Soluble Expression Of Recombinant Tyrosine Decarboxylase And Application In Synthesis Of Tyramine

Tyramine and its derivatives are key intermediates for the synthesis of various physiological and pharmacological active ingredients of pharmaceuticals,health care and cosmetics industries.Tyrosine decarboxylase?TyrDC?catalyzes the decarboxylation of trysoine for the synthesis of tyramine,which is regarded as one of the most promising pathways due to its simple,efficient and environmentally friendly characteristics.In this study,recombinant expression of TyrDC from Lactobacillus brevis in Escherichia coli was investigated to solve the inclusion bodies issue.Our results are as follows:?1?The addition of glucose,fructose,arabinose and mannose into LB medium can effectively improve the soluble expression of recombinant TyrDC?rTyrDC?;?2?Expression of rTyrDC as soluble protein or inclusion bodies have independent mechanisms,acidic pH could promote its soluble expression,whereas carbohydrates such as glucose,fructose or arabinose is indispensable to inhibit the formation of inclusion bodies.Based on above results,the soluble expression of rTyrDC was achieved through addition of glucose(10 g·L^–1,LBG medium)and pH control?6.50–6.80?.Compared with simple pH control?6.50–6.80,LB medium?,the activity of TyrDC in LBG was enhanced from 22.00 to 30.70 kU·L^–1,representing a 40%increase.Also,the specific activity of the lyophilized enzyme powder was enhanced from 12.60 to 46.30 U·mg^–1,increased by 3.67 times.For the synthesis of tyramine from tyrosine using lyophillied rTyr DC powder,the reaction conditions including buffer system,pH,temperature,and surfactant were optimized.The results are as follows:Na₂HPO₄-KH₂PO₄ buffer system,pH 5.50,40°C,and Tween 80 were the optimal conditions.The amount of enzyme required for complete conversion of 100 mmol·L^?1tyrosine in 20 h was 0.125 g·L^–1,and the substrate to catalyst ratio?S/C?and total turnover number?TTN?were 145 g·g^–1 and 116,000.0.50 g·L^–1 rTyrDC was required to convert 200mmol·L^?1 tyrosine in 24 h,with S/C and TTN of 72.50 g·g^–1 and 58,200 respectively.When further increase the substrate concentration to 400 mmol·L^?1,2.50 g·L^–1 enzyme was required to achieve over 99.99%conversion in 24 h,with S/C and TTN of 29.00 g·g^–1 and 23,300.L-tyrosine decarboxylase was engineered by alanine scanning to enhance its activity toward D-tyrosine.Five beneficial mutation sites were identified,M99,S101,Y331,Y395 and S586.Among them,the best variant S101A exhibited 2.06 times activity compared with that of wide-type rTyrDC?WT?.It was also found that the activity of double mutant M99A/S586A was2.73 times of that of WT.The specific activities of purified S101A and M99A/S586A were 0.98and 1.45 U·g^–11 toward D-tyrosine respectively.In summary,highly soluble rTyrDC has been employed in the synthesis of tyramine in a green and efficient way,providing an efficient catalyst for the biosynthesis of tyramine.Five beneficial mutation sites were identified enhancing the activity toward D-tyrosine through alanine scanning,providing hot-spots for the further engineering and laying the theoretical foundation for the construction of a cascade reaction to synthesize chiral amino alcohols.