Secretion of Proteinases with Fibrinolytic Activity by Micromycetes of the Genus <Emphasis Type="Italic">Aspergillus</Emphasis>

Proteolytic activity of extracellular enzymes of 11 strains of different Aspergillus species was studied. Comparison of the enzymatic indices of strains grown on agar medium containing either casein or fibrin allowed the selection of the strain Aspergillus terreus 2 as a promising producer of fibrinolytic proteases. It was found that A. terreus 2 proteinases demonstrated maximum activity at pH 8.0. The highest values of fibrinolytic and total proteolytic activities expressed in U_Tyr (amount of micromoles of tyrosine released from fibrin or casein for 1 min) were 34.0 and 358.3, respectively. Maximum activities were detected when growing the producer on a medium containing only amine nitrogen sources (fish flour hydrolysate and peptone); however, the amount of extracellular protein and the specific fibrinolytic and total proteolytic activities were greater in the medium containing both mineral and amine nitrogen sources (fish flour hydrolysate and sodium nitrate) than in the medium containing only fish flour hydrolysate and peptone as nitrogen sources.     

Extracellular Secretion of Noncatalytic Plant Cell Wall-Binding Proteins by the Cellulolytic Thermophile Caldicellulosiruptor bescii

Caldicellulosiruptor bescii efficiently degrades cellulose, xylan, and native grasses at high temperatures above 70°C under anaerobic conditions. C. bescii extracellularly secretes multidomain glycoside hydrolases along with proteins of unknown function. In this study, we analyzed the C. bescii proteins that bind to the cell walls of timothy grass by using mass spectrometry, and we identified four noncatalytic plant cell wall-binding proteins (PWBPs) with high pI values (9.2 to 9.6). A search of a conserved domain database showed that these proteins possess a common domain related to solute-binding proteins. In addition, 12 genes encoding PWBP-like proteins were detected in the C. bescii genomic sequence. To analyze the binding properties of PWBPs, recombinant PWBP57 and PWBP65, expressed in Escherichia coli, were prepared. The PWBPs displayed a wide range of binding specificities: they bound to cellulose, lichenan, xylan, arabinoxylan, glucuronoxylan, mannan, glucomannan, pectin, oligosaccharides, and the cell walls of timothy grass. The proteins showed the highest binding affinity for the plant cell wall, with association constant (K_a) values of 5.2 × 10⁶ to 44 × 10⁶ M⁻¹ among the insoluble polysaccharides tested, as measured using depletion binding isotherms. Affinity gel electrophoresis demonstrated that the proteins bound to the acidic polymer pectin most strongly among the soluble polysaccharides tested. Fluorescence microscopic analysis showed that the proteins bound preferentially to the cell wall in a section of grass leaf. Binding of noncatalytic PWBPs with high pI values might be necessary for efficient utilization of polysaccharides by C. bescii at high temperatures.     

Purification and Characterization of an Extracellular Protease from Penicillium chrysogenum Pg222 Active against Meat Proteins

An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.     

Extracellular Proteinases of Yeasts and Yeastlike Fungi

Approximately 800 yeasts and other fungi, representing over 70 species, were tested for extracellular caseinolysis. Isolates of a variety of genera, including Aureobasidium, Cephalosporium, Endomycopsis, Kluyveromyces, and numerous sporobolomycetes, demonstrated significant proteolytic activity. Caseinolysis was not necessarily correlated with gelatin liquefaction or with albuminolysis. Numerous fungi showed significant proteolysis at 5 C. The most active organisms were isolates of Candida lipolytica, Aureobasidium pullulans, Candida punicea, and species of Cephalosporium. Taxonomic and ecological implications of proteolytic activity are discussed.     

Secretion of Specific Extracellular Proteins by Somatic Embryos of Picea abies is Dependent on Embryo Morphology

