Optimization of elastolysis conditions and elastolytic kinetic analysis with elastase from Bacillus licheniformis ZJUEL31410

Optimization of elastolysis conditions and elastolytic kinetic analysis with elastase from Bacillus licheniformis ZJUEL31410 first-author †E-mail:qhchenzju@yahoo.com.cngqhe@zju.edu.cn †E-mail:qhchenzju@yahoo.com.cngqhe@zju.edu.cn qhchenzju@yahoo.com.cn corresp ‡Corresponding Author ‡Corresponding Author †E-mail:qhchenzju@yahoo.com.cngqhe@zju.edu.cn †E-mail:qhchenzju@yahoo.com.cngqhe@zju.edu.cn qhchenzju@yahoo.com.cn Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, China Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, China The solubilization of elastin by Bacillus licheniformis elastase cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme-substrate complex has no physical meaning. In this paper we report the optimization of elastolysis conditions and analysis of elastolytic kinetics. Our results indicated that the hydrolyzing temperature and time are very important factors affecting elastolysis rate. The optimized conditions using central composite design were as follows: elastolysis temperature 50 °C, elastase concentration 1×10 4 U/ml, elastin 80 mg, elastolytic time 4 h. Investigation of the effects of substrate content, elastase concentration and pH was also revealed that low or high elastin content inhibits the elastolysis process. Increasing elastase improves elastin degradation, but high elastase may change the kinetics characterization. Alkaline environment can decrease elastin degradation rate and pH may affect elastolysis by changing elastase reaction pH. To further elucidate the elastolysis process, the logistic model was used to elastolysis kinetics study showing clearly that the logistic model can reasonably explain the elastolysis process, especially under lower elastase concentration. However, there is still need for more investigations with the aid of other methods, such as biochemical and molecular methods. Bacillus licheniformis Elastase Elastolysis condition optimization Elastolytic kinetics INTRODUCTION 1 Elastases are heterogeneous with differing substrate specificities and catalytic mechanisms. In fact, enzymes with elastolytic activity can be found in most of the major proteolytic families, including serine, thiol, aspartic, and metallo-enzymes. Despite the differences in catalytic mechanisms, all of these elastases share a common specificity for cleaving peptide bonds associated with hydrophobic or aromatic amino acids (Hall and Czerkawaki, 1961 1961 ). Bacterial elastases belonging to the family of metalloproteinases have been known for many years. These proteinases require Zn atoms for activity and in contrast to the serine proteinases cleave peptide bonds on the amino-terminal side of the amino acid that determines specificity. Elastase from Bacillus thermoproteolyticus and Streptomyces fradiae are four and eight times more active than PPE (pig pancreatic elastase), respectively, making them some of the most potent elastolytic proteinases reported (Mecham et al., 1997 1997 ). Elastins are extracellular matrix proteins responsible for the resilience of tissues such as skin, arteries and lung and are insoluble, hydrophobic and extensively cross-linked protein forming fibers present in variable amounts depending on the tissue. Elastin displays extreme hydrophobicity and extensive cross-linking. Because of these properties, it is quite stable and persistent in tissues unless subjected to the catalytic activity of one or more of a group of proteinases, collectively termed elastases because of their ability to solubilize amorphous elastin (Mecham et al., 1997 1997 ). Solubilization of elastin is a complicated biologic process since the substrate is exceedingly insoluble and is also heterogeneous with respect to enzyme reaction sites. Such a system is far more complex than the reaction of a proteinase with a soluble protein, and classical enzyme kinetics cannot be applied directly to give understanding of the elastolytic mechanisms. Elastin degradation with enzyme is very applicable and useful to food industry and daily use chemicals industry. Elucidation of elastolysis