Novel fibrinolytic enzymes from Virgibacillus halodenitrificans SK1-3-7 isolated from fish sauce fermentation

Virgibacillus sp. SK1-3-7 exhibited the highest fibrinolytic activity among 25 bacterial isolates obtained from fish sauce fermentation. Results of 16S rRNA gene sequence analysis showed 99% homology to Virgibacillus halodenitrificans ATCC 49067. It was, therefore, identified as V. halodenitrificans SK1-3-7. Fibrinolytic enzymes from V. halodenitrificans SK1-3-7 were partially purified using ammonium sulfate fractionation, hydrophobic and ion-exchange chromatographies. The enzymes with molecular weight of 20- and 36-kDa showed fibrinolytic activity on a fibrin zymogram. The enzymes were stable between pH 4 and 10 and below 60 °C. The enzymes were activated by 20 mM CaCl₂ and 0.15 M NaCl. The activity increased with CaCl₂ up to 100 mM and increased with NaCl concentration up to 2 M. In addition, the residual fibrinolytic activity of 61% was found at 4 M NaCl. The enzymes were completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-pNA, suggesting a subtilisin-like serine proteinase. V. halodenitrificans SK1-3-7 enzymes hydrolyzed fibrin to a greater extent than did plasmin. In addition, the enzymes were resistant to pepsin and trypsin digestion. The de novo peptide homology analysis of a 20- and 36-kDa proteinase revealed no matches to bacilli serine proteinases, suggesting that both were novel fibrinolytic enzymes.     

Halobacterium sp. SP1(1) as a starter culture for accelerating fish sauce fermentation

Aims: Application of Halobacterium sp. SP1(1) for the acceleration of fish sauce fermentation. Methods and Results: Traditional fish sauce fermentation was mimicked using Halobacterium sp. SP1(1) as starter culture. Protease activity, peptide release and α‐amino content (parameters used to monitor the progress of the fermentation) were high at day 10 in tests and day 20 in un‐inoculated controls. The total protein and nitrogen contents were also high in tests compared with controls. The amino acid profile observed at the end of fermentation in experimental samples, when compared with the commercial sauce preparation, was found to be better with respect to flavour and aroma contributing amino acids as well as essential amino acid lysine. Microflora analysis of the final fish sauce revealed the absence of any nonhalophilic or halotolerant micro‐organisms. The protease‐producing halophilic isolates obtained from the fish sauce of eviscerated and uneviscerated controls were identified as Halobacterium sp. F1 and F2, respectively, by 16S rDNA sequence analysis. Conclusions: Exogenous augmentation of Halobacterium sp. SP1(1) accelerated the fish sauce fermentation process with an additive effect on the existing natural microflora present in the fish during fermentation. Halobacterium sp SP1(1), therefore, can be used as an important starter culture for accelerating the fish fermentation process, which is attributed to its extracellular protease. Significance and Impact of the Study: The present study is the first report on use of Halobacterium species as a starter culture for accelerating fish sauce fermentation. Use of halobacterial starter cultures may revolutionize the process in fish sauce industries by reducing the fermentation time and making the process more economical with improved nutritive value of product.     

Identification of novel halotolerant bacillopeptidase F-like proteinases from a moderately halophilic bacterium, Virgibacillus sp. SK37

Aims: Virgibacillus sp. SK37 isolated from Thai fish sauce produced numerous NaCl‐activated subtilisin‐like proteinases. Our objectives were to purify, characterize and identify these extracellular proteinases. Methods and Results: Three major subtilisin‐like enzymes including 19, 34 and 44 kDa were partially purified and showed maximum activity at pH 8, 55–60°C, 25–30% NaCl and 70–100 mmol l^?1 CaCl₂. Enzymes showed stability at 0–30% NaCl and <20 mmol l^?1 CaCl₂ and were completely inhibited by phenylmethanesulphonyl fluoride but not by ethylenediaminetetraacetic acid. The isoelectric points of 19‐, 34‐ and 44‐kDa proteinases were at 3·6, 5·2 and 3·8, respectively, based on 2D electrophoresis. Peptide mass fingerprint and de novo peptide homology analysis of tryptic peptides using MALDI‐TOF and LC–MS/MS, respectively, suggested that all three enzymes were novel and homologous to bacillopeptidase F. Conclusions: The three major proteinases are a member of bacillopeptidase F‐like enzymes exhibiting thermophilic and halotolerant characteristics with high stability at 30% NaCl. Significance and Impact of the Study: This is the first report on bacillopeptidase F‐like proteinases in genus Virgibacillus with a distinct halotolerant feature. They showed potential to be a processing aid for food and biotechnological applications, particularly in high salt condition.     

Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol

A newly isolated strain Penicillium sp. GXU20 produced a raw starch-degrading enzyme which showed optimum activity towards raw cassava starch at pH 4.5 and 50 °C. Maximum raw cassava starch-degrading enzyme (RCSDE) activity of 20 U/ml was achieved when GXU20 was cultivated under optimized conditions using wheat bran (3.0% w/v) and soybean meal (2.5% w/v) as carbon and nitrogen sources at pH 5.0 and 28 °C. This represented about a sixfold increment as compared with the activity obtained under basal conditions. Starch hydrolysis degree of 95% of raw cassava flour (150 g/l) was achieved after 72 h of digestion by crude RCSDE (30 U/g flour). Ethanol yield reached 53.3 g/l with fermentation efficiency of 92% after 48 h of simultaneous saccharification and fermentation of raw cassava flour at 150 g/l using the RCSDE (30 U/g flour), carried out at pH 4.0 and 40 °C. This strain and its RCSDE have potential applications in processing of raw cassava starch to ethanol.     

Substrate Specificity and Salt Inhibition of Five Proteinases Isolated from the Pyloric Caeca and Stomach of Sardine

To elucidate the mechanism of hydrolysis of fish muscle proteins by fish proteinases in fish sauce production, each pure preparation of three alkaline proteinases and two acid proteinases from sardine was tested for its ability to hydrolyze various proteins and its stability in the presence of 0 to 25% of NaCl. Each of the alkaline proteinases hydrolyzed casein more rapidly than other proteins. A major alkaline proteinase (III) hydrolyzed sarcoplasmic protein from sardine 5-times faster than other alkaline proteinases. Each of two acid proteinases hydrolyzed hemoglobin and myoglobin more rapidly than the other proteins. After preincubation with 25% NaCl, an alkaline proteinase (III) and an acid proteinase (II) were stable although the other proteinases became unstable. The two proteinases, alkaline proteinase III and acid proteinase II, were also stable for three months after the beginning of fish sauce production. The proteolytic activity of each of alkaline and the acid proteinases was strongly inhibited by more than 15% NaCl; however, minimum inhibition was observed when sardine muscle proteins were used as the substrate.     

鲮鱼皮胶原肽的制备及其抗氧化活性的检测

为了以细菌胞外蛋白酶酶解低值蛋白资源制备抗氧化活性肽以及挖掘新型蛋白酶,采用液体发酵培养的方法对假交替单胞菌Pseudoalteromonas sp.SHK1-2进行发酵产酶,获得胞外蛋白酶粗酶液用于酶解热水法提取的鲮鱼胶原蛋白,通过超滤、sephadex LH-20分子筛层析获得具有DPPH自由基清除能力(35.6%±7%)、氧自由基清除能力(Oxygen radical absorbance capacity,ORAC)及DNA氧化损伤抑制活性的小分子肽,液相色谱质谱联用鉴定该活性肽分子量为776.2 Da,预测氨基酸序列为Thr-Ala-Gly-His-Pro-Gly-Thr-His。ORAC检测验证了人工合成的多肽同样具有抗氧化活性。研究表明细菌胞外蛋白酶在低值资源高值化中具有一定的应用前景,对于新型蛋白酶及其应用的挖掘具有一定的参考意义。     

