Evaluation of Protease-producing Ability of Fish Gut Isolate Bacillus cereus for Aqua Feed

Extracellular protease production by Bacillus cereus isolated from the intestine of fish Mugil cephalus has been investigated in shake-flask experiment using different preparations of tuna-processing waste such as raw fish meat, defatted fish meat, alkali hydrolysate, and acid hydrolysate as nitrogen source. Among the tuna preparations tested, defatted fish meat supported the maximum protease production (134.57 ± 0.47 U ml⁻¹), and 3% concentration of the same was found to be optimum for maximizing the protease production (178.50 ± 0.28 U ml⁻¹). Effect of carbon sources on protease production in the optimized concentration of defatted tuna fish meat revealed that galactose aided the higher protease production (259.83 ± 0.04 U ml^–1) than the other tested carbon sources and a concentration of 1.5% galactose registered as optimum to enhance the protease production (289.40 ± 0.16 U ml⁻¹). The halotolerancy of B. cereus for protease production indicated that 3% of sodium chloride was optimum to yield maximum protease (301.63 ± 0.20 U ml⁻¹). Among the surfactants tested, protease production was high in Triton X 100-added medium (298.63 ± 0.12 U ml⁻¹) when compared to other surfactants, and its optimum concentration recorded was 0.8% (320.57 ± 0.17 U ml⁻¹) for more protease production. Partial characterization of crude enzyme revealed that pH 7.0 (278.90 ± 0.08 U ml⁻¹) and 60°C temperature (332.37 ± 0.18 U ml⁻¹) were optimum for better protease activity by B. cereus.     

Inactivation Kinetics of Enzymes by Using Continuous Treatment with Microbubbles of Supercritical Carbon Dioxide

ABSTRACT Enzyme inactivation using a new apparatus for continuous treatment with microbubbles supercritical carbon dioxide (SC-CO₂) was investigated. D value of a-amylase (5.0±1.2 min) subjected to microbubbles of SC-CO₂ treatment (microbubbles-SCT) at 35 °C, 30 MPa was lower than that (227 ± 15.9 min) subjected to heat treatment (HT) at 70 °C. D value of acid protease was reduced by microbubbles-SCT at 50 °C, 30 MPa (15.4 ± 4.1 min), compared to HT at 50 °C (233 ± 15.2 min). The activation energy for the inactivation of acid protease (135 ± 8.3 kJ mol^-1) by microbubbles-SCT was 1 half of that (259 ± 9.0 kJ mol^-1) by HT. These results indicated that continuous treatment with microbubbles of SC-CO₂ was effective for enzyme inactivation.     

Optimization of process conditions for the production of a prolylendopeptidase by <Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 11414 in solid state fermentation

The effect of 8 factors [(with/without) daily mixing and moisture control, incubation time (t), temperature, ratio between dry substrate mass and bed’s cross section area (MA), inoculum size (spores/g), wheat germ content (WG), initial pH, and moisture content (M)] in the production of a prolyl endopeptidase (PEP) by Aspergillus niger ATCC 11414 in solid state fermentation (SSF) was tested. Contribution of all the factors was significant (p<0.05); main effects were those of MA, t, and M. The 4 interactions that presented high interaction severity indexes involved the WG. Under optimized conditions PEP and protease activity were 9.76±0.06 and 3.6×10⁶±1.5×10⁵ U/kg, respectively. The enzyme was partially purified (ammonium sulfate precipitation, dialysis, DEAE-Sepharose ionexchange); it has a molecular weight of 66 kDa (SDS-PAGE), and maximum activity was exhibited at pH 4 and 50°C. The enzyme is stable in a wide pH range (2.2–10) and at temperatures lower than 70°C.     