is beneficial for some diseases therapy related with elastin. Optimization of batch bioreactors depends on the kinetics of the processes that can become extremely complicated for enzymatic hydrolysis of proteins. The large number of peptide bonds that are cleaved, both in parallel and in series, during the hydrolysis of most polymer molecules sets natural limitation to the possibility of estimating the basic kinetic parameters. This is a general problem in the establishing of kinetic models for the hydrolysis of macromolecular substrates. If the kinetic model is too simple, its inadequate representation of the true mechanism will limit its general applicability, while a complicated model cannot be analyzed statistically with sufficient precision by the often relatively crude kinetic experiments. According to Adler-Nissen, it is generally accepted that serine protease like alcalase, when acting on peptide bonds of protein, generally follow Michaelis-Menten kinetics (Adler-Nissen, 1986 1986 ). However, other kinetics models such as logistic model were also used to describe proteolysis. Considerable efforts were made to screen the elastase-producing strains, to study their pathogenic effect and their characterizations (Tsuzuki and Oka, 1965 1965 ; Tsai et al., 1988 1988 ; Sharon et al., 1997 1997 ; Ozaki and Shiio, 1975 1975 ). Reported studies on the reaction mode between elastin and elastase are rare (Hall and Czerkawaki, 1961 1961 ). The reaction was found to be a complex process of at least three steps (Heather et al., 1999 1999 ). The mechanism of the reaction is still under investigation at present. There are many studies focused on the identification and purification of elastase-producing strains, but few on elastolysis. In a previous work, a new elastase was isolated from Bacillus licheniformis ZJUEL31410 to study its cultivation conditions and fermentation kinetics (Chen et al., 2002 2002 ; 2004 2004 ); this enzyme was a protease with very high elastolytic activity, so it is interesting to further study its structural and functional relationships, including the difference in substrate specificity. In the present work, elastolysis conditions were optimized and the elastolysis kinetics model was preliminarily constructed. MATERIALS AND METHODS 1 Materials 2 Elastase was made from Bacillus licheniformis ZJUEL31410 (stored in my lab) culture liquid with graded salts precipitation (45%~70%). The elastase activity after precipitation was 20000 U/ml (Chen et al., 2003 2003 ). Crude elastin was produced by the method of Partridge and Davis ( 1948 1948 ). Standard elastin was bought from Sigma Company (USA). Coomaas brilliant blue and casein were bought from Shanghai Biochemical Co. Ltd. (China). Congo-red elastin from bovine neck ligament was purchased from Sigma Co. Ltd. (USA). Other chemical agents were analytical grade (Hangzhou, China). Microorganism and media 2 Bacillus licheniformis ZJUEL31410 was isolated and mutated by Zhejiang University, China. This strain was maintained on LB (g/L) slants (peptone 6, yeast extract 2, beef extract 4, NaCl 5, agar 2). The medium for the inoculum contained the above components in addition to the agar. Assay methods 2 1. Enzyme assay 3 Elastolytic activity was assayed by the colorimetric method of Sachar ( 1955 1955 ). Enzyme preparation was incubated with 20 mg of congo-red elastin in 2 ml of 0.2 mol/L boric acid buffer (pH 7.4) with shaking for 20 min at 37 °C. The reaction was stopped by adding 2 ml of 0.7 mol/L sodium phosphate buffer (pH 6.0), and immediately filtered. Absorbency of the filtrate was read at 495 nm against the control (no enzyme). One unit of elastase activity was defined as the amount of enzyme required to solubilize 20 mg elastin-congo red under the tested conditions. 2. Enzyme preparation 3 Crude elastase and purified elastase were prepared by such method. Briefly, Bacillus licheniformis ZJUEL31410 was aerobically cultured in a medium containing glucose and casein and corn steep flour, at 37 °C for 30 h. Ammonium sulfate precipitation (40%~70%) of the culture fluid was performed to obtain the partially purified enzyme, and this fraction was then further purified using Sephadex G-100 (Pharmacia, Co. USA) column chromatography (Chen et al., 2003 2003 ). 