Evidence for extrusion of unfolded extracellular enzyme polypeptide chains through membranes of Bacillus amyloliquefaciens.

The production of extracellular alpha-amylase and protease by protoplasts of Bacillus amyloliquefaciens has been achieved. The production of enzymically active protease was totally dependent on a high concentration of either Mg2+, Ca2+, or spermidine, but production of active alpha-amylase was not. This cation dependence of protease production was seen immediately upon addition of lysozyme to intact cells. The cations could prevent the inactivation of protease and alter the cytoplasmic membrane configuration of protoplasts. Production of active alpha-amylase and protease by protoplasts was totally inhibited by proteolytic enzymes such as trypsin, alpha-chymotrypsin, or the organism's purified extracellular protease. The evidence suggests that these degradative enzymes act specifically on the emerging polypeptide of the extracellular enzyme and that the polypeptide emerges in a conformation different from that of the native molecule.     

Lysosomal nature of hormonally induced enzymes in wheat aleurone cells

The subcellular distribution of the enzymes alpha-amylase, protease and ribonuclease in wheat aleurone layers after treatment with gibberellic acid was determined by differential centrifugation. Of the alpha-amylase 56% was precipitable from cell homogenates, indicating that it is a particulate enzyme. Similar results were recorded with protease. Particulate alpha-amylase showed distinct structural latency, and membrane-rupturing mechanical or chemical treatments were required to release the enzyme in an active form; the results were completely analogous to results with lysosomal enzymes found in animal tissues. The identification of the hormonally induced enzymes as lysosomal suggests that the hormonal mechanism may be more closely associated with extracellular enzyme synthesis rather than with nucleic acid metabolism.     

Production of alpha-amylase in fed-batch cultures of vgb+ and vgb- recombinant Escherichia coli: some observations.

Synthesis and excretion of Bacillus stearothermophilus alpha-amylase is analyzed in fed-batch cultivations of Escherichia coli JM103[pMK79] and E. coli JM103[pMK57], the former strain containing the plasmid-encoded Vitreoscilla hemoglobin (VHb) gene (vgb) and the latter strain being devoid of this gene. Fed-batch operation is observed to be substantially superior to batch operation as concerns the alpha-amylase production rate and the extent of excretion of the enzyme. Faster feeding of a nutrient medium (LB or M9) discourages synthesis of alpha-amylase. While synthesis of alpha-amylase in the vgb(-) strain is discouraged when oxygen availability is reduced, the reverse is the case with the vgb(+) strain, the promotion of alpha-amylase synthesis in the latter strain being linked to the synthesis of VHb. Increased availability of the principal carbon source (glucose) in a defined medium leads to overproduction of both alpha-amylase and VHb under oxygen limitation, which may be responsible for the segregational instability observed with the vgb(+) strain. The very high extents of excretion of alpha-amylase attained in fed-batch cultures are encouraging for downstream processing of the recombinant protein.     

Morphological change and enhanced pigment production of monascus when cocultured with saccharomyces cerevisiae or aspergillus oryzae

When a Monascus isolate, a producer of Monascus pigments, was cocultured with either Saccharomyces cerevisiae or Aspergillus oryzae in a solid sucrose medium, there were significant morphological changes in Monascus culture. Cocultures exhibited cell mass increases of 2 times and pigment yield increases of 30 to 40 times compared to monocultures of Monascus. However, enhanced cell growth, an increase in pigment production, and morphological change did not occur in coculture with Bacillus cereus. Saccharomyces cerevisiae was more effective at enhancing pigment production than Asp. oryzae. Enhanced cell growth and increased pigment production occurred only in conjunction with morphological changes. Culture filtrates of S. cerevisiae were also effective in inducing morphology change in Monascus, similar to culture broths of S. cerevisiae. The hydrolytic enzymes produced by S. cerevisiae, such as amylase, and chitinase, are thought to be the effectors. The commercial enzymes alpha-amylase and protease from Asp. oryzae both caused a morphological change in Monascus and were effective in enhancing pigment production. However, lysozyme, alpha-amylase and protease from Bacillus species, protease from Staphylococcus, and chitinase from Streptomyces were not effective. The hydrolytic enzymes which cause a morphological change of Monascus culture and enhancement of pigment production are thought to be capable of degrading Monascus cell walls. An approximate 10-fold increase in pigment production was observed in liquid cocultures with S. cerevisiae. Copyright 1998 John Wiley & Sons, Inc.     