An alkaline protease from Kocuria kristinae F7: properties and characterization of its hydrolysates from soy protein

An alkaline serine protease (SF1) from Kocuria kristinae F7 was purified. The molecular mass of the SF1 protease was estimated to be 57 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The N-terminal amino acid sequence of the first 14 amino acids of the SF1 protease showed low homology with bacterial proteases, suggesting that the enzyme had not been described previously. The SF1 protease exhibited maximal activity at pH 9.0 and 60 °C. The activity of the SF1 protease was enhanced by the presence of Ca²⁺ and Mg²⁺ ions. It showed high stability in the presence of NaCl and ethanol. Reverse-phase high-performance liquid chromatography (RP-HPLC) analyses indicated that soybean protein isolates treated with the SF1 protease generated four principal new hydrophilic peptides. Mass spectrometry analyses indicate that the distribution of molecular weight of these peptides was from 0.705 to 1.305 kDa. Hydrolysis of soybean protein isolates with the SF1 protease increased the level of total free amino acids, essential amino acids and flavor amino acids. The SF1 protease may decrease the bitterness of soy protein hydrolysates. The results showed that the SF1 protease of Kocuria kristinae F7 appears to be good candidate enzyme for potential application in acceleration of fermented soybean food ripening.     

Optimisation of hydrolysis of purple sea urchin (Strongylocentrotus nudus) gonad by response surface methodology and evaluation of in vitro antioxidant activity of the hydrolysate

BACKGROUND: Hydrolysates prepared from sea urchin (Strongylocentrotus nudus) gonad by enzymatic treatment showed strong 1,1‐diphenyl‐2‐picrylhydrazyl radical scavenging activity and reducing power. RESULTS: Hydrolysis of S. nudus gonad by the commercial protease papain was optimised for maximum degree of hydrolysis (DH) and trichloroacetic acid‐soluble peptide index (TCA‐SPI) using response surface methodology. Results showed that the optimal conditions were the following: temperature of 48.83 °C, pH of 6.92, enzyme‐to‐substrate ratio of 3143 U g^?1, and substrate concentration of 83.5 g L^?1. Under these conditions, a DH of 27.96 ± 0.54% and a TCA‐SPI of 57.32 ± 0.63% were obtained. The hydrolysate prepared in the optimal conditions was fractionated by an ultra‐filtration system and the resultant fraction below 10 kDa was found to effectively scavenge hydroxyl radical (EC₅₀ = 13.29 ± 0.33 mg mL^?1) and hydrogen peroxide (EC₅₀ = 16.40 ± 0.37 mg mL^?1), inhibit lipid peroxidation (EC₅₀ = 11.05 ± 0.62 mg mL^?1), chelate Fe²⁺ (EC₅₀ = 7.26 ± 0.44 mg mL^?1), and protect mice macrophages against death induced by tert‐butyl hydroperoxide. CONCLUSION: Hydrolysates prepared from S. nudus gonad have the potential to be applied as natural antioxidant agents. Copyright © 2012 Society of Chemical Industry     

PURIFICATION AND CHARACTERIZATION OF BACILLUS SUBTILIS JM-3 PROTEASE FROM ANCHOVY SAUCE

Bacillus subtilis JM‐3 was isolated from anchovy sauce naturally fermented in an underground cellar at 15 ± 3C for 3 years. The activity of the B. subtilis protease was highest in the 40–60% ammonium sulfate fraction. The yield of the purified protease was 5.3%, and its purification ratio was 35.6 folds. The molecular weight of the B. subtilis protease was 17.1 kDa, and its K_m and V_maxvalues were 1.75 μg/mL and 318 μM 1/min, respectively. The optimal temperature for protease activity was 60C, but optimal stability temperature was 30C. The optimal pH for protease activity and stability was 5.5. Therefore, the B. subtilis JM‐3 protease was classified as an acid protease. The relative activities of the B. subtilis JM‐3 protease were 69, 21 and 1.3% at 10, 20 and 30% NaCl concentrations, respectively. The best substrate for the B. subtilis JM‐3 protease was benzyloxycarbonyl‐glycine‐p‐nitrophenyl ester followed by bovine serum albumin. p‐Toluene‐sulfonyl‐L‐lysine chloromethylketone was the strongest inhibitor followed by soybean trypsin inhibitor, but N‐ethylmaleimide did not inhibit this enzyme. The B. subtilis JM‐3 protease was therefore presumed to be a trypsin‐like serine protease.     

