| Keratinase is a special enzyme that can hydrolyze keratin substrate, showing great potential in feed additives, laundry, leather, and pharmaceuticals industry. However, low production and poor property of keratinase block its commercial development and industrial application. In order to realize its industrial production and application, an efficiently feather-degrading strain Stenotrophomonas maltophilia BBE11-1 was isolated and medium optimization in shake flask and fermenter level was conducted to improve keratinase production. Protein purification, enzyme characterization, and gene cloning were conducted for keratinase. The heterologous expression system was constructed and rational protein design was used to improve kerainase characterization. The main research results were as follow.(1) Keratinase production strain screening and fermentation optimizationThe strain S. maltophilia BBE11-1 using feather as sole nitrogen source and showing keratinase activity in fermentation broth was isolated from Mashan poultry farms in Wuxi. The single factor experiments, Plackett–Burma experiment, and response surface method were conducted to determine fermentation medium in shake flask: 1.5 g×L-1 Asp, 1.45 g×L-1 soy peptone, 4.25 g×L-1 glucose, 10 g×L-1 wool, 1 g×L-1 K2HPO4, 1 g×L-1 KH2PO4, 1 g×L-1 Na Cl, 100 μl×L-1 Tween-20, original p H 9.0, 23 °C, 200 rpm shaking for 48 h. By three strategies(temperature shift, dissolved oxygen control, and glucose feeding) in 3 L fermenters, keratinase production was improved from 145.2 U×ml-1 to 1282.7 U×ml-1 and shortened fermentation time from 48 hours to 18 hours. In 30 L fermenter, keratinase production reached 1728 U×ml-1 within 18 hours, 32-fold improvement of productivity. Besides, the final fermentation broth with plenty of essential amino acids had great potential in organic fertilizer and feed addtivies application.(2) Keratinase purification and characterization studyThrough hydrophobic interaction column, ion exchange column, and sephadex column, three proteins related to keratin degradation were isolated from S. maltophilia. Two proteins Ker SMD and Ker SMF showed keratinase activity. The N-terminal amino acid sequencing showed that N-terminus of Ker SMD and Ker SMF were LAPNDPYYQQ and LTPNDTRFSE, respectively. Based on codon bias of Stenotrophomonas and N-terminus sequences, the degenerate primers were designed and used to clone keratinase genes from S. maltophilia BBE11-1 genome by thermal asymmetric interlaced(TAIL) PCR. The keratinase genes ker SMD(1905 bp) and ker SMF(1743 bp) were obtained and linked to p ET plasmids for further heterologous expression in Escherichia coli BL21(DE3). It found that culture supernate had keratinase activity, and Ker SMD showed 3-fold keratinase activity than Ker SMF as well as 9-fold half-life(50?°C) compared to Ker SMF.(3) Functional analysis of the C-terminal domain of Ker SMDThe C-terminal domain of keratinase might be related to substrate specificity and has other special functions. It also found that Ker SMD C-terminus could be auto-truncated. In order to learn the effects of C-terminus structure, one β-fold truncation of C-terminus was conducted to obtain various variants V456, V455, V435, V415, V395, V380, V370, and V355. The whole truncation of C-terminus resulted in low collagen activity of V355, which can maintain 40% keratinase activity at high alkalinity(p H 12) and high concentration(4%, w/v) sodium dodecyl sulfonate(SDS) as well as 60% keratinase activity at high salinity(15%, w/v), indicating its great potential in laundry and leather treatment. V380, V370, and V355 increased 1.7-fold activity at 60 °C; V456 could maintain 70% residual activity after 90 min incubation at 60 °C, 20% improvement compared to wild type. The structure models showed that V456 has changed distance and hydrogen bonds of catalytic center sites, improving interaction force and thermostability and thermophilic characterization; V355 used the weak negative charged loop to resist negative detergent SDS above catalytic triangle. All results indicated that C-terminal domain can adjust the catalytic domain structure to affect substrate specificity and resistance.(4) Domain swapping to improve catalytic efficiency and thermostability of Ker SMDAmino acid sequences alignment and protein model analysis showed that Ker SMD and Ker SMF have special N-propeptide and C-terminal domain. Based on catalytic domain of Ker SMD, swapping N/C domains obtained several variants DDF, FDD, FDF, DD and FD. Compared to wild type, DDF showed 500 units increase of keratinase activity(3909 ± 45 U×mg-1) and 54.5% increase of catalytic efficiency kcat/Km. FDF showed 244.6 ±?2 min half-life(60 °C), 5.9-fold of wild type, and 12% increase of keratinase activity at alkaline solution(p H 8.0-12.0), indicating its application in leather and laundry. Structure models showed that keratinase with new domains changed the structure of substrate binding pocket. We proposed that C-terminal domain can adjust pocket size to affect keratinase catalytic properties while Npropeptide assist folding and maturation to realize high catalytic efficiency and heat stability.(5) Molecular modification of S1 pocket to improve Ker SMD catalytic activityAccording to the above research, it found that catalytic activity of Ker SMD was related to S1 pocket comformational change. Crystal structures and amino acid sequences analysis indicated there are mainly four different sites(Ser180, Glu208, Tyr215, and Arg216). Sitedirected mutagenesis with hydrophobic and short-chain amino acids was conducted. It found that Tyr215 site is the most important site for keratinase activity, and Y215 G showed the highest kcat/Km of 365 s-1·m M-1. Saturation mutagenesis on Tyr215 sites indicated that suitable side chain and hydrophobicity in S1 pocket can improve catalytic activity. In order to further optimize S1 pocket structure, combination mutagenesis with sites Ser180, Glu208, and Tyr215 were conducted. S180G/Y215 S showed the highest keratinase activity(4800 ± 138 U×mg-1), and S180G/Y215 A obtained the highest K:C ratio(keratinolytic activity : caseinolytic activity) of 4.5. Besides, Y215 S, Y215 G, and S180G/Y215 S were all thermophilic, and Y215 S showed 7000 U×mg-1 at 70 °C, 2-fold improvement compared to wild type. Protien model structure confirmed that suitable volume size and hydrohopic side chain can work together to contribute the keratine degrading ability. The overexpression and fermentation of keratinase variant S180G/Y215 S were conducted in E. coli. By feeding carbton and inducing at later growth phase, keratinase activity was increased from 400 U×ml-1 of flask shaker to 4500 U×ml-1 of 3-L fermenter, more than 11.25-fold when compared to the original production. |
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