产碱性蛋白酶芽孢杆菌的鉴定

通过测量比较在碱性蛋白平板上产生的蛋白水解圈直径,从土壤中筛选到一株高产蛋白酶菌株Bacillus sp.HFBL0079,根据生理生化特性、16S rDNA序列,鉴定为B.amyloliquefaciens。其最适培养温度为35°C-37°C,最适生长pH 8.0,在特定培养条件下16 h达到稳定期,菌体生长和蛋白酶合成同步进行。以大豆分离蛋白为氮源时发酵液具有最高酶活。发酵液在pH 10时具有最高酶活,表明为碱性蛋白酶。该菌株产生的碱性蛋白酶可水解多种天然蛋白质,对胶原蛋白水解度高于其他蛋白质,对羽毛角蛋白也有一定水解能力,提示该酶具有一定新颖性。     

Enzyme inactivation by ethanol and development of a kinetic model for thermophilic simultaneous saccharification and fermentation at 50 °C with Thermoanaerobacterium saccharolyticum ALK2

Studies were undertaken to understand phenomena operative during simultaneous saccharification and fermentation (SSF) of a model cellulosic substrate (Avicel) at 50°C with enzymatic hydrolysis mediated by a commercial cellulase preparation (Spezyme CP) and fermentation by a thermophilic bacterium engineered to produce ethanol at high yield, Thermoanaerobacterium saccharolyticum ALK2. Thermal inactivation at 50 °C, as shown by the loss of 50% of enzyme activity over 4 days in the absence of ethanol, was more severe than at 37 °C, where only 25% of enzyme activity was lost. In addition, at 50 °C ethanol more strongly influenced enzyme stability. Enzyme activity was moderately stabilized between ethanol concentrations of 0 and 40 g/L, but ethanol concentrations above 40 g/L accelerated enzyme inactivation, leading to 75% loss of enzymatic activity in 80 g/L ethanol after 4 days. At 37 °C, ethanol did not show a strong effect on the rate of enzyme inactivation. Inhibition of cellulase activity by ethanol, measured at both temperatures, was relatively similar, with the relative rate of hydrolysis inhibited 50% at ethanol concentrations of 56.4 and 58.7 g/L at 50 and 37 °C, respectively. A mathematical model was developed to test whether the measured phenomena were sufficient to quantitatively describe system behavior and was found to have good predictive capability at initial Avicel concentrations of 20 and 50 g/L.     

Third form of calcium-activated neutral proteinase from calf brain: purification, partial characterization and comparison of properties with other forms

A third form (CANP3) of calcium-activated neutral proteinase (CANP) has been purified, 3900-fold, to near homogeneity from calf brain cortex. The purification procedure is based on the one recently developed for the purification of CANP1 and CANP2. The molecular weight of CANP3, as judged on SDS-polyacrylamide gel electrophoresis was Mr 78,000. A protein with an apparent Mr 17,000 co-purified with the proteinase. At neutral pH (7.2), it was maximally active at 260 microM CaCl2. In the presence of CaCl2, CANP1 and CANP3 were autolyzed very rapidly, whereas the autolysis of CANP2 was slow and gradual. The autolyzed CANP1 and CANP3 responded differently to CaCl2; CANP1 lost activity completely, whereas CANP3 was fully active at 0.5 microM CaCl2. Despite the opposite behavior of these proteinases in the presence of Ca2+, no significant differences in the peptide maps of the three proteinases were observed. Neurofilaments, neurotubules and myelin basic protein (MBP) were degraded by each of the proteinases. Monoclonal antibodies raised against CANP2 reacted almost equally with CANP1 and CANP3. As with CANP1 and CANP2, leupeptin and sulfhydryl-modifying compounds, NEM and iodoacetic acid, inhibited the activity of CANP3.     