Embryogenic cell lines ofPicea abieswere categorized into twogroups, A and B, based on the morphology of the somatic embryosand the ability of the somatic embryos to proceed through amaturation process when treated with ABA. Group A embryos hada distinct, densely-packed embryonic region whereas group Bembryos had loosely packed cells in their embryonic region.Embryo morphology was shown to be regulated by changes in theplant growth regulators in the culture medium. Treatment withN⁶-benzyladenine stimulated embryos to develop large embryonicregions. The morphology of somatic embryos and especially thatof the embryonic regions was correlated with the presence ofspecific extracellular proteins. Only somatic embryos with denselypacked cells in the embryonic regions secreted proteins withrelative molecular weights of 28, 66 and 85kD. The extracellularprotein of 28kD was isolated and the first 21 amino acids inthe N-terminus were identified. These showed 52–57% identitywith the N-terminal sequence conserved among members of a proteinfamily which includes zeamatin and which have been shown tobe involved in plant anti-fungal mechanisms. Immunological studiesof extracellular chitinases and zeamatin-like proteins, as wellas of activity of extracellular peroxidase, revealed a closecorrelation between the presence of specific chitinases andembryo morphology. Auxin; cytokinin; embryogenic cell lines; embryo morphology; extracellular proteins; Norway spruce; Picea abies; somatic embryos     

Extracellular Enzyme Secretion by Pseudomonas lemoignei

The ability of succinate to repress the secretion of Pseudomonas lemoignei poly-beta-hydroxybutyrate depolymerase was a function of pH. Repression only occurred when the pH of the medium was 7.0 or less. At a higher pH, lack of sensitivity to succinate concentration may have been due to a limited ability to transport succinate. Actively secreting cultures (at pH 7.4) continued to secrete enzyme for approximately 30 min after the pH was rapidly decreased to pH 6.8, even though sufficient succinate was present to repress enzyme synthesis. Similarly, after the addition of rifampin to secreting cultures, there was a 30-min delay before secretion was inhibited. Evidence is presented which suggests that continued secretion may be the result of depolymerase messenger ribonucleic acid accumulation within the cells. Studies with chloramphenicol indicated that de novo protein synthesis is necessary for the secretion of poly-beta-hydroxybutyrate depolymerase and that exoenzyme is not released from a preformed pool. Studies with various inhibitors of protein synthesis indicated that synthesis of exoenzyme is 5 to 10 times more susceptible to inhibition than is the synthesis of cell-associated proteins.     

丝状真菌胞外蛋白高效分泌机制的研究进展

丝状真菌由于其胞外蛋白分泌的高效性,成为生产酶制剂的高效细胞工厂.近年来针对真核生物胞外蛋白分泌途径的研究发现,丝状真菌蛋白的分泌途径相比其他真核生物具有高效分泌的特性.为了研究丝状真菌高效分泌的机制,本文总结了近年来丝状真菌分泌途径的最新研究进展,并且选取了分泌途径中关键环节的数种蛋白进行分析,通过与其他真核生物相关蛋白进行结构与序列比对,推测了丝状真菌胞外蛋白高效分泌的可能机制.     

Classification of Lactobacillus helveticus Strains by Immunological Differences in Extracellular Proteinases

Polyclonal antibodies against an extracellular proteinase of Lactobacillus helveticus CP53 were raised. The antibodies reacted with a 170-kDa enzyme with activity and a 53-kDa protein that seemed to be a degradation product of the 170-kDa proteinase from results of immunoblotting. The antibodies reacted also with a 45-kDa extracellular proteinase of L. helveticus CP790. However, monoclonal antibodies to the CP790 proteinase did not react with the proteinase of L. helveticus CP53. Seventeen strains of L. helveticus were tested for immunological reactivity with the two kinds of antibodies. The strains all had the same reactivity as either strain CP53 or strain CP790. Eleven strains with the 45-kDa proteinase were identified as L. helveticus biovar jugurti because they did not ferment maltose, four other strains with the 170- and 53-kDa proteins were identified as L. helveticus biovar helveticus because they fermented maltose. The remaining two strains dit not fit this pattern; they had both the 170- and 53-kDa proteins, but classification by their sugar utilization showed them to be L. helveticus biovar jugurti.     

Purification and Characterization of Extracellular Proteinases of Aspergillus oryzae

[This corrects the article on p. 507 in vol. 30.].     

