Production Of Neutral Protease Research Articles

Isolation and Purification of Bacillus amyloliquefaciens D1 Protease and Its Application in the Fermentation of Soybean Milk to Produce Large Amounts of Free Amino Acids.

In this study, a strain of Bacillus amyloliquefaciens D1, with a notably high production of neutral protease, was isolated from Morchella crassipes. The protease was purified to 10.4-fold with a specific activity of 4542.9 U/mg and 2.7% recovery. The enzyme was purified by 70% (NH4)2SO4 and DEAE-Cellulose-52 column. The estimated molecular mass of the purified protease obtained by SDS-PAGE was approximately 40kDa. The enzyme was optimally active at pH 6.0 and 50°C. Furthermore, the maximum hydrolysis rate (Vmax) and apparent Michaelis-Menten constant (Km) values of the purified protease were 8.2mg/mL and 65.7µg/(min mL). The enzymatic properties and rapid and efficient purification of Bacillus amyloliquefaciens D1 provide the basis for its potential commercialization and industrial development. Moreover, more essential amino acids, such as isoleucine, leucine, and phenylalanine, would be released when the strain fermented soybean milk, and then a better amino acid profile would be formed in soybean milk. Results suggest that this strain exhibits great potential in fermented soybean milk, and the enzyme could lay a foundation for its industrial application and further research.

Optimization of fermentation conditions for production of neutral metalloprotease by Bacillus subtilis SCK6 and its application in goatskin-dehairing

In this study, a high protease-producing strain was screened by spread plate method and identified by molecular biology and morphological identification. It was identified as Bacillus sp. LCB14. A neutral protease gene was cloned and heterologous expressed by B. subtilis SCK6. Then, the recombinant protease was used to dehair the goat skins. The fermentation conditions of neutral protease production by B. subtilis SCK6 were optimized. The single factor experiments, Plackett–Burma experiment, and response surface method were conducted to determine fermentation medium and culture conditions. The optimized medium contained corn meal 49 g/L, soluble starch 28 g/L, soybean meal 17 g/L, corn steep liquor powder 8 g/L, yeast extract 10 g/L, Na2HPO4 2.3 g/L, KH2PO4 1.9 g/L, MgSO4 0.5 g/L, MnCl2 0.1 g/L and ZnSO4 0.05 g/L. The optimized culture conditions were 35 °C and pH 7.0. Under the optimum conditions, the recombinant strain reached 33467.28 U/mL after 72 hr ferment. Moreover, by fed batch in 30 L fermenters, neutral protease production reached 39,440.78 U/mL and shortened fermentation time from 72 hr to 46 hr. Finally, the crude enzyme was utilized to replace sodium sulfide for dehairing of goatskins. The enzymatic dehaired pelts were white, smooth, and soft; the grain side of enzymatic dehaired pelts were clear; there was no obvious damage to the grain side of enzymatic dehaired pelts by visual observation and tactile test. Furthermore, there were no hair roots, hair follicles and other glands in enzymatic dehaired belts, and the collagen fibers of enzymatic dehaired belt were dispersed well by histological analysis.

Extracellular neutral protease from Arthrospira platensis: Production, optimization and partial characterization

Proteases are industrially important catalysts. They belong to a complex family of enzymes that perform highly focused proteolysis functions. Given their potential use, there has been renewed interest in the discovery of proteases with novel properties and a constant thrust to optimize the enzyme production. In the present study, a novel extracellular neutral protease produced from Arthrospira platensis was detected and characterized. Its proteolytic activity was strongly activated by β-mercaptoethanol, 5,5-dithio-bis-(2-nitrobenzoic acid) and highly inhibited by Hg2+ and Zn2+ metal ions which support the fact that the studied protease belongs to the cysteine protease family. Using statistical modelling methodology, the logistic model has been selected to predict A. platensis growth-kinetic values. The optimal culture conditions for neutral protease production were found using Box-Behnken Design. The maximum experimental protease activities (159.79 U/mL) was achieved after 13 days of culture in an optimized Zarrouk medium containing 0.625 g/L NaCl, 0.625 g/L K2HPO4 and set on 9.5 initial pH.The extracellular protease of A. platensis can easily be used in the food industry for its important activity at neutral pH and its low production cost since it is a valuation of the residual culture medium after biomass recovery.