3. Protein content 3 The protein hydrolysate content was determined by Bradford ( 1976 1976 ) method, using bovine serum albumin (BSA) as the standard. The content of amino of elastin was measured by the method of Lahl ( 1994 1994 ). 4. Experimental methods 3 In the optimization process, the hydrolyzing factors were optimized with statistical design. Then under this optimized condition, the degradation process was investigated after studying the effect of pH, temperature and enzyme concentration on the elastolysis process. In the experiment, the hydrolyzing rate was calculated by the following equation: $$DH = S_{\text{N}} /T_{\text{N}} \times 100\% = \frac{{(OD_{595} + 0.0012) \times 0.5140 \times 100\% }}{{0.0987 \times W_{{\text{substrate}}} }},$$ where DH is degree of hydrolysis; S N is the soluble nitrogen; T N is the total nitrogen; OD 595 is the absorbency density at wavelength 595 nm; W substrate is elastin weight added. Experimental designs 2 In this work the experimental sets were designed with SAS software and the results were analyzed by SAS software also. Response surface methodology (RSM) is an excellent experimental strategy for seeking optimal conditions for multi-variables system, and have been successfully employed for optimizing the medium composition and operating conditions in many bioprocess (Ma and Ooraikul, 1986 1986 ). We used the central composite design based on previous experiment to optimize the following factors. The experiment factor level design is shown in the Table 1 1 in the form of both coded values and natural values. The correspondence between these values can be calculated by: X i =( x i − x i 0 )/ δ i , where X i is the coded value, x i is the corresponding natural value, x i 0 is the natural value at the center of the domain, and δ i is the increment of x i corresponding to 1 unit of X i . X 1 =( x 1 −50)/10; X 2 =( x 2 −20000)/10000; X 3 =( x 3 −80)/30; X 4 =( x 4 −3)/1 Runs Temperature X 1 Elastase con. X 2 Substrate con. X 3 Elastolytic time X 4 OD 595 DH (%) Y ( OD 595 ) 1 1 1 1 1 0.182 32.83 2 1 1 1 −1 0.188 33.90 3 1 1 −1 1 0.077 30.83 4 1 1 −1 −1 0.090 35.95 5 1 −1 1 1 0.169 30.50 6 1 −1 1 −1 0.121 21.90 7 1 −1 −1 1 0.073 29.25 8 1 −1 −1 −1 0.089 35.60 9 −1 1 1 1 0.159 28.71 10 −1 1 1 −1 0.109 19.75 11 −1 1 −1 1 0.094 37.53 12 −1 1 −1 −1 0.067 26.89 13 −1 −1 1 1 0.162 29.24 14 −1 −1 1 −1 0.054 9.90 15 −1 −1 −1 1 0.100 39.90 16 −1 −1 −1 −1 0.093 37.14 17 −2 0 0 0 0.162 40.21 18 2 0 0 0 0.221 54.75 19 0 −2 0 0 0.206 51.05 20 0 2 0 0 0.180 44.65 21 0 0 −2 0 0.064 64.26 22 0 0 2 0 0.206 29.17 23 0 0 0 −2 0.051 12.86 24 0 0 0 2 0.061 15.33 25 0 0 0 0 0.199 49.33 26 0 0 0 0 0.230 56.97 27 0 0 0 0 0.204 50.56 28 0 0 0 0 0.211 52.28 29 0 0 0 0 0.208 51.54 30 0 0 0 0 0.236 58.44 31 0 0 0 0 0.194 51.00 A full second-order polynomial model obtained by a multiple regression technique using the SAS package (SAS Institute, Cary, NC, USA) was adopted to describe the response surface. For three factors, the model obtained is expressed as follows: $$Y = b_0 + b_1 X_1 + b_2 X_2 + b_3 X_3 + b_4 X_4 + b_{12} X_1 X_2 + b_{13} X_1 X_3 + b_{14} X_1 X_4 + b_{23} X_2 X_3 + b_{34} X_3 X_4 + b_{11} X_1^2 + b_{22} X_2^2 + b_{33} X_3^2 + b_{44} X_4^2,$$ where Y is the measured response (elastase activity in this study), b 0 is the intercept term, b 1 , b 2 , b 3 and b 4 are linear coefficients, b 12 , b 13 , b 14 , b 23 and b 34 are the interactive coefficient, b 11 , b 22 , b 33 and b 44 are quadratic coefficients, and X 1 , X 2 , X 3 and X 4 are coded independent variables. RESULTS AND DISCUSSION 1 Optimization of elastolysis conditions with Bacillus licheniformis elastase 2 The degradation conditions are important for proteolysis process. In this part, the effects of elastolytic temperature, elastase activity, elastin content and elastolysis time on elastolysis rate were investigated and optimized by using response surface methodology. The experimental design and its results are shown in Table 1 1 . Based on the analysis results shown in Table 2 2 and Table 3 3 , it was apparent that the second model is very