Ultrasonic-pretreated waste activated sludge hydrolysis and volatile fatty acid accumulation under alkaline conditions: Effect of temperature

The effect of temperature on the hydrolysis and acidification of ultrasonic-pretreated waste activated sludge (WAS) under alkaline conditions was investigated in this study. The experiment temperatures were set at 10, 20, 37, and 55°C. Experimental results showed that the hydrolysis of ultrasonic-pretreated WAS under alkaline conditions increased significantly with temperature from 10 to 55°C, while the volatile fatty acid (VFA) accumulation was not augmented as temperature increased. Among the four temperatures tested, 37°C was the point with the highest VFA accumulation after 72h fermentation. VFA accumulation decreased markedly at 55°C compared to 37°C. Mechanism investigation revealed that among all the temperatures tested, 37°C was the temperature at which consumptions of WAS protein and carbohydrate, activities of key enzymes related to VFA formation and ratio of Bacteria to Archaea all reached the maximum. Due to activities of related microorganisms inhibited by higher temperature (55°C), VFA accumulation decreased at 55°C.     

Effect of calcium on the morphology and functionality of whey protein nanofibrils

Self-assembly of amyloid-like nanofibrils during heating of bovine whey proteins at 80 °C and pH 2 is accelerated by the presence of NaCl and/or CaCl(2), but the rheological consequences of accelerated self-assembly are largely unknown. This investigation focused on the impact of CaCl(2) on the evolution of rheological properties and fibril morphology of heated whey protein isolate (WPI), both during self-assembly at high temperature and after cooling. Continuous rotational rheometry of heated 2% w/w WPI showed a nonlinear effect of CaCl(2) on the viscosity of fibril dispersions, which we attributed to effects on fibril flexibility and thus the balance between intrafibril and interfibril entanglements. Small-amplitude oscillatory measurements made in situ during heating of 10% w/w WPI at 80 °C suggest that CaCl(2) is not involved in either fibril structure or gel structure, and this was confirmed with dialysis experiments.     

Substrate specificity of enzymes from Bacillus mesentericus

The proteolytic enzymes of the sporogenous Bacillus mesentericus strains 64 and 8 were tested for their ability to hydrolyse different protein substrates. The enzymes were isolated using affinity chromatography on bacillichine-silochrome, and eluted with 25% isopropanol in 0.05 M Tris-HCl buffer, pH 8.0-8.4, containing 0.01 M CaCl2. Casein, hemoglobin, elastin, albumin and synthetic peptides, Z-L-Ala-Ala-Leu-pNa and Z-L-Ala-Gly-Leu-pNa, were used as substrates. The activity of esterase was assayed in terms of indophenyl acetate cleavage. The proteinases were compared with terrilytin, a commercial preparation. The proteinase of strain 64 was active in the hydrolysis of casein, hemoglobin and elastin; its specificity was close to that of terrilytin. The proteinase of strain 8 differed from them in a higher thrombolytic and fibrinolytic activity, and had a high esterase activity.     

Optimization of culture conditions for the production of haloalkaliphilic thermostable protease from an extremely halophilic archaeon Halogeometricum sp. TSS101

AIMS: Isolation and screening of extreme halophilic archaeon producing extracellular haloalkaliphilic protease and optimization of culture conditions for its maximum production. METHODS AND RESULTS: Halogeometricum sp. TSS101 was isolated from salt samples and screened for the secretion of protease on gelatin and casein plates containing 20% NaCl. The archaeon was grown aerobically in a 250 ml flask containing 50 ml of (w/v) NaCl 20%; MgCl(2) 1%; KCl 0.5%; trisodium citrate 0.3%; and peptone 1%; pH 7.2 at 40 degrees C on rotary shaker. The production of enzyme was investigated at various pH, temperatures, NaCl concentrations, metal ions and different carbon and nitrogen sources. The partially purified protease had activity in a broad pH range (7.0-10.0) with optimum activity at pH 10.0 and a temperature (60 degrees C). The enzyme was thermostable and retained 70% initial activity at 80 degrees C. Maximum protease production occurred at 40 degrees C in a medium containing 20% NaCl (w/v) and 1% skim milk powder after 84 h in shaking culture. Enzyme secretion was observed at a broad pH range of 7.0-10.0. Addition of CaCl(2) (200 mmol) to the culture medium enhanced the production of protease. Protein rich flours proved to be cheap and good alternative source for enzyme production. Different osmolytes were tested for the growth and production of haloalkaliphilc protease and found that betaine and glycerol enhanced growth without secretion of the protease. Immobilization studies showed that whole cells immobilized in 2% alginate beads were stable up to 10 batches and able to secrete the protease, which attained maximum production within 60 h under shaking conditions. CONCLUSIONS: Halogeometricum sp. TSS101 secreted an extracellular haloalkaliphilic and thermostable protease. The optimum conditions required for maximum production are 20% NaCl, 1% skim milk powder and temperature at 40 degrees C. Addition of CaCl(2) (200 mmol) enhanced the enzyme production. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of haloalkaliphilic protease. SIGNIFICANCE AND IMPACT OF THE STudy: The low cost protein rich flours were used as an alternative carbon and nitrogen sources for enzyme production. Immobilization of halophilic cells in alginate beads can be used in continuous production of halophilic enzyme. The halophilic and thermostable protease from Halogeometricum sp. TSS101 is good source for industrial applications and can be a suitable source for preparation of fish sauce.     