A parametric study ot protease production in batch and fed-batch cultures of Bacillus firmus

Proteolytic enzymes produced by Bacillus species find a wide variety of applications in brewing, detergent, food, and leather industries. Owing to significant differences normally observed in culture conditions promoting cell growth and those promoting production of metabolites such as enzymes, for increased efficacy of bioreactor operations it is essential to identify these sets of conditions (including medium formulation). This study is focused on formulation of a semidefined medium that substantially enhances synthesis and secretion of an alkaline protease in batch cultures of Bacillus firmus NRS 783, a known superior producer of this enzyme. The series of experiments conducted to identify culture conditions that lead to improved protease production also enables investigation of the regulatory effects of important culture parameters including pH, dissolved oxygen, and concentrations of nitrogen and phosphorous sources and yeast extract in the medium on cell growth, synthesis and secretion of protease, and production of two major nonbiomass products, viz., acetic acid and ethanol. Cell growth and formation of the three nonbiomass products are hampered significantly under nitrogen, phosphorous, or oxygen limitation, with the cells being unable to grow in an oxygen-free environment. Improvement in protease production is achieved with respect to each culture parameter, leading in the process to 80% enhancement in protease activity over that attained using media reported in the literature. Results of a few fed-batch experiments with constant feed rate, conducted to examine possible enhancement in protease production and to further investigate repression of protease synthesis by excess of the principal carbon and nitrogen sources, are also discussed. The detailed investigation of stimulatory and repressory effects of simple and complex nutrients on protease production and metabolism of Bacillus firmus conducted in this study will provide useful guidelines for design of bioreactors for production of protease and bulk chemicals by this bacterium.     

Effect of partial pressure of CO2 on the production of thermostable alpha-amylase and neutral protease by Bacillus caldolyticus

Controlling the concentration of dissolved oxygen is a standard feature in aerobic fermentation processes but the measurement of dissolved CO2 concentrations is often neglected in spite of its influence on the cellular metabolism. In this work room air and room air supplemented with 5% and 10% carbon dioxide were used for aeration during the cultivation of the thermophilic microorganism Bacillus caldolyticus (DSM 405) on starch to produce alpha-amylase (E.C. 3.2.1.1) and neutral protease (E.C. 3.4.24.27/28). The increased CO2 concentrations resulted in a 22% raise in activity of secreted alpha-amylase and a 43% raise in protease activity when compared with aeration with un-supplemented room air. There was no effect on the final biomass concentration. Furthermore, the lag-phase of fermentation was reduced by 30%, further increasing the productivity of alpha-amylase production. Determinations of dissolved CO2 in the culture broth were conducted both in situ with a probe as well as using exhaust gas analysis and both the methods of quantification showed good qualitative congruence.     

Isolation, characterization, and identification of Geobacillus thermodenitrificans HRO10, an alpha-amylase and alpha-glucosidase producing thermophile

Thermophilic and amylolytic aerobic bacteria were isolated from soil through a selective enrichment procedure at 60 degrees C with starch as the carbon source. One of the isolates designated as HRO10 produced glucose aside from limit dextrin as the only hydrolysis product from starch and was characterized in detail. The starch-degrading enzymes produced by strain HRO10 were determined to be alpha-amylase and alpha-glucosidase. Whereas the alpha-amylase activity was detected exclusively in the culture supernatant, alpha-glucosidase occurred intracellular, extracellular, or on the surface of the bacteria depending on the growth phase. The optimum temperature and pH required for the growth of strain HRO10 were about 50 degrees C and pH 6.5 to 7.5. The strain used different carbohydrates as the carbon source, but the maximum production of alpha-amylase occurred when 1.0% (w/v) starch or dextrin was used. The use of organic vs. inorganic nitrogen favored the production of alpha-amylase in strain HRO10. The metal ions Li+, Mg2+, and Mn2+ stimulated the production of both enzymes. Identification of strain HRO10 by physiological and molecular methods including sequencing of the 16S rDNA showed that this strain belongs to the species Geobacillus thermodenitrificans. Biochemically, strain HRO10 differs from the type strain DSM 465 only in its ability to hydrolyze starch.     