Purification and Characterization of a Halotolerant Alkaline Serine Protease from Penicillium citrinum YL-1 Isolated from Traditional Chinese Fish Sauce

A halotolerant alkaline serine protease from Penicillium citrinum YL-1 which was isolated from traditional Chinese fish sauce was purified by ammonium sulfate precipitation, dialysis, and DEAE 52-Cellulose column, thereby resulting in a 4.66-fold increase in specific activity (110.68 U/mg). The molecular weight (MW) was estimated to be 32.27 kDa using SDS-PAGE analysis. The protease exhibited optimal activity toward the substrate casein at pH 8.0 at 40°C and was stable at pH 6.0–8.0 and 4–30°C. Activity was inhibited by NaCl and retained at 28.3, 21.4 and 18.1% of the initial activity after incubation for 6 h at 20, 25 and 30% NaCl concentrations, respectively. The enzyme was stimulated by Mn²⁺ and inhibited by K⁺, Ca²⁺, Zn²⁺, Mg²⁺, Fe²⁺, and Fe³⁺. K_m and V_max of the protease for casein were 1.93 mg/ml and 56.81 μg/(min·ml), respectively. Protease activity was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), which confirmed the serine protease nature of the enzyme. The protease can hydrolyze tilapia protein in the absence or presence of NaCl (5–30%), thus suggesting that this protease is more halotolerant than the protease from other bacteria with high salinity resistance based on the current literature. These properties make the halotolerant alkaline serine protease a suitable candidate enzyme for fish protein hydrolysis during fish sauce fermentation.     

INFLUENCE OF HARVEST SEASON ON THE PROTEOLYTIC ACTIVITY OF HEPATOPANCREAS AND MANTLE TISSUES FROM JUMBO SQUID (DOSWICUS GIGAS)

Jumbo squid (Dosidicus gigas) size and protease activities were evaluated after harvest in April (A) and November (N). A were smaller (560±120 g vs 6040±1130 g) and hepatopancreas was a larger percent of body weight (8.3±2.5 vs 4.6±2.1). Mantle from A had lower water (73.9±l.l vs 79.1± 1.2) and higher protein (29.0±1.1 vs 23.1±0.8). Lipid and protein contents of N and A hepatopancreas tissues did not differ (P < 0.05). Azocaseinolytic, trypsin‐like, chymotrypsin‐like, aminopeptidase, and carboxypeptidase activities were detected in mantle (ME) and hepatopancreas extracts (HPE). HPE and ME from N had higher activity than A for all substrates (P < 0.05). With azocasein substrate, HPE activity from A had a pH optimum of 5 ‐ 6 and a temperature optimum of 70C, whereas HPE from Nhad highest activity at pH 9 and 40–70C. ME from A had maximum proteolytic activity at pH 6 and 60C, whereas that from N had maximum activity at pH 10 and 40C. SDS‐PAGE zymograms of HPE from A and N revealed different patterns of activity and 1 and 2 major protease zones, respectively.     

Isoelectric fractionation and some properties of a protease from soyabean seeds

A protease, capable of hydrolysing benzoyl DL -arginine p-nitroanilide(BAPA), and L-amino acid β-naphthylamide derivatives, was purified, by isoelectric focusing in the region pH 3–6, from dormant and 6-day germinated soyabean seeds. The enzyme was focused at pH 4·80. The K_m value using BAPA as substrate was found to be 5·03 × 10⁻⁴M . Maximum activity of the enzyme towards BAPA was obtained in the pH 8·2–8–5 region. Slight activation was observed in the presence of 0·05 M concentration of Ca²⁺ and Mg²⁺ ions. The protease lacked caseinolytic activity, and was not inhibited by Kunitz soyabean trypsin inhibitor.     

Partial purification and characterisation of cysteine protease in wheat germ

BACKGROUND: Proteases have become an essential part of the modern food and feed industry, being incorporated in a large and diversified range of products for human and animal consumption. The objective of this study was to purify and characterise a protease from wheat germ. RESULTS: After purification a single protease of molecular weight 61–63 kDa (determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis) was obtained. The purified protease had optimal activity at 50 °C and maintained its activity completely after incubation at 30 °C for 30 min, while over 47% of the activity was lost after incubation at 80 °C for 30 min. The purified protease had optimal activity and maintained maximum stability at pH 5.5, while the activity decreased after incubation for 30 min at other pH values. The protease was inhibited by Mg²⁺, Mn²⁺, Ba²⁺ and iodacetic acid and stimulated by Li⁺, Ca²⁺, Cu²⁺, β‐mercaptoethanol and dithiothreitol, while Zn²⁺, L ‐cysteine and glutathione had no significant effect on its activity. At pH 5.5 the enzyme had a K_m of 0.562 mg mL^?1 with casein as substrate and showed higher affinity to casein than to bovine serum albumin, ovalbumin and gelatin. CONCLUSION: The purified enzyme from wheat germ was identified as a cysteine protease. Copyright © 2011 Society of Chemical Industry     