Determination of bioactive compounds in fermented soybean products using GC/MS and further investigation of correlation of their bioactivities

The active ingredients and bioactivities (anti-oxidant, anti-tyrosinase, anti-proliferative and estrogenic activities) of soybean and soybean products (Cheonggukjang, Meju, Makjang, Doenjang and soy sauce) produced by different fermentation processes were compared. There were high correlations between active ingredients and bioactivities. Free phenolic acids extracted from soybean products were identified and quantified by gas chromatography/mass spectrometry (GC/MS). Overall, the components and activities in fermented soybean products were different than those in soybeans. Total phenolic content (TPC), protein content (PC) and anti-oxidant activity increased as fermentation time increased. TPC and PC showed strong negative correlations with anti-oxidant activity. Doenjang and soy sauce, two long-term fermented products, showed lower total flavonoid content (TFC) and estrogenic activities than short-term fermented soybean products. This might be explained by the decomposition and hydrolysis of flavonoids due to the long fermentation time and high temperature. Strong anti-proliferative activity against cancer cell lines, which was highly correlated with TFC, was found in Meju and Cheonggukjang. Soybean and all fermented products except Meju exhibited effective tyrosinase inhibitory activities. Fermented products showed stronger estrogenic activity than soybeans, which was highly correlated with syringic acid.     

Lipase from marine Aspergillus awamori BTMFW032: production, partial purification and application in oil effluent treatment

Marine fungus BTMFW032, isolated from seawater and identified as Aspergillus awamori, was observed to produce an extracellular lipase, which could reduce 92% fat and oil content in the effluent laden with oil. In this study, medium for lipase production under submerged fermentation was optimized statistically employing response surface method toward maximal enzyme production. Medium with soyabean meal-0.77% (w/v); (NH(4))(2)SO(4)-0.1m; KH(2)PO(4)-0.05 m; rice bran oil-2% (v/v); CaCl(2)-0.05 m; PEG 6000-0.05% (w/v); NaCl-1% (w/v); inoculum-1% (v/v); pH 3.0; incubation temperature 35°C and incubation period-five days were identified as optimal conditions for maximal lipase production. The time course experiment under optimized condition, after statistical modeling, indicated that enzyme production commenced after 36 hours of incubation and reached a maximum after 96 hours (495.0 U/ml), whereas maximal specific activity of enzyme was recorded at 108 hours (1164.63 U/mg protein). After optimization an overall 4.6-fold increase in lipase production was achieved. Partial purification by (NH(4))(2)SO(4) precipitation and ion exchange chromatography resulted in 33.7% final yield. The lipase was noted to have a molecular mass of 90 kDa and optimal activity at pH 7 and 40°C. Results indicated the scope for potential application of this marine fungal lipase in bioremediation.     