Co-production of alpha-amylase and beta-galactosidase by Bacillus subtilis in complex organic substrates

Various nutrients belonging to three categories, carbon, organic nitrogen and complex organic sources, were investigated for the first time in terms of their effect on the co-production of extracellular thermostable alpha-amylase and beta-galactosidase by Bacillus subtilis, a bacterium isolated from fresh sheep's milk. Among the organic nitrogen sources tested, tryptone and corn steep liquor favored their production. Substitution of soluble starch by various starchy substrates, such as corn flour, had a positive effect on both enzyme yields. Furthermore, a two-fold higher production of both enzymes was achieved when corn steep liquor or tryptone was used in combination with the different flours. Among the divalent cations examined, calcium ions appeared to be vital for alpha-amylase production. The crude alpha-amylase and beta-galactosidase produced by this B. subtilis strain exhibited maximal activities at 135 degrees C and 65 degrees C, respectively, and were also found to be significantly stable at elevated temperatures.     

Pleiotropic phenomena in autolytic enzyme(s) content, flagellation, and simultaneous hyperproduction of extracellular alpha-amylase and protease in a Bacillus subtilis mutant.

A mutant of Bacillus subtilis 6160 that had been isolated by its hyperproduction of alpha-amylase and protease lacked flagella and motility, and its content of autolytic enzyme(s) was reduced to one-third to one-fourth that of the parent. These phenotypic differences were completely co-transferred by the deoxyribonucleic acid (DNA) of the mutant when five DNA recipient strains of B. subtilis were transformed. The revertants, isolated by motility with a frequency of approximately 10(-7), recovered a normal level of autolytic activity and showed reduced productivity of alpha-amylase and protease. This point mutation allowed normal flagellin synthesis, spore formation, and rate of growth. The comparison of cell envelope of the mutant with that of the parent indicated that there was no significant difference except loss of flagella. Therefore the association at the cell surface of a group of extracellular proteins consisting of alpha-amylase, proteases, flagellin, and autolytic enzymes(s) seem to be coordinately regulated by the gene or seem to be affected coordinately by certain undetected alterations of the cell envelope.     

Expression of alpha-amylase in Bacillus licheniformis.

In Bacillus licheniformis, alpha-amylase production varied more than 100-fold depending on the presence or absence of a catabolite-repressing carbon source in the growth medium. alpha-Amylase was produced during the growth phase and not at the onset of the stationary phase. Induction of alpha-amylase correlated with synthesis of mRNA initiating at the promoter of the alpha-amylase gene.     

Studies on catabolite repression in solid state fermentation for biosynthesis of fungal amylases

Catabolite repression by glucose of the biosynthesis of alpha amylase and amyloglucosidase by Aspergillus niger CFTRI 1105 was studied in a solid state fermentation (SSF) and in submerged fermentation (SMF) systems and the results were compared. The addition of glucose did not enhance the production of alpha-amylase and amyloglucosidase in an earlier fermentation system. However, a drastic reduction in alpha-amylase production was observed in submerged fermentation by the addition of 5·0 mg ml⁻¹ glucose and of amyloglucosidase production by 10 mg ml⁻¹ glucose. Glucose concentrations above 50 mg ml⁻¹ completely suppressed the production of both enzymes in the initial hours. In contrast, in the SSF system the repression was negligible, even when the glucose level was raised to 150 mg g⁻¹ wheat bran for both alpha and amyloglucosidase synthesis.     

