| β-glucanase (lichenase, E.C.3.2.1.73) is a major feed enzyme that hydrolyses only beta-1,4-linkages adjacent to beta-1,3-linkages in β-glucans, so it can alleviate the anti-nutritional effect. Most of β-glucanases are from microorganisms. Among them, Clostridium thermocellum lichenase (LicB) had highest thermostability with 80℃ of themostable temperature. So the research was conducted to study its mechanism for the high thermostability, the main research contents and results were as follows:(1) Three constructs derived from lichenase gene(licB) of Clostridium thermocellum obtained by PCR were expressed in Escherichia coli, and their products were characterized and compared. These products corresponded to the mature (licMB) and two truncated (LicMBΔ238-305 and LicMBΔ217-305) forms of lichenases. licMB was mature form of lichenase, composed of 305 amino acids without signal peptide. LicMBA238-305 lacked 68 amino acids at carboxyl-termini which corresponded to the dockerin domain, and LicMBΔ217-305 lacked Thr-Pro-box (linker of 21 amino acids) and dockerin domain. The specific activity of LicMBA238-305 and LicMBA217-305 was higher than that of LicMB by 90.58% and 36.65%. The thermostability of LicMBA238-305 was higher than that of licMB and LicMBΔ217-305. The results demonstrated that the dockerin domain of licB had a negative role on thermostability and enzymatic activity of lichenase, while the Thr-Pro-box had a positive role on thermostability and enzymatic activity. The effect of two regions offset, so LicMBΔ217-305 had similar the enzymatic activity and thermostability to licMB. It can be concluded that the LicMBΔ238-305 with dockerin domain truncated and Thr-Pro-box retained had more specific activity and thermostability than parent enzyme (LicMB), Thr-Pro-box (linker) and dockerin domain were not necessary for enzymatic activity, and catalytic domain conferred the most of activity and thermostability to C. thermocellum lichenase.(2) Seven N-terminal truncated enzymes and three C-terminal truncated enzymes of LICMB-CD(catalytic domain of mature lichenase from C. thermocellum ) wereconstructed. Seven N-terminal truncated enzymes were designated LicMB-CDAN3, LicMB-CDAN5,LicMB-CDAN7,LicMB-CDAN9,LicMB-CDANl 1, LicMB-CDAN13 and LicMB-CDAN25 which were truncated 3,5,7,9,11,13 and 25 amino acids at N-terminus of LICMB-CD, respectively, While three C-terminal truncated enzymes were designated I/cMB-CDAC3, IicMB-CDAC6 and IicMB-CDAC9 which were truncated 3,6 and 9 amino acids at C-terminus of LICMB-CD, respectively. The results showed that compared with the parent enzyme (LICMB-CD), thermostability of LicMB-CDAN3, LicMB-CDAN5, LicMB-CDAN7 and LicMB-CDAN9 decreased to some extent, but not significantly. The residual activity of them were above 70% after incubation at 70℃ for 10 min, and the residual activity of LICMB-CD, LicMB-CDAN3,LicMB-CDAN5, LicMB-CDAN7 and LicMB-CDAN9 were 79.09%s 57.72%,59.86%,37.11% and 11.90% after at 75℃ for 10 min incubation. LicMB-CDAN13 exhibited less thermostability retaining 64.51% of its activity after during incubation at 60℃ for 10 min. LicMB-CDANll and LicMB-CDAN25 had the least thermostability which were stable below 50℃. The residual activity of LicMB-CDAC3, LicMB-CDAC6 and LicMB-CDAC9 were 8.38%, 24.17% and 41.33% during 5 min incubation at 75℃, respectively. The results implied that whether the N-terminal or C-terminal sequence of LicMB-CD partially contributed to its thermostability with additive effect. Among them, the 10-216th amino acids of LicMB-CD were necessary for its keeping high thermostability.