Production of protease by Bacillus subtilis grown on sardinelle heads and viscera flour

Fish flours from Sardinelle (Sardinella aurita) were prepared and tested for protease production by Bacillus subtilis. Protease synthesis was strongly induced when cells were grown in media containing only a combined head and viscera preparation. Sardinelle heads and viscera flour enhanced protease production up to 100% more than commercial peptones. The enhancement could have been due to a high lipid content, which might have contained nutritional factors acting as inducers, since defatting fish meal led to a decrease in protease production.     

Culture requirements for the production of protease by Aspergillus oryzae in solid state fermentation

Summary A number of culture conditions for protease production by Aspergillus oryzae NRRL 2160 on solid substrates were investigated. The pH of the medium and the substrate markedly affected protease production. High protease yield was obtained when the fungus was cultivated for 72–96 h on rice hulls: rice bran (7:3), at an initial pH of 7.0. Maximal protease production was achieved at an initial moisture content of 35–40%, corresponding to a water activity range of 0.982–0.986. Casein and gluten were effective inducers. Polyethylene bags proved to be promising containment systems for solid state cultivation. Offprint requests to: A. M. R. Pilosof     

Kinetic characterization and fed-batch fermentation for maximal simultaneous production of esterase and protease from Lysinibacillus fusiformis AU01

The simultaneous production of intracellular esterase and extracellular protease from the strain Lysinibacillus fusiformis AU01 was studied in detail. The production was performed both under batch and fed-batch modes. The maximum yield of intracellular esterase and protease was obtained under full oxygen saturation at the beginning of the fermentation. The data were fitted to the Luedeking–Piret model and it was shown that the enzyme (both esterase and protease) production was growth associated. A decrease in intracellular esterase and increase in the extracellular esterase were observed during late stationary phase. The appearance of intracellular proteins in extracellular media and decrease in viable cell count and biomass during late stationary phase confirmed that the presence of extracellular esterase is due to cell lysis. Even though the fed-batch fermentation with different feeding strategies showed improved productivity, feeding yeast extract under DO-stat fermentation conditions showed highest intracellular esterase and protease production. Under DO-stat fed-batch cultivation, maximum intracellular esterase activity of 820?×?10³ U/L and extracellular protease activity of 172?×?10³ U/L were obtained at the 16th?hr. Intracellular esterase and extracellular protease production were increased fivefold and fourfold, respectively, when compared to batch fermentation performed under shake flask conditions.     

Regulation of Extracellular Protease Production in Bacillus cereus T: Characterization of Mutants Producing Altered Amounts of Protease

Twenty-nine mutants of Bacillus cereus T were selected on casein agar for their inability to produce large amounts of extracellular protease. They all formed spores, and 27 were also auxotrophs for purines or pyrimidines. Upon reversion to prototrophy, a large fraction regained the capacity to produce protease. Conversely, reversion to normal protease production resulted in loss of the purine or pyrimidine requirement in a large fraction of the revertants. One spontaneous low-protease-producing pyrimidine auxotroph studied in detail grew as well as the wild type and produced spores which were identical to those produced by the wild type on the basis of heat resistance, dipicolinic acid content, density, and appearance in the electron microscope. The rate of protein turnover in the mutant was the same as the wild type. The mutant did grow poorly, however, when casein was the principal carbon source. A mutant excreting 5 to 10 times as much protease as the wild type was isolated as a secondary mutation from the hypoproducer discussed above. Loss of the pyrimidine requirement in this case did not alter the regulation of protease production. Although the secondary mutant grew somewhat faster in most media than the wild type, the final cell yield was lower. The spores of this mutant appeared to have excess coat on the basis of both electron microscopic and chemical studies. There appear to be closely related but distinct catabolic controls for both extracellular protease and spore formation. These controls can be dissociated as for the hypoproducers but can also appear integrated as for the hyperprotease producer.     

