Efficient Expression, Affinity Purification And Stability Of Sarcosine Oxidase

As one of the flavoprotein oxidases, sarcosine oxidase(EC.1.5.3.1,SOX) needs FAD cofactor to catalyze the degradation of sarcosine under oxidation. SOX is one key enzyme in detecting the serum or urine creatinine for diagnosis. In this dissertation, a strategy integrating SOX gene codon and fermentation control protocol was primarily proposed for improving SOX production. Then, an affinity medium was synthesized with 4-amino-pyrrole-2-carboxylic acid and ethylenediamine as ligand and spacer, respectively; and a simple affinity protocol for purifying SOX was explored. The mechanism of thermal unstable problem of Bacillus SOX was analyzed. The enzyme surface was modified with poly-lysine to improve Bs SOX stability. Finally, SOX sequence from a thermophic bacterium was synthesized, expressed, and the sequence alignment of two SOX were analyzed to illustrate the thermostability. The main contents are listed as follows:A strategy was established by integrating codon design and fermentation control to increase SOX yield. The bases of SOX gene had been reasonably substituted based on the codon preference of E. coli, without changing the amino acid sequence of Bs SOX. The codon adaptation index(CAI) relative to E. coli was increased to 0.87; codon optimization rate was about 62.5%. The expressiveness of Bs SOX in E. coli was improved by 33.5%.With lactose and 30℃ as the inducer and induction temperature, respectively, the yields of Bs SOX were 45.7% and 23.4% higher than the value of IPTG inducer and 37℃ induction temperature. Based on the analysis of kinetic parameters with various p H-stat conditions, a better balance between cell growth and expression was achieved by the two stage temperature-p H strategy. In the initial stage of fermentation, 37℃ and p H-stat 7.5 was carried out for ensuring better cell growth; In the induction phase, the operation condition was shifted to 30℃ and p H-stat 6.5 for further accelerating the Bs SOX expression. Compared with results from p H-stat 7.5 and p H-stat 6.5, the yield of Bs SOX(8490 U?L-1) was increased by 63.3% and 34.9%, respectively. And the fermentation period was shortened from 56 h to 40 h. Additonally, the constant feeding strategy based on the two stage temperature-p H strategy was proposed. A more moderate and stable condition was maintained by the constant feeding model, which would be beneficial for further cell growth and Bs SOX expression, and the yield of Bs SOX(12466 U?L-1) was improved by 46.8% higher on the basis of two stage temperature-p H strategy.A special affinity medium was synthesized and one-step purification protocol was further established for efficiently purifying SOX. The result of adsorption analysis showed that Sepharose-ethylenediamine- 4-amino-pyrrole-2-carboxylic acid affinity medium(2367 U?g-1 medium) had the optimum adsorption to SOX among all synthesized affinity mediums. Moreover, the successful coupling of guanine to Sepharose medium was further confirmed by LC-MS. A creative protocol for SOX purification was developed with only one-step by synthesizing Sepharose-ethylenediamine-4-amino-pyrrole-2-carboxylic acid as affinity medium. The activity recovery of the purified SOX was 91.2%. The result of SDS-PAGE showed that one-step purified SOX was electrophoresis purity. The Km and Vmax values of pure Bs SOX for sarcosine were 141.6 mmol·L-1and 0.115 mmol·L-1·min-1, reapectively.The chemical surface modification was used to improve Bs SOX stability based on the exposure of hydrophobic groups, which was the main reason of s SOX thermal instability. The mechanism of thermal unstable problem of Bacillus SOX was analyzed and revealed that mainly involved with the exposure of hydrophobic residues and further degaration ofenzyme moleucles. Its stability was significamtly enhanced by surface modification with poly-lysine. The covalent binding of poly-lysine onto Bs SOX surface was confirmed by MS and FTIR analysis. The corresponding thermodynamic parameter(Tm and △ H)of the modified enzyme(117.98℃and 2916.17 J·g-1) were higher than free enzyme(63.03℃ and 0.1217 J·g-1). Compared to free enzyme, both Km and Vmax values of the modified enzyme were decreased. Nevertheless, the thermal, p H and storage stability of modification SOX were significantly improved. More than 90% ofinitial activity of modified enzyme was maintained at a broad range of p H from 5.0 to 10.0. After being stored at 37℃ for 7 days, 95.1% of modified SOX acitivity was remained compared to only 7.9% of original SOX. Multiple covalent bonds were formed between polylysine molecules and surface carboxyl of Bs SOX. A network protective layer on Bs SOX surface was formed, which was favor to inhibit enzyme loose of Bs SOX natural structures and withstand unfavorable external environment factors.The thermophic SOX sequence was synthesized and the thermostability mechanism of Tr SOX was also explored via analysis of amino acid sequence and intramolecular forces. According to the codon bias in E. coli, the Tr SOX gene sequence was optimized and successfully expressed in E. coli. The soluble expression of Tr SOX was improved significally by co-expression of chaperone Gro ES-Gro EL and Dna K-Dna J-Grp E-Gro ES-Gro EL. The factors for contributing to the thermostability of Tr SOX were discussed, such as amino acids composition, hydrogen bonds, ion pairs and the protein structure features. There were more Ala, Pro, Arg and Glu amino acids in Tr SOX for contributing to the thermostability. Meanwhile, it was the same for hydrogen bonds and ion pairs. There were about 68.2 pairs hydrogen bonds, 179 hydrophobic amino acids and 23 ion pairs in Tr SOX. Intertwined structure was formed between above non-covalent bonds. And it played an important role in the thermostability of Tr SOX by promoting interaction of Tr SOX sencondary structure and increasing the tightness of Tr SOX.