Fitness Cold Protease And The Molecular Basis Of Adaptation In Temperature Protease Temperature And Its High-value Processing Of Marine Biological Protein Resources

Part Ⅰ, The molecular base of temperature adaptation of cold-adapted protease MCP-01 and mesophilic protease BP-01Marine cold-adapted microorganisms and enzymes have been studied since 1999 in our lab. A cold-adapted bacterium named Pseudoalteromonas. sp. SM9913 was isolated from 1855 meter deep-sea sediment by Xiu-Lan Chen et al. SM9913 secreted a typical cold-adapted protease MCP-01, which had high catalytic efficiency at low temperature (Chen et al, 2002, Chen et al, 2003a; Chen et al, 2003b). The gene of MCP-01 was cloned and sequenced by Jun Pan. Based on these researches, compared with another mesophilic protease BP-01,which belonged to the same protease family with cold-adapted protease MCP-01. the relationship between structure and function of MCP-01 was studied, and the molecular structure base of cold-adapted protease MCP-01 for high catalytic efficiency, low stability and high thermolability was revealed.The mesophilic protease BP-01 was secreted by a mesophilic strain Bacillus subtilis SM98011. In this article, the gene of mesophilic BP-01 was cloned and sequenced. According to the nucleotide sequence of its gene, the amino acid sequence of BP-01 was deduced and compared with that of cold-adapted protease MCP-01, which was deduced according to the nucleotide sequence of its gene by Pan Jun. The result showed that they had little identity at their amino acid sequences. The 3D structure models of their catalytic domains were constructed by an automated homology-modeling server, respectively. According to their 3D structure models, the catalytic domains of two proteases all belonged to the type subtilase-S8. Their catalytic cavities were surrounded by α -helixes and β -folds, and the three amino acids Asp, Ser, His, which took part in catalyzing were all in the catalytic cavity.These results indicated that the catalytic mechanism of protease MCP-01 and BP-01 ^was the same. According to the result of homology-modeling, there are lower content of a -helixes and higher content of P-turns and random in MCP-01 than in BP-01, which led to its higher flexibility and lower stability in structure. Such a flexible structure favored its catalytic activity at low temperature, and was its molecular base for high catalytic efficiency at low temperature. This was confirmed by the CD spectra of the two proteases. The temperature leading to loss of 50% secondary structure of protease MCP-01 and BP-01 were 49.5°C and 60 °C, respectively, indicating that cold-adapted protease MCP-01 need less energy during its thermo-structure-change and was easy to change its structure. In brief, The high flexibility of cold-adapted protease MCP-01 caused by reduce of a -helixes and increase of p-turns and random in its structure was its molecular base for its high catalytic efficiency, low stability and high thermolabilityConformation changes of protease BP-01 and MCP-01 in the presence of protease inhibitor, PMSF, EDTA and SDS was studied. According to CD spectra, PMSF had little effect on the structures of the two proteases, although it could completely inhibit their activities. EDTA had no effect on the structure and activity of BP-01, and had remarked effect on the structure and activity of MCP-01. When EDTA was added to MCP-01 solution, the activity of MCP-01 was completely inhibited and a -helixes obviously reduced according to CD spectra. So Ca2+ is very important for the maintance of the flexible conformation of MCP-01.GuHCl could cause structure change of proteins through scrabbling for the combined-H2O in protein moleculars. In the presence of GuHCl, the fluorescence intensity of protease BP-01 and MCP-01 both decreased with GuHCl concentration increase. The Xmax of BP-01 showed a smaller red shift, but the tanax of MCP-01 showed a bigger red shift, nearly 20nm. The CD spectra of the two proteases indicated that GuHCl could drastically destroy the secondary structures of MCP-01 and BP-01, but the concentrations needed were different. The half-unfolding concentration C\a of GuHCl for MCP-01 and BP-01 were 0.3M and 1.8M, respectively. The concentrationof lost activities of two proteases in the presence of GuHCl was 0.5M and 3M, respectively. The results indicated the MCP-01 was more sensitive to denaturants than BP-01. Morever, it suggested that MCP-01 had a more flexible conformation than BP-01, resulting in its less stable structure and total lost of its conformation and activities at a very low denaturant concentration.The effects of three different buffers, all at 40 mmol/L concentrations, on thermostability of protease MCP-01 and BP-01 were studied. The thermostability of MCP-01 increased successively in the following order: carbonate Applied research of cold-adapted protease and mesophilic protease in marine bio-protein resource utilization and taste improvement.1 -. High valuing development of low-value marine proteinsA rapid and sensitive method for the determination of the angiotensin-converting enzyme (ACE) inhibitory reaction via capillary electrophoresis has been established and used for high throughput screening of marine protein materials enriched with ACE inhibitory peptides. Samples from twelve marine organisms or parts were