| Halohydrin dehalogenases(HHDHs), which occur in the biodegradation pathways of halogenated compounds, can catalyze the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins(ring-closure reaction) to yield the corresponding epoxides and its reverse reaction(ring-opening reaction) to produce β-substituted alcohols. Those enzymes are attractive industrial biocatalysts for synthesizing a series of important chiral building blocks for fine chemicals and pharmaceuticals.Halohydrin dehalogenase from Agrobacterium radiobacter AD1(HheC) has been cloned, sequenced and studied extensively. The enzyme displays a broad substrate range with regards of both ring-closure and ring-opening reactions. Sequence alignment of six well-known HHDHs reveals that HheC has 10 amino acids longer C-terminal region than other HHDHs, which might contribute to the large functional difference among the enzymes of these three HHDH groups. Previous studies suggested that such a C-terminal region have an important effect on the activity of Hhe C, however, no detailed information related to the regulation mechanism of HheC’s C-terminal region is available. In this study, two efficient multi-sites evolution strategies were adopted to explore the specific functions and regulation mechanism of Hhe C’s C-terminal region. This work could greatly facilitate both theoretical study and industrial application of HheC. The main content of this thesis is as follows:(1) In order to determine the specific functions of C-terminal region on the catalytic activity and thermostability of HheC, the last ten residues(M245~E254) were subjected to truncated mutagenesis, resulting in ten truncated variants(Δ1~Δ10). The results showed that the activity and/or thermostability of those truncated variants decreased dramatically compared to wild-type HheC. Furthermore, the catalytic activity of the truncated variants Δ7~Δ10 could not be accurately determined due to the fact that their activity are almost completely lost. The truncated mutagenesis results indicated that the C-terminal region play an important role in both the activity and thermostability of HheC.(2) In order to gain beneficial replacements on C-terminal region for improving the activity and/or thermostability of Hhe C, these residues were subjected to the single-site saturation mutagenesis, resulting in 20 single-site variants. The results showed: residues W249 and M252~E254 were crucial for regulating enzyme activity and thermostability, respectively. In addition, residues P253 and E254 were confirmed to regulate the enzyme thermostability independently without affecting the catalytic activity of Hhe C. Moreover, homology structural models of single-site variants were constructed and analyzed. The results showed that hydrogen bond network of Hhe C’s C-termnial region might contribute to the overall conformational stabilization and play crucial roles for maintaining and regulating the enzyme thermostability of Hhe C.(3) In this study, efficient multi-site evolution strategies were designed and applied for further exploring and regulating the catalytic functions of HheC’s C-terminal region. Initially, a DC-Analyzer-facilitated combinatorial strategy for rapidly evolving functional enzymes with multiple discontinuous mutagenesis sites was designed based on an iterative saturation mutagenesis and DC-Analyzer software. The DC-Analyzer is a useful tool for designing a set of degenerate codons that cover all the user-defined amino acids sets without any inherent amino acid biases and redundancy. DC-Analyzer-facilitated combinatorial strategy had been successfully applied to improve the enzyme activity of HheC. After screening about 900 clones, the best variant which displayed a 9.3-fold higher in kcat value than that of wild-type enzyme was obtained. However, when the mutated sites are located contiguously or in a proximity mode, large numbers of degenerate primers are needed by using DC-Analyzer software. For this, a MDC-Analyzer-facilitated combinatorial strategy is developed for rapidly evolving functional enzymes when the multiple continuous and/or adjacent mutagenesis sites were targeted. The MDC-Analyzer is an online software tool for rapidly and automatically designing a codon set of optimal degenerate primers according to user-defined mutagenesis scheme for constructing high-quality multi-sites mutagenesis libraries. MDC-Analyzer-facilitated combinatorial strategy had been successfully applied to improve the enzyme activity and thermostability of HheC. The best variants DG9 and ZB8 displayed a 5.9-fold higher in kcat value and a 2.3-fold higher in τ1/2(55 °C) value than that of its parent templates, which were obtained after screening about 1,151 and 384 clones respectively. The results clearly indicated that those two combinatorial strategies are simple and efficient for obtaining functional enzymes and could serve as a general tool for evolving novel biocatalysts.(4) To obtain desired enzyme with improvement in both activity and thermostability, the beneficial replacements at positions 245, 249, 252, 253 and 254 of HheC’s C-terminal region were rapidly combined by using efficient multi-site evolution strategies. Initially, positions 245, 249 and 252 were combined to explore their composite effect by using the DC-Analyzer-facilitated combinatorial strategy. After screening 135 clones, one positive variant DL10(W249P/M252L) was obtained, displaying about 5.0-fold higher kcat value and 3.1-fold higher τ1/2, 55 °C value than that of wild-type Hhe C. The results indicated that W249 P and M252 L affected the activity and thermostability of HheC in an additive fashion. In order to further enhance the thermostability of positive variant DL10, positions 253 and 254 were introduced in DL10 variant by using the MDC-Analyzer-facilitated combinatorial strategy. After screening 36 clones, three positive clones were selected for their enhanced thermostability compared to that of its parent template DL10 variant, displaying about 4.5- to 5.7-fold higher τ1/2(55 °C) values without compromising their enzyme activity than that of variant DL10. The most thermally stable variant PX14(W249P/M252L/P253D) exhibited a 17.8-fold higher in half-life and a 4.0-fold higher in kcat value than that of the wild-type Hhe C. These results revealed that the enzyme surface residues P253 and E254 could regulate the thermostability without compromising the activity of Hhe C. Modeling structures analysis showed those three positive variants gained two more hydrogen bonds formed between residues at positions 253 and 254 as compared to the template DL10. The new formed hydrogen bonds might contribute to the overall conformational stabilization of these positive variants, leading to the improvement of their thermostability. These efficient variants display great potential for their industrial application as biocatalysts.In summary, this study systematically explored the functions and regulation mechanism of C-terminal region of halohydrin dehalogenase from Agrobacterium radiobacter AD1 on the activity and thermostability by using the efficient multi-site combinatorial strategies. Multiple positive variants with improvement of both activity and thermostability were obtained. This study provided valuable information on HheC’s C-terminal region related to the activity and thermostability, which could greatly facilitate both theoretical study and industrial application of HheC. |