PURIFICATION OF A PROTEASE FROM AN ALKALOPHlLIC BACILLUS SUBTILIS CHZ1 ISOLATED FROM A ZIMBABWEAN HOT SPRING

A proteolytic enzyme produced by Bacillus subtilis CHZ1 was purified using ammonium sulfate precipitation, gel filtration and cationic exchange on S‐Sepharose fast flow column chromatography. Production of the protease was higher when the Bacillus strain was cultured in a synthetic medium, M162, supplemented with 0.3% (w/v) organic compared to inorganic nitrogen sources. Enzyme production was growth dependent and production was highest when tryptone was used as the nitrogen source. When run on SDS‐PAGE gel, the purified enzyme gave a 35 kDa band, suggesting that it consisted of one polypeptide chain. High enzyme activity was observed in the pH range of 6–10 with a maximum value at pH 8.0 when 0.5% (w/v) azocasein was used as the substrate. Optimum temperature for protease activity was found to be 60–80C, and the enzyme had considerable thermal stability for 5.5 h retaining about 90% activity after 5.5 h. At 2.5 mM concentration, PMSF, Ag⁺ and Hg⁺ inhibited activity of the protease. Metal cofactors like Mn²⁺, Mg²⁺ and Fe²⁺ increased the enzyme activity by about 20%. Zn²⁺, Cu²⁺ and Ca²⁺ did not affect the enzyme's activity. The pH and thermal stability as well as high specific activity of this enzyme can be exploited for industrial applications.     

菌酶协同发酵水解大米蛋白ACE抑制肽及其活性的研究

使用植物乳杆菌2-18和枯草芽孢杆菌Y、枯草芽孢杆菌Y4-2联合蛋白酶共同水解大米蛋白,测定大米蛋白的水解溶出率及脱除液的血管紧张素转换酶(angiotensin I-converting enzyme,ACE)抑制活性。在利用菌酶联合水解大米蛋白的组合中,植物乳杆菌2-18+风味蛋白酶/菠萝蛋白酶+胃蛋白酶/酸性蛋白酶组合对大米蛋白的水解溶出率最高,蛋白脱除率为(91.32±1.60)%。大米蛋白水解溶出液中必需氨基酸占总氨基酸含量的39.62%。通过对分子量分布分析,大米蛋白水解溶出液的多肽分子量分布主要在1 kDa～1.5 kDa部分。经植物乳杆菌2-18+风味蛋白酶/菠萝蛋白酶+胃蛋白酶/酸性蛋白酶组合对大米蛋白的水解溶出液ACE抑制率达(91.95±1.63)%,具有良好的ACE抑制活性。     

Purification of a novel protease enzyme from kesinai plant (Streblus asper) leaves using a surfactant–salt aqueous micellar two-phase system: a potential low cost source of enzyme and purification method

Serine protease from kesinai leaves was purified for the first time by a surfactant–polymer aqueous micellar two-phase system. The effectiveness of different types and concentrations of non-ionic surfactants (Pluronic series and X-114) on the partitioning behaviour of the protease was evaluated. The results showed that the enzyme preferentially partitioned into the bottom surfactant-rich phase, while the hydrophilic amino acid preferred the top aqueous phase. This distribution of the enzyme is due to the hydrophobic interaction of the serine protease with the hydrophobic lid of the micelle core in the bottom phase. The influence of different types of salts (K₂SO₄, KH₂PO₄, KCl and KNO₃) on the purification and selectivity of the enzyme was determined. The protease partitioning in the bottom phase increased in the presence of KNO₃, which confirmed that the salt was able to improve the protein solubility in bottom phase and increase the hydrophobic interaction between the two phases. In addition, the protease from the bottom phase was re-extracted to a new aqueous phase solution to remove and recycle the surfactant. Addition of potassium thiocyanate led to the partitioning of the enzyme in top aqueous phase due to high ionic strength of SCN^?, which forced the lighter micellar phase toward the upper position of the system. A high purification factor (10.3) and yield of 92 % of the enzyme were achieved in a solution of 31 % of Pluronic L61 using 0.3 % KNO₃ and 50 % crude feedstock at pH 7.0.     