selected for use in protein hydrolyzation: shrimp (Acetes chinensis) with low commercial value; scallops; oysters; shark meat; six kinds of seafood waste including codfish bone, codfish skin, mackerel head, mackerel bone, herring skin, and herring bone; marine red alga Polysiphonia urceolata; and blue-green alga Spirulina platensis.Protein from each organism or specified component was degraded with four proteases: Flavourzyme, Protamex, Alcalase and SM98011 produced by the strain Bacillus sp. SM98011. ACE inhibitory activity of the hydrolysates was determined by capillary electrophoresis. Detection was complete within 5 minutes at 228 nm. Time to determine the IC50 values of the 48 hydrolysates was remarkably reduced with the use of capillary electrophoresis. IC50 values of the hydrolysates were in the range of 0.17-501.7 mg/ml. Among them, shrimp, shark meat, mackerel bone, Polysiphonia urceolata and Spirulina platensis were beneficial materials for the production of ACE inhibitory peptides by proteolysis. Selected proteases also affected ACE inhibitory activity of the hydrolysates. Hydrolysates digested with the proteases Protamex and SM98011 had higher ACE inhibitory activity than those digested with Flavourzyme and Alcalase. Our results indicate that capillary electrophoresis is a superior method for the rapid screening of hydrolysates from marine proteins enriched in peptides with ACE inhibitory activity.Acetes chinensis was digested by protease produced by Bacillus sp. SM98011. The angintensin I-converting enzyme (ACE) inhibitory activity of hydrolysate was investigated with IC50 value of 0.98 mg/ml. IC50 value of the ultrafiltrate with MW cut-off 3000Da decreased to 0.22 mg/ml. Peptide fractions in the ultrafiltrate was separated with Sephadex G-15 gel chromatography. The fractions, which showed the high ACE-inhibitor activity, were further purified by reverse-phase high-performance liquid chromatography. Five ACE inhibitory peptides were identified and three of them were novel. Their sequences were FCVLRP, IFVPAF and KPPETV, with IC50 values of 12.3 uM, 3.4uM and 24.1 uM, and recovery of 30 mg/lOOg, 19 mg/lOOg and 33 mg/lOOg, respectively.Acetes chinensis was digested by BP-01 and filtered through a 3 kDa ultrafiltration membrane. Biological functions of the hydrolysate and ultrafiltrate were then assayed. The analyses showed that 41% of the oligopeptides in the ultrafiltrate had a molecular mass lower than 3 kDa. The anti&xidant activities of the hydrolysate and ultrafiltrate were determined through the scavenger activity of the hydroxylradical, with inhibitions of 42.38% and 67.95%, respectively. The hydrolysate and ultrafiltrate also had good Angiotensin-I-converting enzyme (ACE) inhibitory activity, with IC50 values of 0.98 mg/ml and 0.22 mg/ml, respectively. In addition, Chitin and chitosan were recovered from the hydrolytic sediment using a much smaller volume of strong acids and bases than is normally needed. With this method, we have shown that Acetes chinensis can be utilized to generate a high value-added product, and have revealed its hidden potential in the production of functional foods and ACE inhibitory peptides.Based on these researches in lab, middle-scale experiment and industrial scale production of preparing ACE inhibitory peptides by hydrolyzing Acetes chinensis with proteases were finished. With strain Bacillus subtilis SM98011, middle-scale production of mesophilic protease BP-01 was done in 200 L fermentation vessel. The activity of protease BP-01 in ferment solution reached 4000 U/ml. Middle-scale and industrial hydrolyzation of Acetes chinensis by protease BP-01 was done in 100L and 10,000L reaction vessels, respectively. The dry product was powder with orange color and a little shrimp smell. It was easy to be solved in water and its solution was clear and with a little orange color. The IC50 values of the products from the 100L and 10,000L reaction vessel were determined and their oligopeptide composition was analyzed with HPLC. The IC50 values of the products from the 100L reaction vessel and 10,000L reaction vessel were 0.96mg/ml and 0.97mg/ml respectively, which was the same as that of hydrolysate in lab (0.98mg/ml). The peptide spectra of the products from the 100L reaction vessel and 10,000L reaction vessel had good repetitions with that of hydrolysate in lab. Protein content and peptide lower than 3000 Da from the 10,000L reaction vessel were 85.6% and 48.7%, respectively. Middle-scale experiment and industrial production of preparing ACE inhibitory peptides by hydrolyzing Acetes chinensis laid a good foundation for its industrialization.2. Application of cold-adapted protease MCP-01 in taste Improvement ofrefrigerated foodThe cold-adapted protease produced by a deep-sea cold-adapted bacterium Pseudoaltermonas sp. SM9913 and the mesophilic protease produced by a mesophilic bacterium Bacillus subtilis SM98011 were sprayed onto the surfaces of marine fish, pork and shrimp meat, respectively, and then stored at 0 °C for 6 days. The amounts of free amino acids in the hydrolysates of samples were determined. The results showed that the samples treated with MCP-01 released more more taste amino acids and essential amino acids than those treated with BP-01 at 0 "C. Therefore, the cold-adapted protease had potential in improving the taste of refrigerated food.