The influence of storage conditions of tuna viscera before fermentation on the chemical, physical and microbiological changes in fish sauce during fermentation

Effect of storage condition of tuna viscera on chemical, physical and microbiological changes of its sauce were monitored. Results based on microbial counts, protein degradation products, total volatile base (TVB), and trimethylamine (TMA) contents, showed that tuna viscera stored at room temperature underwent more deterioration than that kept in ice, especially with increasing storage time. As a result, fish sauce obtained from tuna viscera stored at room temperature for a longer time rendered the greater amino nitrogen, TVB, TMA contents as well as browning intensity. However, storage conditions had no marked effect on overall changes in chemical, physical and microbiological characteristics of sauce generated during fermentation. Additionally, fish sauce produced from tuna viscera kept at room temperature comprised lower histamine content than that prepared from fresh or ice-stored viscera. Therefore, tuna viscera stored at room temperature for up to 8h could be used for the production of fish sauce with no detrimental effect on the quality.     

Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce

A gram-positive thermotolerant bacterium, designated strain RKK-04, was isolated from a fermented Thai fish sauce broth as it demonstrated high proteolytic activity. A phylogenetic analysis based on comparisons of 16S rRNA gene sequences showed that strain RKK-04 is Bacillus licheniformis. The proteolytic enzyme, which was purified 80-fold with 18% yield, has a molecular mass of 31 kDa and an isoelectric point higher than 9.3. The optimum pH and temperature of the enzyme activity were found to be 10.0 and 50°C, respectively. The addition of diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride completely inhibited enzymatic activity. These results showed that the enzyme is a subtilisin-like alkaline serine proteinase. On the other hand, the enzyme exhibited unique cleavage sites in oxidized insulin B-chain that differed from those of other subtilisin-like proteases. High enzymatic activity was also retained under high salt conditions (30% NaCl). The myosin heavy chain of fish protein was completely digested by reaction with this enzyme. Thus the halotolerant proteinase from B. licheniformis RKK-04 is a key enzyme for fish sauce fermentation.     

A statistical approach for the enhanced production of alkaline protease showing fibrinolytic activity from a newly isolated Gram-negative Bacillus sp. strain AS-S20-I

An alkaline-fibrinolytic protease-producing bacterial strain (AS-S20-I) isolated from a soil sample in Assam was a Gram-negative rod and grown at temperatures ranging from 25 to 55 °C, and pH 6.5 to 11.0. Taxonomic identification of isolated strain by polyphasic approach (phenotypic characterization, chemotaxonomic properties, and ribotyping data of the strain) suggested that it belongs to the genus Bacillus, for which the name Bacillus sp. strain AS-S20-I (MTCC 8961) was proposed. The initial screening by using Plackett-Burman's design demonstrated that among the tested factors, casein, ammonium sulphate and pH of the medium significantly (p < 0.05) enhanced the protease (fibrinolytic enzyme) yield in submerged fermentation. Further optimization of fibrinolytic protease production by Bacillus. sp. strain AS-S20-I in SmF by applying RSM was achieved as 749.0 × 10(3)UL(-1) in the presence of 3.0% (w/v) casein and 0.12% (w/v) ammonium sulphate at pH 10.9 and 45 °C. This was a 4.0-fold increase in yield compared with that obtained before applying the Plackett-Burman and RSM experimental design. The protease preparation preferentially degraded the fibrin (specific activity 2408.0 ± 70.0 U mg(-1); mean ± S.D.) suggesting that its future application in pharmaceutical industry as thrombolytic and anticancer drugs is highly promising.     

Influence of Metronidazole, CO, CO(2), and Methanogens on the Fermentative Metabolism of the Anaerobic Fungus Neocallimastix sp. Strain L2

The effects of metronidazole, CO, methanogens, and CO(2) on the fermentation of glucose by the anaerobic fungus Neocallimastix sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H(2), acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When Neocallimastix sp. strain L2 was cocultured with the methanogenic bacterium Methanobrevibacter smithii, the fermentation pattern changed in the opposite direction: H(2) and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO(2) in the gas phase resulted in increased H(2) and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in Neocallimastix sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.