significant for this response ( α =0.00001). And the model is also very stable and appropriate ( α =0.0180). The regression results (Table 2 2 and Table 3 3 ) indicated that the most important factors for elastolysis were temperature ( X 1 ), substrate ( X 3 ) and elastolytic time ( X 4 ); elastase concentration ( X 2 ) is not the most insignificant for elastolysis. This second model was determined mainly by the quadratic term of four factors. Based on the above results, the enzyme concentration has little impact on elastin degradation; hydrolyzing time, temperature and elastin content were the most effective factors for elastolysis process. Source Sum of square Degree of freedom Mean square F -value Significant level X 1 0.0072 1 0.0072 12.27766 0.00294 X 2 0.0003 1 0.0003 0.47661 0.49986 X 3 0.0553 1 0.0553 94.17236 0.00000 X 4 0.0050 1 0.0050 8.58966 0.00979 X 1 2 0.0068 1 0.0068 11.64804 0.00356 X 2 2 0.0063 1 0.0063 10.79094 0.00466 X 3 2 0.0398 1 0.0398 67.78160 0.00000 X 4 2 0.1317 1 0.1317 224.17510 0.00000 X 1 X 2 0.0006 1 0.0006 1.07530 0.31517 X 1 X 3 0.0060 1 0.0060 10.28240 0.00550 X 1 X 4 0.0048 1 0.0048 8.15471 0.01145 X 2 X 3 0.0038 1 0.0038 6.43422 0.02199 X 2 X 4 0.0012 1 0.0012 2.01596 0.17484 X 3 X 4 0.0057 1 0.0057 9.67768 0.00672 Sources Sum of square DF MS F -value Significant level Regression 0.1051 14 0.0075 12.80000 0.00001 Residual 0.0094 16 0.0006 Lack of fit 0.0079 10 0.0008 3.24900 0.01750 Error 0.0015 6 0.0002 Total 0.1145 30 Fig. 1 1 indicated the effect of four factors on elastolysis rate. The results of response surface analysis showed that at the point of maximal elastolysis rate, the elastolysis conditions were as follows: elastolysis temperature 50 °C, elastase activity 10 000 U/ml, elastin content 80 mg, elastolytic time 4 h. A verifying experiment conducted at the combination of optimal elastolytic conditions showed that the selected model is very suitable for explaining the actual elastolysis process. Effect of different Bacillus licheniformis elastase activity on elastolysis process 2 The above results showing that elastase concentration had little effect on elastolysis mean that elastase did not play certain role in elastin degradation. Because of the dissolubility of elastin in elastolysis process, the elastolysis of elastin is a very complex system, especially for elastase active site and elastin reaction site. This reaction system is more complex than that of soluble substrates and enzymes. Consequently, it is not easy to explain the reaction mechanism by using classical kinetics. For these reasons, this part mainly discusses the effect of enzyme concentration on elastolysis process. Fig. 2 2 shows the experimental results. It was found that the elastase concentration had significant impact on elastolysis rate. However, elastase activity had no effect on elastolysis mode. With increasing reaction time, the elastolysis rates were not the same among different enzymes concentrations. However, elastase concentration had no significant impact on elastin degradation mechanism. According to the result of Fig. 2 2 , it was difficult to describe and explain elastolysis process with using the classical kinetics model such as Monod equation. Under the low elastase concentrations, the elastolysis kinetics demonstrated the first-order type, or else the opposite. Under high elastase concentrations, the change point all appeared at the reaction time 180 min of the elastolysis curves. During the reaction time from 180 to 240 min, the reaction rate decreased slowly, and after that the degradation rate increased rapidly. Impact of different substrate content on elastolysis process 2 The effect of different elastin content on elastolysis process was investigated in this part. The experimental result of Fig. 3 3 shows that adding elastin significantly affected elastolysis process. High or low elastin amount greatly inhibits elastin degradation. It was supposed that lower substrate amounts likely are not enough for enzyme absorption and degradation; and that too high substrate amount may possibly have inhibitory effect on elastolysis. This phenomenon had been experimented on and verified in the research