Effect of environmental conditions on the production of two extracellular proteolytic enzymes byVibrio SA1

The production of two extracellular proteases, an endopeptidase and an aminopeptidase, by the marine bacteriumVibrio SA1 was studied in batch cultures. The production of the proteases was induced during growth of the organism in peptone media and by several amino acids during growth in minimal media. It was repressed by easily metabolisable carbon compounds such as glucose, lactate and succinate during growth in peptone media. During growth in a lactate basal medium, phenylalanine was one of the best inducers and this amino acid was therefore used in further experiments. That lactate dit not repress the synthesis of the proteases during growth in the lactate basal medium supplemented with 2mm phenylalanine as an inducer, appeared to be a consequence of the low iron content of this medium. Growth curves ofVibrio SA1 on such media showed a period of linear growth during which protease production was observed. When the iron concentration was made sufficiently high to prevent linear growth, the synthesis of the proteases remained repressed. Apparently by imposing an iron limitation on the organism, catabolite repression by lactate was relieved. Similarly, when growth was limited by very low values of the dissolved oxygen tension in the medium, a high rate of protease synthesis was found which was immediately repressed when the oxygen limitation was released. The results indicate that the growth rate and/or a factor associated with the energy metabolism play a role in the regulation of the synthesis of the enzymes.This study was supported by the Foundation for Fundamental Biological Research (BION), which is subsidised by the Netherlands Organization for the Advancement of Pure Research (ZWO).     

Studies on the biosynthesis of extracellular proteases by bacteria. I. Serratia marcescens,synthetic and gelatin media

Apparently the extracellular protease of S. marcescens is present in protein- or protein derivative-free media only when the conditions favor an abundant growth and bacterial conglomerates are formed, which possibly could be explained by assuming that the proteins of dead bacteria or proteic metabolic products act as inducers of protease formation. Dilute solutions of gelatin induce the production of proteolytic activity after a short period of time and its rapid increase before the initial number of bacteria has been duplicated. Calcium is necessary to preserve the activity of the enzyme formed in synthetic media but not that produced in gelatin.     

一株海洋细菌产碱性蛋白酶的研究

对1株产碱性蛋白酶的海洋细菌Pseudomonas sp.7-11发酵产生碱性蛋白酶的条件和酶的调控机制进行了初步研究。结果显示,7－11为好氧菌,具有嗜低温特性,且适宜在偏碱性的条件下生长并产酶,该菌产生碱性蛋白酶表现出初级动力学持征;酷蛋白培养基适合菌株产酶;K^ 对于菌株产酶有关键作用;复合氨基酸对产酶有抑制作用;初步显示,7－11产生蛋白酶受诱导及阻遏两种方向的调控。     

Characterization of Aeromonas hydrophila strains and their evaluation for biodegradation of night soil

Among 67 psychrotrophic bacterial isolates of Leh, India screened for production of hydrolytic enzymes at 10 °C, four belonging to Aeromonas hydrophila were characterized and evaluated for biodegradation of night soil. All strains produced metalloproteases on a variety of carbon and nitrogen sources. Strains LA1 and LA15 also produced α-amylase and PC5 both α- & β-amylase. No amylase was produced by PN7, however it produced lipase. Casein and glucose induced maximum enzyme activity (protease and amylase) in LA15 and PC5, respectively. In LA1, maximum induction of protease was observed with casein and of amylase with maltose. Corn oil/tributyrin served as the best inducers for protease and lipase production by PN7. A. hydrophila strains were found to be psychrotrophic with optimum growth and enzyme activity at 20 and 37 °C, respectively. Maximum biodegradation of night soil was observed by strain LA1 at 5–20 °C. This revised version was published online in July 2006 with corrections to the Cover Date.     

Protease production by the ontjom fungus,Neurospora sitophila

Summary The production of protease and mycelium byNeurospora sitophila cultured on solid and liquid potato dextrose media was studied. Maximal activity of protease extracted from 4-day-old cultures occurred at pH 6.5 when an unfractionated peanut (groundnut) protein substrate was used. The greatest protease activity and mycelium production occurred during the first day of the 4-day test period. Potato dextrose media containing more than 0.2 M NaCl resulted in decreased protease and mycelium production, while tapioca starch was without affect at concentrations up to 1.4%. Addition of up to 0.3 M sucrose to growth media greatly stimulated protease production and mycelial growth. Maximal proteolytic activity was observed in extracts from mycelium cultured in potato dextrose media adjusted from pH 6.0 to 7.5. Activity was greater when soluble peanut protein was used as a substrate, compared to unfractionated or globulin protein substrates.     

