Co-Innovation Center for Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, China.
Jiangsu Provincial Key Lab for the Chemistry and Utilization of Agro-Forest Biomass, Nanjing Forestry University, Nanjing, 210037, China.
Appl Biochem Biotechnol. 2023 Oct;195(10):5933-5949. doi: 10.1007/s12010-023-04374-4. Epub 2023 Feb 1.
Lipase B from Candida antarctica (CALB) is an important biocatalyst with many potential applications. However, original CALB is usually with lower enzyme activity and also costly to produce from Candida antarctica; hence, it is often necessary to prepare recombinant CALB through gene heterologous expression. In this research, seven promoters and five signal peptides were compared respectively for expressing codon-optimized CALB in Pichia pastoris, and then recombinant P. pastoris containing 3 copies of calb gene were obtained by screening with high concentrations of antibiotics under the condition of the optimal combination. In a 1.3-L bioreactor, the maximum CALB activity and total protein content reached 444.46 ± 18.81 U/mL and 5.41 ± 0.1 g/L, respectively, after about 9 days of incubation in FM22 medium, which were 34 times and 20 times higher than the initial strains, respectively. In addition, the obtained CALB was used to catalyze the transesterification of acidified gutter oil with methanol, suggesting a promising pathway to convert waste or low quality of bio-oil feedstocks with high amount of free fatty acids into biodiesel by using recombinant CALB as catalyst. The results can provide with a good reference for efficient expression of CALB and enhancing lipase production in P. pastoris. It is supposed to bring with new possibility for the bio-production of other valuable proteins.
南极假丝酵母脂肪酶 B（CALB）是一种具有多种潜在应用的重要生物催化剂。然而，原始的 CALB 通常酶活较低，而且从南极假丝酵母中生产成本也较高；因此，通常需要通过基因异源表达来制备重组 CALB。在这项研究中，分别比较了七种启动子和五种信号肽，用于在毕赤酵母中表达密码子优化的 CALB，然后在最优组合条件下，通过高浓度抗生素筛选获得了含有 3 个 calb 基因拷贝的重组毕赤酵母。在 1.3-L 生物反应器中，在 FM22 培养基中培养约 9 天后，CALB 的最大酶活和总蛋白含量分别达到 444.46±18.81 U/mL 和 5.41±0.1 g/L，分别是初始菌株的 34 倍和 20 倍。此外，所得 CALB 用于催化酸化地沟油与甲醇的转酯化反应，表明通过使用重组 CALB 作为催化剂，将富含游离脂肪酸的废油或劣质生物油原料转化为生物柴油是一种很有前途的途径。该结果可为 CALB 的高效表达和提高毕赤酵母中的脂肪酶产量提供良好的参考，为其他有价值蛋白质的生物生产带来新的可能性。