on pig pancreatic elastase. The phenomenon’s causative reason was that PE (pancreatic elastase) has two substrate-absorbing active sites. With the first site having higher substrate-absorbing capacity that requires lower substrate content than the second site, so its reaction kinetics conforms to the general kinetics system; with the second site having lower substrate-absorbing capacity that requires more substrates in the reaction process, which certainly inhibit the first site’s decrease in the elastolysis rate (Heather et al., 1999 1999 ). The maximal elastolysis rate was observed at the reaction time 250 min for three treatments. But before 60 min of reaction time, the reaction kinetics of these three substrates showed first-order mode; after that the reaction kinetics did not conform to the classical kinetics model. With increasing substrate concentration, the reaction process is not well explained by the classical kinetics model, such as Michaelis-Menten kinetics. Effect of different pH on elastolysis process 2 At the optimal point of pH, all reactions showed the maximal reaction rate. When this point is higher or lower than the optimal point, the maximal reaction rate both will decrease. pH is an important factor that be helpful in providing much more proofs for explaining its reaction mechanism. Considering this point, the effect of pH on elastolysis rate was researched. Experimental results are shown in Fig. 4 4 . From Fig. 4 4 , it is apparent that pH has significant impact on elastolysis process. The elastolysis rate decreases with increasing pH; and at pH 9.0 was apparently lower than that at pH 7.4 and 8.0. The maximal reaction rate appeared at 240 min under pH 7.4 and pH 8.0. The elastolysis curves were the same before the 240 min reaction time under pH 7.4 and pH 8.0; after that odd change occurred. High pH may likely cause the enzyme to denature or partially stop reacting verified in some researches (Du et al., 1991 1991 ). For this elastase studied, the optimal reaction pH was found to be 7.4 in my previous study (Chen et al., 2003 2003 ), which accorded with the elastolysis change in this study. Study on the kinetics model of elastolysis 2 Elastin is a very dissolving protein that is not easily degraded by proteases and other methods. So the attack process of elastase on elastin and elastolysis could not be described and explained by the classical hydrolyzing kinetic law. So the degradation mechanism was not easy to be understood. With using the new technology and experimental methodology, the elastolysis mechanisms will be gradually understood, so elastolytic kinetics can possibly be investigated. A simplified model of elastin solubilization can be considered to proceed according to the equation: E+S→ES→→→E+P. Lonky and Wohl ( 1983 1983 ) postulated the existence of two types of binding sites for HLE (human leukocyte elastase) on elastin. The mechanism of binding to the first class of site was thought to be electrostatic, which was permanent but did not result in catalysis. The second class was thought to be located in the alanine-rich hydrophobic cross-linked areas of elastin and to lead to catalysis (Heather et al., 1999 1999 ). Based on the studies reported, the logistic kinetics model was generally adopted to describe the proteolysis process, especially for macromolecular substrates (Mecham et al., 1997 1997 ). Fig. 5 5 and Table 4 4 show the experimental results of the kinetics model analysis, respectively. Results clearly showed that the kinetics model selected was suitable for explaining the elastolysis process as the enzyme concentration decreased. The statistical data in Table 4 4 apparently showed that the logistic model was very significant and very appropriated ( P =0.001, R 2 > 0.93). The logistic model is not good for explaining the actual elastolysis process as increasing the elastase concentration, which still is under investigation. The experimental results were also rechecked in Fig. 5 5 . From the simulation figures of real value and predicted value, it was found that the logistic model could better describe the elastolysis process when enzyme concentration is low. Elastolysis is a