein profiles of Beauveria bassiana and their relationship with virulence against Helicoverpa armigera

Intracellular total soluble proteins of Beauveria bassiana are believed to play an important role in virulence against insect hosts. Thirty B. bassiana isolates collected from different geographical regions and host ranges were characterised by total soluble proteins present in cells, using the SDS–PAGE technique to differentiate the isolates based on virulence and host insect origin. In vitro analysis of total soluble protein profiles of 30 isolates was studied to understand the relationship of isolates with their host of origin and virulence against Helicoverpa armigera. There was a positive relationship between virulence and host origin. All the non-virulent isolates are grouped together. Similarly, highly virulent isolates against H. armigera were grouped together. The relationship between total soluble proteins and pathogenicity was positively correlated. Thirty isolates shared only 22% similarity in their protein profiles.     

Characterization of an insoluble adenosine deaminase complexing protein from human kidney

Evidence for the presence of an insoluble form of adenosine deaminase complexing protein in human kidney has been obtained. An initial study demonstrated that binding of monomeric adenosine deaminase to particulate material from kidney was saturable and could be blocked by preincubating the enzyme with soluble complexing protein. Treatment of particulate material with deoxycholate, followed by immunoassay of the detergent extract, confirmed the presence of an insoluble form of complexing protein in the kidney. Several other human organs examined by this technique contained smaller amounts of insoluble complexing protein. Complexing protein isolated from the soluble and particulate fractions of kidney homogenates were found to be structurally similar. The proteins had the same subunit M_r and showed complete crossreactivity with antiserum to soluble complexing protein. Indirect immunoperoxidase staining of renal cortical tissue revealed that complexing protein was concentrated in the brush border of the proximal tubules. These results indicate that (a) the soluble and insoluble forms of complexing protein from human kidney may be products of the same gene(s) and (b) a portion of the complexing protein in human kidney is bound to the brush border membranes of cells lining the proximal tubules.