Purification and characterization of four keratinases ... - ResearchGate

More documents

Recommendations

Info

F. Xie et al. / Bioresource Technology 101 (2010) 344–350 347 Concentrated culture Sephacryl S-200 Peak I DEAE FF Peak II DEAE FF Peak III Peak IV Octyl FF Octyl FF Octyl FF KI KII KIII KIV Fig. 2. Purification strategy for extracting keratinases from Streptomyces sp. strain 16. kDa 110 M 1 2 3 4 Table 1 Activity determination in different components treated by ultrafiltration. 1 2 3 66 Protein (lg/ml) 244 256 1.5 Keratinolytic activity (U/ml) 37 42 0 Specific activity (U/mg protein) 151.6 164.1 0 45 35 1. The retentate by 100 kDa ultrafiltration. 2. The retentate by 10 kDa ultrafiltration. 3. Filtrate by 10 kDa ultrafiltration. 25 18 14 Fig. 3. SDS–PAGE analysis of the purified keratinases (KI, KII, KIII, and KIV) produced by Streptomyces sp. strain 16. The molecular marker, KI, KII, KIII, and KIV are located in lane M, 1, 2, 3, and 4, respectively. Superose 12 10/300 GL column. The results provided a molecular weight estimate of 203.2 kDa for KI, 100.8 kDa for KII, 31.8 kDa for KIII, and 19.2 kDa for KIV. This indicated that KI is a homo-octamer, KII a homo-dimer, and KIII a monomer and KIV a monomer. To further verify that KI is an active homo-octomer, ultrafiltration was conducted. Retentate with a molecular weight of 100 kDa, retentate with a molecular weight of 10 kDa, and filtrate with a molecular weight of 10 kDa or less were sequentially collected and analyzed (Table 1). Most of the enzyme activity was detected in retentate with a molecular weight of 100 kDa and a molecular weight of 10 kDa. However, no activity emerged in the filtrate with a molecular weight of 10 kDa. 3.3. Influence of temperature and pH on the keratinolytic activity of KI, KII, KIII, and KIV The four purified keratinases had high activities between pH 8.0 and 10.0, with maximal activity at pH 9.0. The four keratinases showed low activities at pH 6.0 and 12.0. The effect of pH 7.0 on each of the four keratinases was different, the keratinase activities for KI and KIV were more than 50% of their maximal activities, but KII and KIII activities were less than 30% of their maximal activities. At pH 11.0, KI showed low keratinase activity, while KII, KIII, and KIV showed more than 50% of their maximal activities. The four keratinases showed the same pH stability. More than 85% of the maximal activities were retained between pH 8.0 and 10.0. The optimal temperature for keratinases KI, KII, and KIII was about 50 °C, while the optimal temperature was about 60 °C for keratinase KIV. KI, KII, and KIII activity dropped fast at temperatures below 40 °C and above 60 °C. For KIV, the activity at 80 °C was approximately half of its maximal activity, which was at 60 °C. All four keratinases retained more than 70% of their maximal activities in the temperature range of 40–60 °C after 1 h incubation. KIV was more stable than the other three keratinases. 3.4. Effects of chemicals on keratinolytic activities of KI, KII, KIII and KIV Protease inhibitors, reducing agents, and metal ions were used to investigate their effects on the enzyme activities (Table 2). All four enzymes were significantly inhibited by the serine protease inhibitor PMSF at 1 mM. This indicates that all of the enzymes belong to the serine protease family. Moreover, the residual activities of the four enzymes were evidently not affected by cysteine protease inhibitor pCMB. However, metalloprotease inhibitor EDTA strongly inhibited KI, KII, and KIII, although it did not inhibit the activity of KIV. For all four keratinases, significant increases in keratinolytic activity were observed by adding reducing agents DTT, ascorbic acid, and sodium sulfite. Carbonyl group modifier EDAC had stimulative effects on the activities of KI, KIII, and KIV. The keratinolytic activities of KI, KIII, and KIV increased by 36%, 100%, and 32%, respectively. The divalent metal ion Ca 2+ showed a stimulative
348 F. Xie et al. / Bioresource Technology 101 (2010) 344–350 Table 2 Effects of the different reagents on the keratinolytic activity of the four keratinases. Compound Concentration (mM) Residual activities (%) effect on KI, an inhibitory effect on KIII, and no effect on KII and KIV. The metal ion Mg 2+ had an inhibitory effect on KII and KIII, and no effect on KI and KIV. 3.5. Substrate specificities of KI, KII, KIII, and KIV The ability of the four enzymes to hydrolyze proteinaceous substrates was examined with casein, NHFS, keratin azure, collagen, cock feathers, and human hair (Table 3). Results showed that the four enzymes exhibited high activities toward casein and NHFS, however, NHFS could not be solubilized by any one of the four keratinases. All four purified enzymes showed higher activities with soluble proteinaceous substrates such as casein than with insoluble proteinaceous substrates such as NHFS. The four keratinases showed low activities toward keratin azure and collagen. For all four keratinases, no activity was observed with human hair and cock feathers as substrates. 3.6. N-terminal sequence N-terminal amino acid sequencing of the purified keratinases resulted in unequivocal determination of the first 10 amino acid residues of the four keratinases (Table 4), with the exception of position 10 in KIII, where no assignment could be made. When compared to the National Center for Biotechnology Information (NCBI) protein database by BLAST search, the KI, KII, and KIII sequences did not show significant homology to known microbial peptidases. However, the N-terminal sequence of KIV was identical to that of the 17.6 kDa protease (Sinha et al., 1991) and SFase-2 (Kitadokoro et al., 1994) from S. fradiae and it showed 70% homology to SGPA from S. griseus (Johnson and Smillie, 1974). 