Binding of Extracellular Matrix Proteins by Enterococci

Forty-four enterococcal strains isolated from human clinical specimens were investigated for binding of ¹²⁵I-labeled fibronectin, vitronectin, thrombospondin, lactoferrin, and collagen type I and IV, and for cell surface hydrophobicity. Most strains expressed low binding of iodine-labeled human fibronectin, collagen I and IV, and higher binding of human vitronectin, human lactoferrin, and human thrombospondin. Bacteria grown in Todd-Hewitt broth exhibited increased binding to vitronectin and thrombospondin. In particle agglutination assays (PAA), Enterococcus faecalis strains reacted strongly with coated latex beads in contrast to E. faecium strains, which generally did not react. The ability of enterococci to bind ECM proteins was affected by heating and proteolytic digestion, suggesting that some protein-binding components become surface exposed after treatment with proteases. The binding of ¹²⁵I-labeled proteins to E. faecalis strain E70 was inhibited when cells were preincubated with unlabeled proteins. Preincubating cells with sulfated polymers such as dextran sulfate (M _r 5000 and 8000), pentosan sulfate and heparin decreased binding of vitronectin, lactoferrin, and thrombospondin. The binding of lactoferrin and thrombospondin was also decreased when bacteria were preincubated with galactose, fucose, and mannosamine, but not with mannose. All of 30 E. faecalis strains expressed pronounced surface hydrophobicity, but 10 of 14 E. faecium strains showed hydrophilic cell surface. Received: 22 April 1996 / Accepted: 29 June 1996     

Purification and Characterization of Extracellular Proteinases of Aspergillus oryzae

The extracellular proteinases of Aspergillus oryzae EI 212 were separated into two active fractions by (NH4)2SO4 and ethanol fractionation followed by diethylaminoethyl-Sephadex A-50 and hydroxyapatite chromatography. The molecular weight was estimated by gel filtration to be about 70,000 and 35,000 for proteinases I and II, respectively. Optimum pH for casein and hemoglobin hydrolysis was 6.5 at 60 C for proteinase I and 10.0 at 45 C for proteinase II, and for gelatin hydrolysis it was 6.5 at 45 C for both enzymes. The enzymes were stable over the pH range 6 to 8 at 30 C for 60 min. The enzyme activity for both the proteinases was accelerated by Cu2+ and inhibited by Fe2+, Fe3+, Hg2+, and Ag+. Halogenators (e.g., N-chlorosuccinimide) and diisopropyl fluorophosphate inhibited proteinase II. Sulfhydryl reagents such as p-chloromercuribenzoate and iodoacetate inhibited proteinase I. Sulfhydryl compounds accelerated the action of both enzymes.     

Synthesis and Secretion of Proteinases by Bacillus intermedius in the Late Stages of Sporulation

In the late stages of sporulation, cells of Bacillus intermedius 3-19 secreted into the medium two proteinases, glutamyl endopeptidase and subtilisin, whose maximum activities were recorded in the 40th and 44th hours of growth, respectively. By estimating -galactosidase activity as a marker of cytoplasmic membrane integrity, it was revealed that the accumulation of these proteinases in the medium was a result of their secretion and not of lysis of the cell envelope. Concentrations of peptone and inorganic phosphate ensuring the maximum production of the enzymes were established. Ammonium ions were shown to inhibit the production of proteinases by the mechanism of repression by nitrogen metabolites.     

Production of patulin by Micromycetes

In the context of the screening for antifungal and antibacterial molecules among the Micromycetes, we investigated the ability of 850 strains grown in liquid medium to produce patulin. The compound was produced by 58 fungi, most of which had not been mentioned in the literature, especially Mucorales and Fungi Imperfecti. Biological tests showed that dermatophytes are the most sensitive to this molecule. It also appears that the origin of the producing strains influences their toxin producing activity.     

Cross Protection against Influenza A Virus by Yeast-Expressed Heterologous Tandem Repeat M2 Extracellular Proteins

The influenza M2 ectodomain (M2e) is well conserved across human influenza A subtypes, but there are few residue changes among avian and swine origin influenza A viruses. We expressed a tandem repeat construct of heterologous M2e sequences (M2e5x) derived from human, swine, and avian origin influenza A viruses using the yeast expression system. Intramuscular immunization of mice with AS04-adjuvanted M2e5x protein vaccines was effective in inducing M2e-specific antibodies reactive to M2e peptide and native M2 proteins on the infected cells with human, swine, or avian influenza virus, mucosal and systemic memory cellular immune responses, and cross-protection against H3N2 virus. Importantly, M2e5x immune sera were found to confer protection against different subtypes of H1N1 and H5N1 influenza A viruses in naïve mice. Also, M2e5x-immune complexes of virus-infected cells stimulated macrophages to secrete cytokines via Fc receptors, indicating a possible mechanism of protection. The present study provides evidence that M2e5x proteins produced in yeast cells could be developed as a potential universal influenza vaccine.     

