Han Zichun, Long Liangkun, Ding Shaojun
The Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Jiangsu Key Lab for the Chemistry & Utilization of Agricultural and Forest Biomass, College of Chemical Engineering, Nanjing Forestry University, Nanjing, China.
Front Microbiol. 2019 Aug 13;10:1869. doi: 10.3389/fmicb.2019.01869. eCollection 2019.
HsCCO and RbCCO from and were selected and characterized from five putative bacterial carotenoid cleavage oxygenase gene sequences, due to merits in expression solubility and catalytic properties. Both enzymes can convert 4-vinylguaiacol and isoeugenol to vanillin. HsCCO showed maximum activity at 40°C and pH 7.0 and was stable at pH 6.5-10 and temperature around 25°C, retaining over 90 and 80% of initial activity, respectively. RbCCO showed maximum activity at 35°C and pH 9.0 and was stable at pH 6-11 and temperatures of 25-30°C, retaining over 80% of initial activity. The kinetic constants of HsCCO for isoeugenol and 4-vinylguaiacol were 1.55 and 1.65 mM and were 74.09 and 27.91 nmol min mg, respectively. The kinetic constants of RbCCO for isoeugenol and 4-vinylguaiacol were 2.24 and 0.85 mM and were 76.48 and 19.96 nmol min mg, respectively. The transformed cells harboring converted isoeugenol and 4-vinylguaiacol at molar conversion yields of 80 and 55% and the maximum vanillin concentrations were up to 1.22 and 0.84 g L, respectively. Comparably, the molar conversion yields of the transformed cells harboring against isoeugenol 4-vinylguaiacol were 75 and 58%, and the maximum vanillin yields were up to 1.14 and 0.88 g L, respectively.
从五个假定的细菌类胡萝卜素裂解双加氧酶基因序列中选择并鉴定了来自[具体来源1]和[具体来源2]的HsCCO和RbCCO，原因在于它们在表达溶解性和催化特性方面具有优势。两种酶都能将4 - 乙烯基愈创木酚和异丁香酚转化为香草醛。HsCCO在40°C和pH 7.0时表现出最大活性，在pH 6.5 - 10以及约25°C的温度下稳定，分别保留超过90%和80%的初始活性。RbCCO在35°C和pH 9.0时表现出最大活性，在pH 6 - 11以及25 - 30°C的温度下稳定，保留超过80%的初始活性。HsCCO对异丁香酚和4 - 乙烯基愈创木酚的动力学常数Km分别为1.55和1.65 mM，Vmax分别为74.09和27.91 nmol min mg。RbCCO对异丁香酚和4 - 乙烯基愈创木酚的动力学常数Km分别为2.24和0.85 mM，Vmax分别为76.48和19.96 nmol min mg。携带[具体基因1]的转化细胞将异丁香酚和4 - 乙烯基愈创木酚的摩尔转化率分别为80%和55%，香草醛的最大浓度分别高达1.22和0.84 g/L。相比之下，携带[具体基因2]的转化细胞对异丁香酚和4 - 乙烯基愈创木酚的摩尔转化率分别为75%和58%，香草醛的最大产量分别高达1.14和0.88 g/L。