牡蛎致敏蛋白初步分离和酶解处理对其抗原性的影响

分离获得太平洋牡蛎致敏蛋白,考察酶解法对牡蛎致敏蛋白抗原性的影响。方法 利用硫酸铵沉淀和葡聚糖凝胶G-75分离牡蛎致敏蛋白,聚丙烯酰胺电泳分析获得蛋白的分子量大小,使用木瓜蛋白酶、中性蛋白酶和动物蛋白水解酶(3 000 U/g)酶解牡蛎致敏蛋白,OPA法测定水解度,间接竞争ELISA法测定酶解产物的抗原性。结果 分离到分子量约为40和98 kD的牡蛎致敏蛋白,经木瓜蛋白酶酶解的致敏蛋白特征条带消失,酶解产物水解度为(31.87±0.309)%,动物蛋白酶和中性蛋白酶对牡蛎致敏蛋白水解度分别为(24.13±0.153)%和(12.43±0.115)%,但致敏蛋白特征条带未完全消失;间接竞争ELISA检测表明3种蛋白酶均能不同程度地降低牡蛎致敏蛋白的抗原性或使致敏性基本消除。结论 蛋白酶酶解法可有效水解牡蛎致敏蛋白,从而降低其抗原性,其中木瓜蛋白酶效果明显。     

Wheat-bug damage in New Zealand wheats: Some properties of a glutenin hydrolysing enzyme in bug-damaged wheat

Some properties of a glutenin hydrolysing enzyme present in bug (Nysius huttoni) damaged wheat (Triticum aestivum) were examined using a modified SDS sedimentation test reported previously. The enzyme appears to be a water-soluble alkaline protease with an activity optimum at pH 9.0. It is relatively heat stable, but the temperature optimum for activity is quite low (35–40°C). The enzyme is not inhibited by EDTA or N-ethylmaleimide, but is inhibited by the metal ions Co²⁺, Mn²⁺ and Fe²⁺.     

Purification and characteristics of serine protease from the head of pacific white shrimp

Serine protease from the head of Pacific white shrimp was purified by the following techniques: ammonium sulfate fractionation, Q-Sepharose HP ion exchange chromatography, and Sephadex G-100 gel filtration. The molecular weight was estimated as 32.8 kDa using SDSPAGE. The optimum pH and temperature of the enzyme for the hydrolysis of casein were determined to be 10.0 and 40°C. It was stable at pH range from 8.0 to 11.0 and had good thermal stability. Pb²⁺, Ca²⁺, Mg²⁺, Cu²⁺, and Mn²⁺ could active the enzyme certainly when Zn²⁺ and Hg²⁺ strongly inhibited the activity. The enzyme was inhibited by the general serine protease inhibitor (PMSF) and the specific trypsin inhibitors (TLCK, SBTI). The modification of various amino acid modifiers for the purified enzyme determined that the enzyme active center included tryptophan, histidine, and serine, moreover, arginine had a certain relationship with the enzyme activity.     

Effect of cellulase/glucanase/xylanase (Roxazyme G2) enzymes combination on nutrients utilisation of vegetable meal (Amaranthus cruentus) fed as sole dietary protein source in rat assay

Amaranthus cruentus vegetable meal (ACVM) had 23% crude protein. Ca, Na, K, Mg and Fe were abundant at 2.0 g kg^?1, 7.1 g kg^?1, 4.8 g kg^?1, 2.5 g kg^?1, 1109 mg kg^?1, respectively. P‐phosporous, oxalates and tannins were noticeable. Lysine, methionine and cystine were limiting. Weight gain for rats on the reference (casein) diet 2 at 6.30 g ±2.87 was highest (P < 0.05) followed by diet 6 (12% ACVM with enzyme supplementation) at 5.01 g ±2.42. Feed intakes were similar (P > 0.05) for rats on the reference diet and for rats on 10% and 12% with/without enzyme supplementation ranging from 42.90 g ± 4.52 in reference diet to 45.12 g ± 3.64. Nitrogen retention was highest for rats on reference diet but similar (P > 0.05) to rats on 12% enzyme supplemented diet at 0.53 ± 1.12 and 0.53 ± 2.10, respectively. Other investigated protein evaluation parameters revealed similar results among rats kept on reference diet and the rats on ACVM based diets with enzyme supplementations. Enzyme supplementation had a complimentary role in ACVM nutrition in rat trial.     