Some observations on protease production in continuous suspension cultures of Bacillus firmus

Invariance of culture conditions in steady state continuous cultures make these a very valuable tool to study the influence of various culture parameters on cell growth and synthesis of primary and secondary metabolites. The result of a parametric study on production of protease in continuous suspension cultures of Bacillus firmus NRS 783 are reported in this article. This strain is a superior producer of an alkaline protease with major application in the detergent industry. The parameters investigated include dilution rate and concentrations of yeast extract, ammonium, and inorganic phosphate in the bioreactor feed, glucose being the principal carbon source in all experiments. The regulatory effects of the key culture parameters on cell growth, synthesis and secretion of protease, and production of acetic acid are investigated. The relations among the specific cell growth rate, specific utilization rates of the principal carbon, nitrogen, and phosphorous sources, and specific production rates of two nonbiomass products, viz., acetic acid and protease, are examined, and the effects of the manipulated culture parameters on these relations, specific protease activity, and yields of cell mass, protease, and acetic acid on the basis of the principal carbon, nitrogen, and phosphorous sources are studied. An increase in dilution rate led to increases in specific utilization rates of the principal carbon, nitrogen, and phosphorous sources and specific production rates of acetic acid and protease and decreases in bulk activities/concentrations of the three products (acetic acid, cell mass, and protease). As a result, the productivities of the three species were maximized at an intermediate dilution rate. Increased supply of yeast extract (a rich source of amino acids, proteins, and vitamins, besides being an additional source of carbon, nitrogen, and phosphorus) promoted cell mass formation but reduced protease production per unit cell mass. Increased supply of nitrogen and phosphorous sources stimulated protease synthesis up to certain threshold levels and repressed the enzyme synthesis beyond the threshold levels. With increased supply of the nitrogen source, the phosphorous source was more efficiently utilized for cell growth and protease synthesis. Stable maintenance of continuous cultures of B. firmus over prolonged period is demonstrated in this study. (c) 1993 John Wiley & Sons, Inc.     

Regulation of Exocellular Proteases in Neurospora crassa: Induction and Repression of Enzyme Synthesis

Neurospora crassa strain 74A grown on Vogel's medium containing bovine serum albumin (BSA) as principal carbon source secretes proteolytic enzymes which appear in the culture filtrate. Low concentrations of sucrose (0.1%) are necessary for growth from conidia, as conidia will not germinate on BSA alone. Once growth is initiated, however, protease production begins and at 5 to 6 hr growth and enzyme production are parallel. Higher concentrations of sucrose (0.5-2%) repress protease synthesis. Other metabolizable materials (sugars, amino acids, peptide mixtures) also repress protease synthesis. Some sugars will not sustain growth but allow germination and full induction of protease in the presence of protein. A material found in culture fluids of cells during induction of protease synthesis when added to repressed cultures causes a five-fold increase in the amount of protease production, although this is still approximately half that of normally induced cells. This material appears to be produced by induced cells in as little as 2 hr of culture, which is before detectable levels of protease can be found. It is heat-stable, of low molecular weight, and is not a simple product of protein digestion by the N. crassa proteases.     

Relationship between morological variability of Bacillus subtilis R-623 and biosynthesis of hydrolytic enzymes

By synthetic sorbent chromatography the influence of Bacillus subtilis R-623 morphology on the qualitative and quantitative composition of alpha-amylase and proteases was studied. It was found that morphological variants of natural variability of Bac. subtilis R-623, alpha-amylase producer, differed in their cultural, morphological and physiological properties as well as in the amount of hydrolytic enzymes synthesized per unit of the cultural medium. Cells of P variant produced the highest quantity of alpha-amylase (314 U/lm) and cells of P and M variants synthesized the greatest amount of proteases (4.3 and 4.0 U/ml, respectively). Quantitative variations of alpha-amylase and proteases were measured in the cultural medium of morphological variants of Bac. subtilis R-623 during cultivation. Qualitative composition of those enzymes was determined when their content in the cultural medium was at the highest level. R variant synthesized alpha-amylase and protease, P and S variants alpha-amylase and two proteases, and P and S variants alpha-amylase and two proteases, and M variant one protease.     