(3) Ten mutants of LICMB-CD mutated at 10th or/11th position were constructed. They were designated LicMB-CDAlOE, LicMB-CDVllP, LicMB-CDAlOEVllP, LicMB-CDAlOG, LicMB-CDVllA, LicMB-CDAlOGVllA, LicMB-CDAlOV, LicMB-CDVllW,LicMB-CDA10VVllW. The results showed ten mutants all had less thermostability than the parent enzyme (LICMB-CD). The residual activity of LzcMB-CDAlOE, LicMB-CDAlOG and ZzcMB-CDAlOV were 23.48%,50.60% and 84.61% after incubation at 75 ℃ for lOmin, The residual activity of Z/cMB-CDVllP, IicMB-CDVllA and 6I/cMB-CDVllW were 12.85%,93.67% and 92.94%; The residual activity of Z/cMB-CDAlOEVl IP, Z/cMB-CDAlOEVl IT,L/cMB-CDAlOGVllA and I/cMB-CDAlOVVllW were 83.34%, 95.00%, 60.53% and 0%, respectively. The loss of thermostability was resulted from the structure change (such as more cavity, bumps between amino acids and less hydrophobility), which was caused by the change of properties of mutated and mutate amino acids. The results implied that the properties of the amino acids at 10th and 11th of LicMB-CD could affect its thermostability. The Ala at 10th and Val at 11th are more suitable for higher thermostability of LicMB-CD.(4) Eight hybrid enzymes derived from LicMB-CD and Bacillus P-glucanase were constructed. They were designated H-N7C3-B, H-N7C8-B, H-N10C3-B, H-N10C8-B, H-N16C3-B, H-N16C8-B, H-N104C3-B and H-N104C8-B (the number after N means the number of N-terminal amino acids from LicMB-CD, the number after C means the number of C-terminal amino acids from LicMB-CD) The results showed that the thermostability of all hybrid enzymes were higher than the parent enzyme (Bacillus p-glucanase) except H-N7C3-B. H-N7C8-B, H-N10C3-B, H-N10C8-B, H-N16C3-B and H-N16C8-B showed the retaining 38.30%, 24.00%, 38.00%, 44.33% and 71.67% of their activities during 5 min incubation at 60 °C, while H-N104-B, H-N104C3-B and H-N104C8-B showed 38.42%, 41.91% and 74.17%%. The results implied that 1-7* amino acids at N-terminal and 214-215th at C-terminal sequence of LicMB-CD had no effect on the thermostability of Bacillus p-glucanase. The amino acids of 16D, 210E and 211Y partially contributed to its thermostability. The effect of 1-16l amino acids at N-terminal and sequence of LicMB-CD on thermostability of P-glucanase was almost equal to that of 1-104th amino acids. The thermostability of H-N104C3-B was higher than that of Bacillus p-glucanase, but much less than that of LicMB-CD. It meant that the thermostability of LicMB-CD was mainly determined by the 97 amino acids at its C-terminal sequence.(5) Five mutants derived from H-N104C8-B were constructed. They were designated HN104C8-m 197-202(S197RN199DA201RN202T), HN104C8-m 189-193-(T189KS193E), HN104C8-ml65-172(V165KT169ET172R), HN104C8-T144E and HN104C8-Y124WG128E. Among them, the thermostability of HN104C8-ml 89-193-(T189KS193E) and HN104C8-ml65-172(V165KT169ET172R) were much higher than that of the parent enzyme (H-N104C8-B). The thermostable temperature of them was 70-75 °C which was 10-15°C higher than that of the parent enzyme. Analysis of the state of the mutated and mutate amino acids showed that the amino acids which were on the surface of enzyme protein before and after mutation had more effect on thermostability. 193E and 169E were key amino acids determining thermostability.Results above implicated: (1) the catalytic domain of LicMB conferred the most of activity and thermostability to C. thermocellum lichenase. (2) whether the N-terminal or C-terminal sequence of LicMB-CD partially contributed to its thermostability with additive effect. The 10-216th amino acids at C-terminal of LicMB-CD were necessary for retaining high thermostability. (3) the properties of the amino acids at 10th and 11th of LicMB-CD could affect its thermostability. Ala at 10th and Val at 11th are more suitable for higher thermostability of LicMB-CD. (4) the thermostability of LicMB-CD were mainly determined by the 97 amino acids at its C-terminal sequence.(5) 193E and 169E were key amino acids determining thermostability of LicMB-CD.
|
PDF Full Text Request