Search for Micromycetes Producing Extracellular Catalase and Study of Conditions of Catalase Synthesis

Production of extracellular catalase by microscopic mycelial fungi (255 strains) belonging to different taxonomic groups was studied. Producers of extracellular catalase were found among fungi of the genera Penicillium, Talaromyces, and Aspergillus. Strains of the genus Penicillium were the most active producers. The formation of catalase depended on the initial pH, carbon and nitrogen sources and their ratio, and the content of microelements in the medium. The yield of extracellular catalase produced by the strains selected (P. chrysogenum, P. funiculosum, P. Pinophilum, and P. minioluteum) was not less than 400–1400 U per ml culture liquid.     

Metabolism of vanillic acid by Micromycetes

The ability of 953 strains of Micromycetes to grow with vanillic acid (0.5 g/l) was investigated. Toxicity assays were performed on malt extract/agar medium, while consumption was estimated by growing fungi on solid synthetic medium with vanillic acid as sole carbon source. More than half of the tested strains grew in both conditions. After cultivation on solid media, 296 strains were selected and cultivated in liquid synthetic medium. These experiments allowed division of the Micromycetes into different groups according to their consumption of the phenolic compound and the appearance of new metabolites. Results were related to the taxonomic position of the strains.     

Specific Secretion of Proline-Rich Proteins by Salt-Adapted Winged Bean Cells

Six proteins, designated SAP1 through SAP6, were secreted specificallyby salt-adapted cells of winged bean (Psophocarpus tetragonolobus)in suspension cultures. The amino-terminal amino acid sequencesof SAP2 (57 kDa), SAP4 (21 kDa), SAP5 (19 kDa) and SAP6 (17kDa) were homologous to the sequences of proline-rich proteins,indicating that proline-rich proteins are secreted specificallyby these salt-adapted cells. In addition, the amino-terminalamino acid sequence of SAP2 was identical to that of SAP4, andthe amino-terminal sequence of SAP5 was identical to that ofSAP6. Secretion of SAP2 was significantly enhanced by additionof AlCl₃ but not of KCl, LiCl, CaCl₂, MgCl₂, mannnitol or sucroseto suspension cultures. Furthermore, secretion of SAP4, SAP5and SAP6 was stimulated by addition of abscisic acid to cultures,suggesting that these proteins might be secreted in responseto salt or osmotic stress. (Received September 12, 1994; Accepted January 20, 1995)     

Heterologous Expression and Extracellular Secretion of Cellulolytic Enzymes by Zymomonas mobilis