Secreted amylolytic enzymes from Schwanniomyces occidentalis: purification by fast protein liquid chromatography (FPLC) and preliminary characterization

Amylolytic enzyme preparations are used extensively for the liquefaction and saccharification of starch in the production of ethanol and SCP (single cell protein). We report the first purification of two amylolytic enzymes from the yeast Schwanniomyces occidentalis using fast protein liquid chromatography (FPLC) in a two step process: size exclusion (Superose 12) followed by anion exchange (Mono Q). The procedure is amenable to direct scale up processes. The enzymes glucoamylase (E.C. 3.2.1.2) and alpha-amylase (E.C. 3.2.1.1) were found in the cell free supernatant of S. occidentalis when grown on a variety of carbon sources. The enzymes are substrate induced and catabolite repressed. Both amylolytic enzymes were purified from three separate culture broths containing either starch, maltose or cellobiose and their physical properties compared. Native molecular masses of glucoamylase and alpha-amylase were determined to be 122,000 +/- 28,000 daltons and 47,000 +/- 11,000 daltons, respectively, while subunit size was approximated at 143,000 +/- 2,000 daltons and 54,500 +/- 1,000 daltons, respectively. Both proteins are N-glycosylated with carbohydrate representing 10-15% of the total mass. The correlation of native mass and denatured subunit structure, while not identical due to slight aberrant behavior on gels and columns as a result of glycosylation, suggest that both proteins exist as monomeric polypeptides. Isoelectric points for both proteins under native conditions could not be determined since alpha-amylase failed to enter native polyacrylamide gels. However, a pI for glucoamylase of 6.2 +/- 0.2 (native) and a pI for alpha-amylase of 6.3 +/- 0.3 (in 6M urea) were determined. Glucoamylase and alpha-amylase specific activities (for the homogeneous proteins) were determined to be 48-67 x 10(3) units/mg and 214-457 x 10(3) units/mg respectively. We could find no apparent differences in either glucoamylase or alpha-amylase proteins obtained from three separate cultures which had been grown on different carbon sources. The purification method we have utilized is easily scaled up to larger protein concentrations, and provides a rapid procedure for analyzing and purifying these amylolytic enzymes.     

Carbon sources affect metabolic capacities of Bacillus species for the production of industrial enzymes: theoretical analyses for serine and neutral proteases and alpha-amylase

The metabolic fluxes through the central carbon pathways were calculated for the genus Bacillus separately for the enzymes serine alkaline protease (SAP), neutral protease (NP) and alpha-amylase (AMY) on five carbon sources that have different reduction degrees (gamma), to determine the theoretical ultimate limits of the production capacities of Bacillus species and to predict the selective substrate for the media design. Glucose (gamma=4.0), acetate (gamma=4.0), and the TCA cycle organic-acids succinate (gamma=3.5), malate (gamma=3.0), and citrate (gamma=3.0) were selected for the theoretical analyses and comparisons. A detailed mass flux balance-based general stoichiometric model based on the proposed metabolic reaction network starting with the alternative five carbon sources for the synthesis of each enzyme in Bacillus licheniformis that simulates the behaviour of the metabolic pathways with 107 metabolites and 150 reaction fluxes is developed. Highest and lowest specific cell growth rates (&mgr;) were calculated as 1.142 and 0.766h(-1), respectively, when glucose that has the highest degree of reduction and citrate that has the lowest degree of reduction were used as the carbon sources. Highest and lowest SAP, NP and AMY synthesis rates were also obtained, respectively, when glucose and citrate were used. Metabolic capacity analyses showed that the maximum SAP, NP, and AMY synthesis rates were, respectively, 0.0483, 0.0215 and 0.0191mmolg(-1)DWh(-1) when glucose uptake rate was 10mmolg(-1)DWh(-1) and specific growth rate was zero. The amino acid compositions and the molecular weights of the enzyme influence the production yield and selectivity. For SAP and NP oxaloacetate and pyruvate, for AMY oxaloacetate appear to be the critical main branch points. Consequently, for SAP and NP syntheses the fluxes towards the alanine group and aspartate group, and for AMY synthesis the flux towards the aspartate group amino acids need to be high. The results encourage the discussion of the potential strategies for improving productions of SAP, NP and AMY.     

Regulation of hut enzymes and intracellular protease activities in Vibrio alginolyticus hut mutants

The production of alkaline protease, collagenase and histidine utilization (Hut) enzymes by Vibrio alginolyticus wild-type, hutH1 and hutU1 strains was investigated. Alkaline protease synthesis was stimulated by histidine and urocanic acid in the wild-type and hutU1 strains. The hutH1 mutant alkaline protease production was stimulated by urocanic acid and not by histidine. The Hut enzymes in the wild-type strain were coordinately induced by histidine. Urocanase and formimino-hydrolase were induced by histidine in the hutH1 mutant which lacked histidase and was not able to convert histidine to urocanic acid. Collagenase production in peptone medium was inhibited in the hut mutants. It is concluded that in V. alginolyticus urocanic acid regulates alkaline protease synthesis but that the Hut enzymes are induced by histidine. The involvement of the Hut genetic system in the regulation of alkaline protease and collagenase synthesis is discussed.     