Purification and characterization of an alkali-thermostable β-mannanase from Bacillus nealsonii PN-11 and its application in mannooligosaccharides preparation having prebiotic potential

An alkali-thermostable β-mannanase from Bacillus nealsonii PN-11 was purified 38.96-fold to homogeneity with specific activity of 2,288.90 ± 27.80 U mg^?1 protein and final recovery of 8.92 ± 0.09 %. The purified β-mannanase was an extracellular monomeric protein with a molecular mass of 50 kDa on SDS–PAGE. The first 20 N-terminal amino acid sequence of mannanase enzyme was MVVKKLSSFILILLLVTSAL. The optimal temperature and pH for enzyme were 65 °C and 8.8, respectively. It was completely stable at 60 °C for 3 h and retained >50 ± 1.0 % activity at 70 °C up to 3 h. The β-mannanase was highly stable between pH 5–10 and retained >85 % of the initial activity for 3 h. The metal ions Ni⁺², Co⁺², Zn⁺² and Mg⁺² enhanced the enzyme activity. The enzyme remained stable after 3 h of preincubation with most of the tested organic solvents. According to substrate specificity study, the purified mannanase had high specificity to locust bean gum which was degraded mainly to mannooligosaccharides (MOS) like mannotriose, mannotetraose and mannopentose. These MOS enhanced the growth of Lactobacillus casei but inhibited the growth of Salmonella enterica indicating potential prebiotic properties. The properties of the purified β-mannanase from B. nealsonii PN-11 make this enzyme attractive for biotechnological applications.     

Acid phosphatase in the tubers of Dioscorea species and its purification from the white yam (D. rotundata poir)

Acid phosphatase activity was determined in 15 cultivars from four species of yam. A 12-fold purification of the enzyme from Dioscorea rotundata (cv. chikakwondo) gave a homogeneous preparation as demonstrated by polyacrylamide gel electrophoresis. This enzyme preparation has an apparent molecular weight of 115 000 ±2000 and an optimum activity at a pH of 5·20 and a temperature of 50°C. The K_m of the enzyme is 3·81 mM with disodium p-nitrophenylphosphate (p-NNP) as a substrate. The energy of activation, heat of activation, energy of inactivation and heat of inactivation are 7·0, 6·4, 4·41 and 4·34 kcal M^?1, respectively. Although it has very little activity with most organic phosphoric acid esters, it is significantly inhibited by Ca²⁺, Hg²⁺ and EDTA and activated by Mg²⁺. The enzyme has a half-life of 50,17 or 13 days, respectively, when stored at 6-8°C, 0°C or room temperature (29±2°C).     

Purification and characterization of cathepsin B from the gut of the sea cucumber (<Emphasis Type="Italic">Stichopus japonicas</Emphasis>)

Cathepsin B from the gut of sea cucumber (Stichopus japonicas) was purified 81-fold with a 3% recovery by ammonium sulfate fractionation and a series chromatography on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-Gel 3000 SWxl. The purified protein appeared as a single band on Native-PAGE but showed 2 bands of 23 and 26 kDa on SDS-PAGE. The optimum activity was found at pH 5.5 and 45°C. The enzyme was stable at pH 4.5–6.0 and the thermal stability was up to 50oC. The enzyme was strongly inhibited by E-64, iodoacetic acid, and antipain, demonstrating it is a cysteine protease containing sulfhydryl groups. Cu²⁺, Ni²⁺, and Zn²⁺ could strongly inhibit the enzyme activity. The amino acid sequences of the purified enzyme were acquired by mass spectrometer, which did not show any homology with previously described cathepsins, suggesting it may be a novel member.