Development of the strategy known as consolidated bioprocessing (CBP) involves the use of a single microorganism to convert pretreated lignocellulosic biomass to ethanol through the simultaneous production of saccharolytic enzymes and fermentation of the liberated monomeric sugars. In this report, the initial steps toward achieving this goal in the fermentation host Zymomonas mobilis were investigated by expressing heterologous cellulases and subsequently examining the potential to secrete these cellulases extracellularly. Numerous strains of Z. mobilis were found to possess endogenous extracellular activities against carboxymethyl cellulose, suggesting that this microorganism may harbor a favorable environment for the production of additional cellulolytic enzymes. The heterologous expression of two cellulolytic enzymes, E1 and GH12 from Acidothermus cellulolyticus, was examined. Both proteins were successfully expressed as soluble, active enzymes in Z. mobilis although to different levels. While the E1 enzyme was less abundantly expressed, the GH12 enzyme comprised as much as 4.6% of the total cell protein. Additionally, fusing predicted secretion signals native to Z. mobilis to the N termini of E1 and GH12 was found to direct the extracellular secretion of significant levels of active E1 and GH12 enzymes. The subcellular localization of the intracellular pools of cellulases revealed that a significant portion of both the E1 and GH12 secretion constructs resided in the periplasmic space. Our results strongly suggest that Z. mobilis is capable of supporting the expression and secretion of high levels of cellulases relevant to biofuel production, thereby serving as a foundation for developing Z. mobilis into a CBP platform organism.The biological conversion of lignocellulosic biomass to ethanol represents a potential major source of future domestic transportation fuels, but the current cost of converting biomass to fermentable sugars still needs to be reduced further (12). Most current strategies for ethanol production via biochemical conversion of lignocellulosic feedstocks utilize simultaneous saccharification and fermentation (SSF) or simultaneous saccharification and cofermentation (SSCF) processes (8, 21, 22). The process configuration known as consolidated bioprocessing (CBP) (20) would alleviate the financial strain of producing saccharolytic enzyme cocktails by combining the necessary steps for ethanol production as the action of one microorganism.A particularly attractive microbial candidate for the development of a CBP microorganism is the Gram-negative fermentative bacterium Zymomonas mobilis. Z. mobilis has been studied for its exceptionally high ethanol production rate, yield, and tolerance to the toxicity of the final product (15-17, 20, 31-33, 35, 43). In addition, Z. mobilis has the ability to ferment sugars at low pH and has a naturally high tolerance to many of the inhibitory compounds found in hydrolysates derived from lignocellulosic biomass (45, 46) Furthermore, the use of the Entner-Doudoroff pathway (37) allows Z. mobilis to achieve the near-theoretical maximum ethanol yields during fermentation while achieving relatively low biomass formation. Accordingly, Z. mobilis has been used successfully in SSF and SSCF processes (14, 24, 36). Additionally, Z. mobilis has been successfully engineered to ferment the pentose (C₅) sugars, xylose (45) and arabinose (10).A necessary prerequisite to establishing Z. mobilis as a CBP host is the ability to achieve high levels of cellulolytic enzyme expression. However, there is not yet a strong consensus on how to achieve maximal heterologous protein expression in Z. mobilis. Multiple groups have attempted heterologous expression of numerous genes, including cellulolytic enzymes in Z. mobilis with various degrees of success (6, 7, 9, 19, 27, 42, 44). Unfortunately, there are no obvious correlations between the expression strategies employed compared to the results obtained. Intriguingly, however, when researchers used the tac promoter (P_tac) to drive expression of native Z. mobilis genes, they were able to express several genes to extremely high levels (2). The results from this study (2) suggest that while the potential to achieve high levels of heterologous cellulase expression in Z. mobilis certainly exists, the ability to do so on a consistent basis will need further investigation.While achieving high-level expression of cellulases is an important hurdle to overcome in the development of the CBP technology, it is imperative that these enzymes additionally be translocated to the extracellular medium in order to directly contact the lignocellulosic substrate. The most obvious means by which to achieve this translocation is by harnessing the host cell''s protein secretion apparatus. There is, however, in general, very little fundamental knowledge regarding the capacity of Z. mobilis to secrete proteins. There is only one account to our knowledge of fusing secretion signals native to Z. mobilis onto proteins from exogenous sources, where extracellular secretion of a recombinant β-glucosidase reached only 11% of the total amount of enzyme synthesized (43).We initially report the finding that several Z. mobilis strains natively produce an endogenous activity against carboxymethyl cellulose (CMC) and that this activity can be detected extracellularly. Together, these results suggest that Z. mobilis may be adept at producing and secreting cellulolytic enzymes, and as this attribute is essential for a CBP organism, Z. mobilis serves as an ideal candidate for further investigation.We next describe the expression of two cellulolytic enzymes (E1 and GH12) in both Escherichia coli and Z. mobilis. E1 (locus tag Acel_0614) and GH12 (locus tag Acel_0619) are both from the acidothermophile Acidothermus cellulolyticus and are representative of glycoside hydrolase families 5 and 12, respectively (10a, 38a). E1 is an endo-1,4-β-glucanase, and GH12 is an uncharacterized enzyme that has a very high sequence identity to the GH12 domain of GuxA (Acel_0615) from A. cellulolyticus. GuxA has activities against a wide variety of substrates, including carboxymethyl cellulose, arabinoxylan, xylan, and xyloglucan (W. Adney, unpublished results). While GH12 has yet to be fully characterized, we used homology modeling (3) to predict the enzyme class of GH12 and found that it strongly resembles an endo-1,4-β-glucanase. These enzymes were chosen because of their relatively low molecular weight, high stability, and activity over a broad temperature and pH range using only the catalytic domains (Adney, unpublished). We report the successful expression of both enzymes in Z. mobilis by addressing several variables related to gene expression. Additionally, the use of codon optimization was explored as a way of enhancing heterologous expression in Z. mobilis. After successfully demonstrating the intracellular expression of E1 and GH12 in Z. mobilis, we further show that Z. mobilis is capable of secreting these proteins extracellularly through the use of native secretion signals predicted to utilize two separate protein translocation pathways in Z. mobilis, the SecB-dependent and twin arginine translocation (TAT) pathways. This finding should prove valuable beyond the production of cellulases and could include all classes of recombinant proteins.