Purification and characterization of neutral protease from Aspergillus oryzae Y1 isolated from naturally fermented broad beans

The strain Y1, with a notably high production of neutral protease, was isolated from naturally fermented broad beans and subsequently identified as Aspergillus oryzae, through the analysis of its morphology characteristics and 18S rDNA sequence. Naturally fermented broad beans are the main raw material in Sichuan broad-bean sauce. The neutral protease from Aspergillus oryzae Y1 was purified using ammonium sulphate precipitation and DEAE-Sepharose Fast Flow chromatography, which resulted in a 10.0-fold increase in the specific activity (2264.3 U/mg) and a recovery rate of 21%. The estimated molecular mass of the purified protease was approximately 45 kDa. The optimal pH and temperature of the purified protease were 7.0 and 55 °C, respectively. The heat resistance of the purified protease was significantly higher than the commercial protease. The effect of metal ions on the activity of the purified protease approximated that of commercial neutral protease. Furthermore, the maximum hydrolysis rate (Vmax) and apparent Michaelis–Menten constant (Km) values of the purified protease were 256.4103 μg/mL min and 20.0769 mg/mL, respectively. The purified protease had a higher affinity for the substrate than the commercial neutral protease. All the results suggest that this neutral protease exhibits the potential for application in industry due to its good resistance to high temperatures and wide range of acids and bases.

A Statistical Approach for Production Improvement of a Neutral Protease From a Newly Isolated Strain of Aeromonas Hydrophila

Neutral proteases have a large variety of industrial applications. Therefore, the optimization of their production for the large-scale commercial purpose is critical. The present study was aimed to the optimization of nutritional and physical conditions affecting neutral protease production of Aeromonas hydrophila MSB16, not previously reported for this genus. Four key determinants (pH, temperature, glucose and peptone concentration), were selected based on the results of the one-factor-at-a-time (OFAT) method and then optimized by the response surface methodology (RSM). Maximum enzyme production (229.72 U/ml) which was 2.3 folds higher than that of the OFAT method (99.25 U/ml) and 3.15 times higher than unoptimized conditions (72.86 U/ml) was observed at 30 °C, pH 7, 4.5% w/v glucose and 4.5%w/v peptone by using a rotatable central composite design (RCCD). The results showed that the OFAT and RSM strategies were a useful screening and optimization methods for enhancing the protease production of the strain MSB16, respectively.

Engineering of a Bacillus amyloliquefaciens Strain with High Neutral Protease Producing Capacity and Optimization of Its Fermentation Conditions.

The neutral protease has high potential for industrial applications, and attempts to improve enzyme expression level have important application values. In the present study, a neutral protease-encoding gene, Banpr, was cloned from Bacillus amyloliquefaciens strain K11, and a genetic manipulation method specific for this difficult-to-transform strain was developed for the high-level expression of neutral protease. The recombinant plasmid pUB110-Banpr was constructed in Bacillus subtilis strain WB600 and then transformed into strain K11 under optimized conditions. A positive transformant 110N-6 with the highest protease secreting capacity on skim milk plates and great genetic stability for more than 100 generations was selected for further study. Optimization of the fermentation conditions increased the enzyme activity of strain 110N-6 to 8995 ± 250 U/ml in flask culture and 28084 ± 1282 U/ml in 15-l fermentor, which are significantly higher than that of the native strain K11 and industrial strain B. subtilis AS.1398, respectively. The high expression level and extreme genetic stability make B. amyloliquefaciens strain 110N-6 more favorable for mass production of neutral protease for industrial uses.

Optimization of neutral protease production fromBacillus subtilis: Using agroindustrial residues as substrates and response surface methodology

Statistically based experimental designs were applied to optimize the fermentation medium and cultural conditions for the maximization of neutral protease using three agroindustrial residues (cassava pulp, soybean meal, and wheat bran) and Bacillus subtilis DES-59. The Plackett-Burman design was used to evaluate the effects of variables such as the concentration of substrates, initial pH, shaker's rotating speed, temperature, inoculum size, and incubation time. Among the eight parameters, three significant variables (cassava pulp, soybean meal, and inoculum size) were selected for the optimization study, in which a central composite design was used to optimize the concentrations of cassava pulp and soybean meal and inoculum size and investigate the interactive effects of the three variables. The optimal parameters obtained from response surface methodology are 37.78 g/L of cassava pulp, 15 g/L of soybean meal, and 6.5% (v/v) of inoculum size, respectively, resulting in a maximum neutral protease activity of 4107 ± 122 U/mL.