4. Discussion KI KII KIII KIV Control 0 100 100 100 100 pCMB 1 92 86 96 95 EDAC 1 136 107 200 132 PMSF 1 33 0 0 0 EDTA 1 36 7 0 97 DTT 1 195 157 170 256 Ascorbic acid 10 145 107 370 155 Na 2 SO 3 1 177 160 130 180 CaCl 2 10 114 86 20 82 MgCl 2 10 86 28 60 87 In our previous studies, Streptomyces sp. strain 16 was found to completely degrade NHFS, and keratinases were produced when Table 3 Substrate specificities of the four keratinases. Substrate Residual activity * (%) KI KII KIII KIV Casein 100 100 100 100 NHFS 93 79.1 89 82 Keratin azure 3.7 0.8 1.0 6.5 Collagen 3.7 1.2 1.0 30 Cock feather 0 0 0 0 Human hair 0 0 0 0 * The proteolytic activity toward casein of each enzyme was used as 100%. Activity on other substrate was compared with it. strain 16 was cultured in a medium that contained NHFS. The addition of yeast extract to the medium was helpful to increase of biomass and enzyme production. A zymogram of culture filtrates showed that complete degradation of NHFS was due to the presence of multiple keratinases. In order to investigate the mechanism of degradation, we purified the keratinases from the culture broth. The results showed that four keratinases secreted by Streptomyces sp. strain 16 were responsible for the degradation of NHFS in the medium. In the purification procedure, we also tried to purify the fifth peptidase appeared on the zymogram gel. Unfortunately, we failed to isolate this enzyme, perhaps due to its low quantity. Most microbial keratinases are inducible enzymes, as reported by Cheng et al. (1995). Various keratinous materials such as chicken feather, feather meal, wool, and bovine hair have been used as inducers of keratinases (De Toni et al., 2002; Ignatova et al., 1999; Kumar et al., 2008; Nam et al., 2002). NHFS belongs to soft keratins from the human sole, which was first used as a keratinase inducer. Proteolytic enzymes have dominated the detergent enzyme market. Nonetheless, there is always a need for new enzymes with novel properties that can further widen the scope of enzyme-based detergents. Keratinases could help in the removal of keratinous soils that are often encountered in the laundry and on which most normal peptidases fail to act. In this study, the keratinases produced by Streptomyces sp. strain 16 can efficiently decompose NHFS, therefore, these keratinases may be candidate enzymes to be used as additives to the detergents. Similar to most keratinases reported in the literature, keratinase KIV is a monomer. However, KII is composed of two subunits in its native form and the molecular mass of each subunit is approximately 50 kDa. Interestingly, KI is composed of eight subunits in its native form with a total molecular weight of 203.2 kDa. This is the first report of a peptidase with eight subunits. A few keratinases of high molecular weight with multimeric structures and multiple domains have been reported. Nam et al. isolated a native-feather-degrading thermophilic anaerobic bacterium identified as Fervidobacteium islandicum from a geothermal hot stream (Nam et al., 2002). A homo-multimeric membrane-bound keratinolytic peptidase was purified from the cell extracts of the anaerobe. Its molecular weight was estimated to be greater than 200 kDa with a 97 kDa subunit (Nam et al., 2002). Fervidolysin is a keratinase (with a molecular weight of 73 kDa) as an immature form from Fervidobacterium pennivorans (Kluskens et al., 2002). The 1.7 Å resolution crystal structure of fervidolysin showed that the peptidase is composed of four domains: a catalytic domain (CD), two b-sandwich domains (SDs), and the propeptide (PD) domain (Kim et al., 2004). Keratinases of high molecular weight may consist of multi-domain structures or oligomeric proteases, which contribute to their substrate specificities (Kim et al., 2004). Accordingly, the oligomeric nature of KI and KII is also postulated to contribute to the substrate specificities. Several Streptomyces keratinases have been classified as serine proteases (Bockle and Muller, 1997; Bressollier et al., 1999). Kerotinolytic activities of studied enzymes were significantly inhibited by PMSF. Consequently, keratinases KI, KII, KIII, and KIV were classified as serine proteases. KI was slightly more stable at the same concentration of PMSF, perhaps due to its homo-multimeric form. Meanwhile, EDTA also showed similar effects on the keratinolytic activity of KI, KII, and KIII suggesting that they are metal ion related enzymes. The activity of KIV was not affected by EDTA. Although KI, KII, and KIII shared the same optimal temperature, and optimal pH of the four keratinases were identical, the effects of pH and temperature on the activities of the four keratinases actually varied. According to the difference in molecular mass, characteristics, and the distinct differences in N-terminal sequences, the four
Page 1 and 2: Bioresource Technology 101 (2010) 3
Page 3: 346 F. Xie et al. / Bioresource Tec
Page 7: 350 F. Xie et al. / Bioresource Tec

Purification and characterization of four keratinases ... - ResearchGate

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?