烷烃溶剂对耐有机溶剂极端微生物地衣芽孢杆菌YP1产胞外蛋白酶的影响

考察了添加5%(V/V)浓度的正庚烷、正辛烷、正癸烷、十二烷、十四烷、十六烷等烷烃溶剂对耐有机溶剂极端微生物地衣芽孢杆菌(Bacillus licheniformis)YP1的生长及产胞外蛋白酶的影响.结果表明5%(V/V)浓度的各种烷烃溶剂对YP1蛋白酶的稳定性及菌体生物量均无显著影响,正庚烷、正辛烷、正癸烷等溶剂显著抑制YP1产蛋白酶,而十二烷、十四烷,十六烷能提高YP1产蛋白酶1倍以上.发酵液中十四烷的浓度(1%-8%,VIV)与蛋白酶的活力呈正相关性,添加十四烷后发酵过程中蛋白酶活力的显著增加出现在菌体生长的对数后期.培养过程中添加十四烷能导致YP1菌体形态显著变小.首次报道了烷烃溶剂对极端微生物产蛋白酶的影响.     

烷烃溶剂对耐有机溶剂极端微生物地衣芽孢杆菌YP1产胞外蛋白酶的影响

考察了添加5％(V/V)浓度的正庚烷、正辛烷、正癸烷、十二烷、十四烷、十六烷等烷烃溶剂对耐有机溶剂极端微生物地衣芽孢杆菌(Bacillus licheniformis)YP1的生长及产胞外蛋白酶的影响。结果表明5％(V/V)浓度的各种烷烃溶剂对YP1蛋白酶的稳定性及菌体生物量均无显著影响, 正庚烷、正辛烷、正癸烷等溶剂显著抑制YP1产蛋白酶, 而十二烷、十四烷、十六烷能提高YP1产蛋白酶1倍以上。发酵液中十四烷的浓度(1％－8％, V/V)与蛋白酶的活力呈正相关性, 添加十四烷后发酵过程中蛋白酶活力的显著增加出现在菌体生长的对数后期。培养过程中添加十四烷能导致YP1菌体形态显著变小。首次报道了烷烃溶剂对极端微生物产蛋白酶的影响。     

Pongamia pinnata seed cake: a promising and inexpensive substrate for production of protease and lipase from Bacillus pumilus SG2 on solid-state fermentation

The production of a protease and a lipase from Bacillus pumilus SG2 on solid-state fermentation using Pongamia pinnata seed cake as substrate was studied. The seed cake was proved to be a promising substrate for the bacterial growth and the enzyme production. The initial pH, incubation time and moisture content were optimized to achieve maximal enzyme production. Maximum protease production was observed at 72 h and that of the lipase at 96 h of incubation. The production of protease (9840 U/g DM) and lipase (1974 U/g DM) were maximum at pH 7.0 and at 60% moisture content. Triton X-100 (1%) was proved to be an effective extractant for the enzymes and their optimal activity was observed at alkaline pH and at 60 C. The molecular mass of the protease and lipase was 24 and 40 kDa, respectively. Both the enzymes were found to be stable detergent additives. The study demonstrated that inexpensive and easily available Pongamia seed cake could be used for production of industrially important enzymes, such as protease and lipase.     

Bradyrhizobium japonicum nodD1 can be specifically induced by soybean flavonoids that do not induce the nodYABCSUIJ operon.

Besides genistein and daidzein, which are active inducers of the nodYABCSUIJ operon in Bradyrhizobium japonicum, soybean seeds also excrete compounds that are not inducers of the nodYABCSUIJ genes but enhance induction of this operon in the presence of a suboptimal genistein concentration. This synergism was studied in detail, and specific compounds were identified in seed exudate which specifically induce the nodD1 gene but not the nodYABCSUIJ operon. Therefore, our current hypothesis is that the observed synergism is caused by a specific induction of nodD1. The specific nodD1 inducers from soybean seed extract have been purified and characterized chemically. They appear to be derivatives of genistein, glycitein, and daidzein with glucose, malonyl, and acetyl groups attached. Both root and seed exudate appear to contain these compounds, with the seed being the major source. No hydrolysis of these compounds to their aglycone forms was detected in the presence of B. japonicum. A model for nod gene induction in B. japonicum is discussed.     