Statistical optimization of culture medium for neutral protease production by Aspergillus oryzae. Comparative study between solid and submerged fermentations on tomato pomace

A comparative study of the production of neutral protease was carried out by the cultivation of Aspergillus oryzae NRRL 2220 in solid-state fermentation (SSF) and submerged fermentation (SmF) using tomato pomace based medium. For this purpose, medium optimization was achieved using two experimental designs. The first, corresponded to the Plackett and Burman design with N=8 experiments and k=N−1 factors; five real (wheat bran, casein, NH4NO3, NaCl and ZnSO4) and two errors. Statistical analysis of the results allowed the selection of two factors having a significant effect on enzyme production (casein and NaCl in SSF, wheat bran and NaCl in SmF). Optima of the selected factors have been determined through a second experimental design, the central composite design of Box and Wilson with two factors; 19.79g/L casein and 0.92g/L NaCl for SSF and 17.92g/L wheat bran and 1.18g/L NaCl for SmF were found. The optimal production time of the neutral protease determined from the kinetic study was 96h leading to 21309U/g in SSF and 2343.5U/g in SmF. The comparison of the proteolytic activities of the optimized media demonstrated a ratio of 9 between SSF and SmF, showing the efficiency of the solid-state fermentation compared to the submerged fermentation. The results confirmed the high biotechnological potential of this fungal strain for neutral protease production in solid-state fermentation. Additionally, the utilization of tomato pomace constitutes an efficient and inexpensive agro industrial substrate for protease production via the SSF approach and a suitable mean for its valorization and to reduce its ecological impact.

Production and partial characterization of neutral protease by an indigenously isolated strain of Aspergillus tubingensis NIICC-08155

An indigenously isolated strain of Aspergillus tubingensis NIICC-08155 from soil of teak dominated vegetation of kusmi forest at Gorakhpur was subjected to neutral protease production and its partial characterization. The maximum production of neutral protease i.e. 68.50 U/ml was attained after 96h of incubation. The crude preparation of protease showed reasonable activity at temperature range of 40 to 60 °C with maximum activity at 40 oC and had a maximum enzyme activity at pH 6.4. The Km value of the enzyme was found to be 45.0 mg/ml and the molecular weight as determined by SDS-PAGE was approximately 45 kDa. The enzyme was completely inhibited by DTT and EDTA.

Optium Conditions for the Production of Neutral Protease from local strain Aspergillus niger var cabonarius

The optimum conditions for the production of neutral protease from local strain Aspergillus niger var carbonarius by solid – state fermentation system (Wheat bran) moisted with 0.2 M phosphate buffer (PH7.0) . the hydration ratio was 1:5 (V:W) . the concentration of inoculum was 1×106 spores per 10 gram of solid materials , initial P H 6.5 and 96 hours of incubation period at 30? C .the enzyme activity was 1300 unit / ml and specific activity was 1550 unit / mg protein .

Optium Conditions for the Production of Neutral Protease from local strain Aspergillus niger var cabonarius

The optimum conditions for the production of neutral protease from local strain Aspergillus niger var carbonarius by solid – state fermentation system (Wheat bran) moisted with 0.2 M phosphate buffer (PH7.0) . the hydration ratio was 1:5 (V:W) . the concentration of inoculum was 1×106 spores per 10 gram of solid materials , initial P H 6.5 and 96 hours of incubation period at 30? C .the enzyme activity was 1300 unit / ml and specific activity was 1550 unit / mg protein .

Production of laccase by membrane-surface liquid culture of Trametes versicolor using a poly( l-lactic acid) membrane

Membrane-surface liquid culture (MSLC) is a promising method for the bioproduction of highly aerobic filamentous fungi [A. Ogawa, A. Yasuhara, T. Tanaka, T. Sakiyama, K. Nakanishi, Production of neutral protease by membrane-surface liquid culture of Aspergillus oryzae IAM2704, J. Ferment. Bioeng. 80 (1995) 35–40]. This paper reports on the production of laccase by Trametes versicolor on a microporous membrane of poly( l-lactic acid) (PLLA), which can be biodegraded via composting after use. The membrane was stable as a support for 24 days at 30 °C. During the first 9 days in MSLC, the fungus produced half as much laccase as it did in liquid-surface culture (LSC); however, the mycelium on the membrane was able to be re-used five times for laccase production. The laccase production was accelerated in the repeated use of the culture while the mycelium in LSC ceased to produce the enzyme. This study shows that compostable PLLA microporous membranes can be used for enzyme production by MSLC of filamentous fungi.