very complex process that is not yet fully understood. Consequently, research on the kinetics model was obviously not easy. However, adopting the simple kinetics model is likely appropriate for explaining its hydrolyzing process and its elastolysis mechanism to some degrees. Elastase con. Logistic equation Significant level ( α ) R 2 1 X 2 =66.9326/(1+exp(2.0054−0.050122 X 1 )) 0.00018 0.9428 2 X 2 =49.6763/(1+exp(2.3651−0.026594 X 1 )) 0.00004 0.9653 3 X 2 =41.8260/(1+exp(2.4346−0.017815 X 1 )) 0.00018 0.9435 4 X 2 =38.7245/(1+exp(2.7929−0.026562 X 1 )) 0.00002 0.9719 5 X 2 =16.7551/(1+exp(3.0898−0.020048 X 1 )) 0.00001 0.9778 6 X 2 =14.3158/(1+exp(6.8225−0.031428 X 1 )) 0.00000 0.9942 CONCLUSION 1 Elastin is the extracellular matrix protein that imparts elastic recoil to tissues. Its cross-linked nature and extreme hydrophobicity make it one of the most stable proteins in the body. This paper first discussed the optimization of elastolysis conditions and the elastolysis kinetics using Bacillus licheniformis elastase. Experimental results indicated that the optimized elastolysis conditions were as follows: temperature 50 °C, elastase activity 1×10 4 U/ml, elastin 80 mg, elastolysis time 4 h. Hydrolyzing temperature and substrate amounts were found to be the most important factors affecting elastolysis. Different elastase concentrations had significant impact on the elastolysis process. At higher elastase concentration, the elastolysis process cannot suitably be explained by logistics model. The high and low substrate addition both inhibits the elastolysis process. However, under three substrates the maximal elastolysis rate occurred at 250 min. It implied that elastolysis might have a special mechanism that determines its elastolysis process. Based on the above studies, it is supposed that the binding time of elastase and elastin is not affected by the mechanism, but the binding rates of elastin and elastase were changed in this case. Baici ( 1992 1992 )’s studies showed that following initial complex formation there is a local structural change in elastin that alters the enzyme-substrate interaction and allows catalysis to proceed. It is supposed that elastin structural change is very important for elastolysis to proceed. In this work the elastolytic kinetics model was also investigated. Heather et al.( 1999 1999 ) attempted to apply elements of classical Michaelis-Menten kinetics to the study of elastolysis. Some quantitatively useful measures were obtained, but their results had no clear-cut physical meanings because empirical fits of their model to experimental results were acceptable only for selected reaction conditions (Heather et al., 1999 1999 ). Consequently, the logistic model was used to describe and explain the elastolysis process. The experimental results showed that this model explained well the real process of elastin degradation, especially under low enzyme concentration. However, some inroads have to be made into the theoretical understanding of enzyme kinetics in heterogeneous reaction systems when one or more reactants are spatially constrained. So much more studies have to be conducted in this field. References J Adler-Nissen . Enzymic Hydrolysis of Food Protein , 1986 . : London Elsevier Applied Science Publishers , . Enzymic Hydrolysis of Food Protein A Baici . C Grassi , J Travis , L Casali , 等 . Current Topics in Rehabilitation . Biochemistry of Pulmonary Emphysema , 1992 . : Berlin Spring-Verlag , 81 - 91 . . Current Topics in Rehabilitation MM Bradford . A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding . Anal Biochem , 1976 . 72 ( 1-2 ): 248 942051 DOI: 10.1016/0003-2697(76)90527-3 http://doi.org/10.1016/0003-2697(76)90527-3 . A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding QH Chen , GQ He , AMA Molhtar . Optimization of medium composition for the production of elastase by Bacillus sp. EL31410 with response surface methodology . Enzyme Microb Technol , 2002 . 30 ( 5 ): 667 - 672 . DOI: 10.1016/S0141-0229(02)00028-5 http://doi.org/10.1016/S0141-0229(02)00028-5 . Optimization of medium composition for the production of elastase by Bacillus sp. EL31410 with response surface methodology QH Chen , GQ He , YL Wu . Screening of elastase-producing strains and primary studies on elastase fermentation conditions . Journal of Zhejiang University (Agriculture and Life Science) , 2003 . 