Solid state fermentation: acid protease production in controlled CO2 and O2 environments

The effect of the partial pressure of O(2) and CO(2) on the acid protease production in solid state fermentation by Aspergillus niger on wheat bran was studied. A fermentation system was used, which allowed on-line reactor measurements and continuous data acquisition of pH, temperature, gas flow, pressure drop and CO(2) production. Six paired combinations of CO(2) and O(2) concentrations were studied. The results showed a direct relationship between pressure drop, production of CO(2) and temperature increase. The pH evolution patterns were similar in all cases but different if the measurements were made on-line or on a liquid homogenate of the fermented substrate. Acid protease production was increased when the gas had 4% CO(2), (vol/vol), and it reached its highest level, a 43% increase over air, with a mixture of 4% CO(2) and 21% O(2). The protease production was strongly related to the mold metabolic activity as represented by the total CO(2) evolved.     

Genetics of Extracellular Protease Production in SACCHAROMYCOPSIS LIPOLYTICA

Mutants of Saccharomycopsis lipolytica with reduced ability to produce zones of clearing on skim-milk agar plates were isolated and their properties studied. For 18 mutants it was possible to score unambiguously segregants of crosses between these mutants and wild type for extracellular protease production. These mutants all produce reduced levels of extracellular protease in liquid culture. The mutations are recessive and are in nuclear genes. The 18 mutations define 10 or 11 complementation groups, no two of which are closely linked. Mutants in four of the complementation groups also produced reduced levels of extracellular RNAse, and the reduced levels of extracellular protease and RNAse production segregate together. Five of the mutants exhibited reduced mating frequency, and one mutant was osmotic remedial for extracellular protease production. These results demonstrate that many genes can affect extracellular protease production. Besides mutations in the structural gene and in regulatory genes, mutations are likely to be in genes involved in steps common to the production of several extracellular enzymes or in genes coding for cell wall or membrane components necessary for extracellular enzyme production.     

Alterations of interferon production in a mouse model of thermal injury

The effect of thermal injury on the response of interferon (IFN) production in vivo and in vitro after stimulation with eight representative inducers was investigated in a mouse model. The response of mice to immune IFN (IFN-gamma) inducers, staphylococcal enterotoxin A, concanavalin A, and a specific antigen for BCG-sensitized lymphocytes (purified protein derivative) was impaired after a 30% total body surface area third-degree burn. Suppression of IFN-gamma production was observed at day 2 and persisted until day 7 after burn. Decreased IFN-gamma production correlated closely with the percentage of total body surface area burned. When virus type IFN (IFN-alpha/beta) inducers, Newcastle disease virus, polyriboinosinic-polyribocytidylic acid, 10-carboxymethyl-9-acridanone, and E. coli endotoxin, were administered to mice, no change in IFN response was observed after thermal injury. Similar results were obtained when spleen cells obtained from thermally injured mice were stimulated with IFN-gamma inducers in vitro. These studies suggest that although the capacity for IFN-alpha/beta production remains intact in thermally injured mice, IFN-gamma production may be selectively decreased in burned animals and in their spleen cells.     