Rice bran as a substrate for proteolytic enzyme production

Rice bran was used as the substrate for screening nine strains of Rhizopus sp. for neutral protease production by solid-state fermentation. The best producer, Rhizopus microsporus NRRL 3671, was used for optimizing the process parameters for enzyme production. Fermentation carried out with 44.44 % initial moisture content at a temperature of 30 C for 72 h was found to be the optimum for enzyme secretion by the fermenting organism. While most of the carbon supplements favored enzyme production, addition of casein resulted in a marginal increase in protease yield. Fermentation was then carried out under optimized conditions to obtain the crude extract of the enzyme, which was partially purified by precipitation and dialysis. A 3-fold increase in the enzyme purity was achieved in this manner. The enzyme was found to be a metalloprotease, being activated by Mn2+, with maximal activity at a temperature of 60 C and pH 7.0.

Comparative evaluation of neutral protease production by Aspergillus oryzae in submerged and solid-state fermentation

A comparative study was carried out on the production of neutral protease using agro-industrial residues as substrate in solid-state fermentation (SSF) and submerged fermentation (SmF). Seven fungal cultures, viz. three strains of Aspergillus oryzae and four strains belonging to Penicillium sp, P. funiculosum, P. funiculosum, P. pinophilum, P. aculeatum were evaluated using a plate assay for enzyme production, which showed a strain of A. oryzae NRRL 1808 as the most useful culture. Several agro-industrial residues (wheat bran, rice husk, rice bran, spent brewing grain, coconut oil cake, palm kernel cake, sesame oil cake, jackfruit seed powder and olive oil cake) were screened for neutral protease production in SmF and SSF. In both systems, wheat bran was the best substrate. Best results in SSF were obtained in a medium having an initial moisture content of 43.6%, when inoculated with 1 ml of spore suspension (8 × 10 8 spores) and incubated at 30 °C for 72 h (31.2 U enzyme per gram of fermented substrate – U/gds). SmF medium (pH 7.5) containing 2% (w/v) wheat bran, when inoculated with 3 ml of spore suspension and incubated at 30 °C and 180 rpm for 72 h gave maximum enzyme yield of 8.7 U/gds. A comparative evaluation of protease yield by the two fermentation systems showed 3.5-fold more enzyme production in SSF, clearly demonstrating the superiority of SSF over SmF.

Production of neutral protease by membrane-surface liquid culture of Aspergillus oryzae IAM2704

A membrane-surface liquid culture (MSLC), in which microorganisms were grown on a microporous membrane surface exposed to the air with the other side of the membrane in contact with liquid medium, was applied to the production of neutral protease from Aspergillus oryzae IAM2704. The amount of protease produced by batch MSLC was much higher than that produced by shaking culture, liquid-surface culture, or agar plate culture. In MSLC the amount of protease produced per milligram of dry cells as well as the amount of protease produced were dependent particularly on the glucose concentration. By decreasing the glucose concentration from 1.5% to 0.2%, the amount of protease produced and its specific amount were increased by 60% and around 7 times, respectively. Using a medium containing 0.2% glucose and 0.4% casein, long-term repeated-batch production of the enzyme was conducted by exchanging the medium every 12 to 48 h with a fresh one. Protease production continued in more than 15 successive batches, although the amount of enzyme produced in each batch gradually decreased. When the medium was exchanged every 24 h, the degree of the decrease was the smallest, and the cumulative amount of enzyme produced was more than 20 and 400 times those obtained in the batch MSLC and shaking culture, respectively.

Oversecretion of the Neutral Protease from Bacillus subtilis in Lactococcus lactis spp. lactis JF254

A thermolytic lactococcal strain that oversecretes neutral protease was constructed. Plasmid profiles and proteolytic activities confirmed that this strain acquired pMG36enpr containing the cloned neutral protease gene from Bacillus subtilis. The oversecreting neutral protease transformant exhibited a halo of white precipitate when it was grown on transparent citrate milk agar and completely solubilized 11% NDM at 21°C after 5 d. Western blot confirmed that the neutral protease halo transformant secreted more neutral protease than did the normal neutral protease transformant. A biochemical and molecular understanding of the mechanism of neutral protease production in the halo transformant could form the basis for the construction of lactococcal strains capable of secreting large amounts of enzymes or other polypeptides that are important to the dairy and food industries.