59 ( 1 ): 59 - 62 . (in Chinese) . Screening of elastase-producing strains and primary studies on elastase fermentation conditions QH Chen , GQ He , P Schwarz . A kinetic model for elastase production by Bacillus sp. EL31410 . Journal of Agricultural and Food Chemistry , 2004 . 52 ( 11 ): 3356 - 3359 . 15161197 DOI: 10.1021/jf0303161 http://doi.org/10.1021/jf0303161 . A kinetic model for elastase production by Bacillus sp. EL31410 JZ Du , BG Shu , XC Wei . Enzyme Structure and Reaction Mechanism , 1991 . : Beijing Peking University Press , 104 - 180 . (in Chinese) . Enzyme Structure and Reaction Mechanism DA Hall , JW Czerkawaki . The reaction between elastase and elastic tissue . Biochem J , 1961 . 80 128 - 136 . 13710758 . The reaction between elastase and elastic tissue MM Heather , GW Howard , AO Caroline , AS Robert , JC Edward . Interaction between leukocyte elastase and elastin: quantitative and catalytic analyses . Biochimica et Biophysica Acta , 1999 . 1430 ( 2 ): 179 - 190 . 10082946 . Interaction between leukocyte elastase and elastin: quantitative and catalytic analyses R Lahl . Enzymatic production of protein hydrolysates for food uses . Food Technol , 1994 . 138 68 - 71 . . Enzymatic production of protein hydrolysates for food uses SA Lonky , H Wohl . Regulation of elastolysis of insoluble elastin by human leukocyte elastase: stimulation by lysine-rich ligands, anionic detergents, and ionic strength . Biochemistry , 1983 . 22 ( 15 ): 3714 - 3720 . 6412747 DOI: 10.1021/bi00284a027 http://doi.org/10.1021/bi00284a027 . Regulation of elastolysis of insoluble elastin by human leukocyte elastase: stimulation by lysine-rich ligands, anionic detergents, and ionic strength AYM Ma , B Ooraikul . Optimization of enzymatic hydrolysis of canola meal with response surface methodology . J Food Proc Preserv , 1986 . 10 99 - 113 . . Optimization of enzymatic hydrolysis of canola meal with response surface methodology RP Mecham , TJ Broekelmann , CJ Fliszar , SD Shapiro , HG Welgus , RM Senior . Elastin degradation by matrix metalloproteinases . J Biol Chem , 1997 . 272 ( 29 ): 18071 - 18076 . 9218437 DOI: 10.1074/jbc.272.29.18071 http://doi.org/10.1074/jbc.272.29.18071 . Elastin degradation by matrix metalloproteinases H Ozaki , I Shiio . Purification and properties of elastolytic enzyme from Flavobacterium immotum . J Biochem , 1975 . 77 171 - 180 . 236995 . Purification and properties of elastolytic enzyme from Flavobacterium immotum SM Partridge , HF Davis . The chemistry of connective tissues . Biochemical J , 1948 . 43 387 . The chemistry of connective tissues LA Sachar . Photometry method for estimation of elastase activity . Proc Soc Expeti Biol Med , 1955 . 90 5323 - 5325 . . Photometry method for estimation of elastase activity SR Sharon , LR Jennifer , NB Bryan . Identification of an elastolytic protease in stationary phase culture filtrates of M . tuberculosis . FEMS Microbiology , 1997 . 151 ( 1 ): 59 - 64 . DOI: 10.1111/j.1574-6968.1997.tb10394.x http://doi.org/10.1111/j.1574-6968.1997.tb10394.x . Identification of an elastolytic protease in stationary phase culture filtrates of M. tuberculosis YCH Tsai , RY Jung , SF Lin . Production and further characterization of an alkaline elastase production by alkalophilic Bacillus strain YaB . Appl Environm Microbial , 1988 . 54 ( 1 ): 3156 - 3161 . . Production and further characterization of an alkaline elastase production by alkalophilic Bacillus strain YaB H Tsuzuki , T Oka . Pseudomonas aeruginosa elastase: isolation, crystallization and preliminary characterization . J Biol Chem , 1965 . 8 3295 - 3303 . . Pseudomonas aeruginosa elastase: isolation, crystallization and preliminary characterization 10.1631/jzus.2006.B0482 Project (No. Y304203) supported by the Natural Science Foundation of Zhejiang Province, China 2005-08-03 2005-11-13 2006-05-12 2022-01-05 Copyright © 2006, Journal of Zhejiang University Science 2006 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 07