Peptone Induction and Rifampin-Insensitive Collagenase Production by Vibrio alginolyticus

Vibrio alginolyticus produces an extracellular collagenase which requires specific induction by collagen or its high-molecular-weight fragments. Peptone also induces collagenase during the late exponential and early stationary growth phases. The peptone inducers have been shown to have a broad molecular weight range between 1,000 and 60,000. The peptone inducers supported slow growth of V. alginolyticus when supplied as the sole nitrogen source in minimal medium. Digestion of the peptone inducers with purified V. alginolyticus collagenase resulted in a decrease in their inducing ability, whereas digestion with trypsin or alpha-chymotrypsin did not. This indicated that induction by the inducers required the presence of collagenase-sensitive bonds. Prolonged digestion of the inducers with collagenase did not completely eliminate the inducing ability of the inducers. The peptone inducers acted as inhibitors of collagenase. A minimal medium induction system has been developed which involves resuspending cells at high density in a medium containing succinate, (NH(4))(2)SO(4), KH(2)PO(4), and the peptone inducer. Cells grown in minimal medium induce earlier than cells grown on peptone, Casamino Acids, or tryptone. Collagenase production was shown to occur for 30 to 60 min in the presence of rifampin at levels which completely inhibit the incorporation of [(3)H]uracil into trichloroacetic acid-precipitable material. Chloramphenicol completely and immediately abolished collagenase production, which together with labeling studies has confirmed that collagenase production involves de novo synthesis of the enzyme. Both glucose and Casamino Acids repressed collagenase production, although synthesis of the enzyme continued for 30 to 60 min after their addition. The repression of collagenase production by glucose and Casamino Acids was more severe than the inhibition of enzyme formation due to addition of rifampin.     

Stimulation of protease production byAspergillus oryzae with oils in continuous culture

Summary The effect of soy sauce oil and various other oils on protease production by Aspergillus oryzae NISL 1913 was studied in chemostat cultures (dilution rate=0.02 h^–1). Soy sauce oil was consumed as a carbon source by the cells and also accelerated protease production. When soy sauce oil was used as sole carbon source, the specific protease production rate was 2.89 protease units·(mg dry weight of mycelium)^–1·h^–1, which was threefold higher than that with starch. The specific protease production rate with linoleic acid, oleic acid, Tween 80 and soybean oil exhibited similar values to that with soy sauce oil but the fatty acids with carbon chains shorter than six, such as caproic acid and acetic acid, did not stimulate protease production. The oils did not cause an increase in other exocellular enzymes such as -amylase, indicating that the protease production was selectively stimulated by the oils. Offprint requests to: Y. Fukushima     

Novel inducers derived from starch for cellulase production by Trichoderma reesei

Soluble inducers derived from starch were investigated for cellulase production by T. reesei. Various methods of starch treatment were compared. Acid-hydrolysed starch was found to be most effective. When 1·0 g starch was employed for cellulase production, the cellulase so produced after 6 days of fermentation, with supplementation of 1% (0·01 ml/g paper) β-glucosidase, saccharified more than 15 g shredded waste office paper in 9·34% suspension, resulting in 10 g fermentable sugars, 72% of which was glucose. The effectiveness of the starch-derived inducers was compared with that of lactose, pure cellulose, CMC, xylan and prehydrolysate of pine wood. The effects of calcium chloride and sorbose addition on cellulase production, and the kinetics of cellulase production by the starch-derived inducers are presented.     

Increasing <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> laccase production by culture medium optimization and copper/lignin synergistic induction

Laccases have great biotechnological potential in diverse industries as they catalyze the oxidation of a broad variety of chemical compounds. Production of laccases by basidiomycetes has been broadly studied as they secrete the enzymes, grow on cheap substrates, and they generally produce more than one isoenzyme (constitutive and/or inducible). Laccase production and isoenzyme profile can be modified through medium composition and the use of inducers. The objective of this work was to increase laccase production by Pleurotus ostreatus CP-50 through culture medium optimization and the simultaneous use of copper and lignin as inducers. Increased fungal growth was obtained through the use of a factorial fractional experimental design 2^6–2 where the influence of the nature and concentration of carbon and nitrogen sources was assessed. Although specific laccase production (U/mg biomass) decreased when malt extract medium was supplemented with carbon and nitrogen sources, fungal growth and laccase volumetric activity increased four and sixfold, respectively. The effect of media supplementation with copper and/or lignin on laccase production by P. ostreatus CP-50 was studied. A positive synergistic effect between copper and lignin was observed on laccase production. Overall, the use of an optimized medium and the simultaneous addition of copper and lignin improved growth, laccase volumetric activity, and process productivity by 4-, 60-, and 10-fold, respectively.