Production of neutral protease from Aspergillus oryzae by a novel cultivation method on a microporous membrane

A novel cultivation method using a microporous membrane (membrane-surface liquid culture) was developed, in which moulds are grown on the membrane surface with its opposite side being in contact with a liquid medium. The amount of neutral protease from Aspergillus oryzae produced was more than 10 times higher than that produced by the conventional liquid culture.

Nucleotide sequence and possible functional mechanism of the transcriptional activator gene ( preL) for neutral protease from Lactobacillus sp.

A positive regulatory gene ( preL) from Lactobacillus sp. no. 1, which enhanced production of neutral protease (NprL), was cloned in Bacillus subtilis DB104 together with the nprL structural gene. When nprL was expressed with the regulatory gene ( preL) on the same plasmid, extracellular protease production in a 12 h culture was 28-fold higher (14.2 U/ml) than protease production without preL expression (0.5 U/ml). The nucleotide sequence of the preL was determined and the deduced amino acid sequence revealed that PreL was relatively homologous (33%) to that of a positive regulatory protein (NprA) for protease production from Bacillus stearothermophilus, whose function is similar to that of preL. Northern hybridization analysis revealed that preL, in multicopy, enhanced transcription of the nprL gene. A consensus DNA binding motif (helix-turn-helix) was found within the amino acid sequence of the PreL. These results suggest that preL is a transcriptional activator of nprL. Indeed, a possible target sequence of PreL with a stable palindromic structure containing putative — 10 region was found in the promoter region of nprL. It is speculated that PreL melts this palindromic structure and assists RNA polymerase in binding effectively to the nprL promoter. Furthermore, a similar palindromic structure was also found in the promoter region of preL itself. Enhanced production of NprL in the stationary phase may possibly be due to this auto-activation by preL.

Cloning and characterization of a pair of novel genes that regulate production of extracellular enzymes in Bacillus subtilis

Two novel Bacillus subtilis genes that regulate the production of several extracellular enzymes were clones and characterized. These two genes are organized as part of an operon. When cloned in a multicopy plasmid, the first gene (tenA, transcription enhancement) stimulates alkaline protease production at the transcriptional level. The second gene (tenI) exerts an opposite effect to reduce alkaline protease production. The production of neutral protease, levansucrase, and alkaline protease can be stimulated up to 11- to 55-fold. Thus, tenA is a new member of the deg (regulatory genes for degradative enzymes) family in B. subtilis. A functional degS product is required to observe the stimulatory effect from tenA. Between the promoter and the ribosome-binding site of tenA, there exists a terminatorlike structure. Deletion of this structure doubles the expression of tenA. Neither tenA nor tenI is essential for cell growth and the production of extracellular enzymes. However, inactivation of these genes causes a delay in sporulation. This operon is located close to tre on the genetic linkage map. The overall organization of this operon and its relationship with other known regulatory factors in the deg family are discussed.

Transforming growth factor-β is a potent inhibitor of IL-1 induced protease activity and cartilage proteoglycan degradation

Treatment of chondrocytes in culture with interleukin-1 results in the production of neutral proteases that cause the degradation of the large aggregating proteoglycan. TGF-beta is a pleiotropic growth factor that has been shown to induce differentiation of cartilage and, in some cases, was able to inhibit the IL-1-dependent processes. In this report, we examined whether TGF-beta could block the IL-1 induced catabolic effects on chondrocytes. After treatment with IL-1 beta (30 ng/ml), rabbit articular chondrocytes produced approximately 2 units of neutral protease activity. Under identical conditions, TGF-beta 1 alone did not induce any protease activity. However, a combination of IL-1 and TGF-beta resulted in a dramatic reduction in the level of protease activity. The inhibitory effect of TGF-beta was also observed at the level of proteoglycan incorporation into the extracellular matrix. The IL-1 treated chondrocytes failed to incorporate proteoglycans into their extracellular matrix. However, addition of TGF-beta in the presence of IL-1 resulted in partial reversal towards a normal extracellular matrix. These studies indicate that TGF-beta can block and at least partially inhibit the catabolic effects of IL-1 on chondrocytes.