Genetic Control of the Rate of α-Amylase Synthesis in Bacillus subtilis

The level of extracellular alpha-amylase (EC 3.2.1.1) of Bacillus subtilis Marburg was increased about fivefold by introducing the amyR marker from B. natto 1212 through transformation. amyR2 of B. natto 1212 has been assumed to determine a high level of alpha-amylase of the organism. The gene acts specifically on alpha-amylase synthesis but not on the production of other extracellular enzymes. alpha-Amylase of an amyR2-carrying strain was found to be quite similar to that of an isogenic amyR1-carrying strain in the thermostability and electrophoretic behavior of whichever amylase the strain produces. Marburg-type alpha-amylase (amyEm) or B. natto-alpha-amylase (amyEn). Anti-amylase serum titration indicates that a high level of the enzyme activity in the amyR2-carrying strain is caused by the existence of more enzyme rather than the presence of an enzyme having higher efficiency. This is supported further by the fact that amyR controls the synthesis of the amyE gene product in mutant M9, which synthesizes a temperature-sensitive-alpha-amylase, and in mutant M07, which secretes cross-reacting material. The results indicate that amyR regulates the rate of alpha-amylase synthesis.     

Enhanced production of alpha-amylase in fed-batch cultures of Bacillus subtilis TN106[pAT5

Growth of Bacillus subtilis TN106[pAT5] and synthesis of plasmid-encoded protein (alpha-amylase) are investigated in batch, continuous, and fed-batch cultures using a defined medium containing glucose and/or starch as the carbohydrate source. The batch culture studies reveal that reduced availability of arginine hampers growth of recombinant cells (which lack an arginine synthesis gene) but promotes production of alpha-amylase and substitution of glucose by starch as the carbohydrate source leads to slower growth of recombinant cells and increased production of alpha-amylase per unit cell mass. Retention of recombinant cells over prolonged periods in continuous cultures is not possible without continuous application of antibiotic selection pressure owing to segregational plasmid instability. Fed-batch experiments with constant volumetric feed rate demonstrate that alpha-amylase production is enhanced at lower feed concentration of starch (sole carbohydrate source) and lower volumetric feed rate. Such slow addition of starch is however not conducive for growth of recombinant cells. The expression of the thermostable alpha-amylase gene carried on the recombinant plasmid pAT5 (derived from a plasmid isolated from a thermophilic bacterium) is promoted at higher temperatures, while growth of recombinant cells is depressed. In all batch and fed-batch experiments, production of alpha-amylase is observed to be inversely related to growth of recombinant cells. The efficacy of two-stage bioreactor operations, with growth of recombinant cells being promoted in the first stage and alpha-amylase production in the second stage, in attaining increased bulk alpha-amylase activity is demonstrated. (c) 1993 John Wiley & Sons, Inc.     

Expression of Bacillus amyloliquefaciens amylase and Vibrio alginolyticus protease A fusion genes.

Previously we reported [Deane, S. M., Maharaj, R., Robb, F. T. & Woods, D. R. (1987) Journal of General Microbiology 133, 2295-2302] that the production of a Vibrio alginolyticus SDS-resistant alkaline serine protease (Pro A) cloned in Escherichia coli was characterized by a 12 h delay between the synthesis of an inactive precursor and secretion of active Pro A. Replacement of the V. alginolyticus promoter region by the alpha-amylase promoter region from Bacillus amyloliquefaciens resulted in the simultaneous synthesis and secretion of Pro A in E. coli. The V. alginolyticus pro A gene cloned on a shuttle vector did not produce active Pro A in Bacillus subtilis. Although Pro A has a typical Gram-positive signal sequence, it was not functional in B. subtilis. Replacement of the Pro A signal sequence with the alpha-amylase signal sequence resulted in the production of active Pro A in B. subtilis.