Zheng Fengzhen, Liu Junquan, Basit Abdul, Miao Ting, Jiang Wei
Beijing Advanced Innovation Center for Food Nutrition and Human Health, State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.
Front Microbiol. 2018 Dec 14;9:3016. doi: 10.3389/fmicb.2018.03016. eCollection 2018.
α-L-arabinofuranosidase (ARA) with enhanced specific activity and in large amounts, is needed for a variety of industrial applications. To improve ARA production with engineered methylotrophic yeast , a genetically modified gene from ND-1 was investigated. Through codon optimization and rational replacement of α-factor signal peptide with the native propeptide (MFSRRNLVALGLAATVSA), ARA production was improved from 2.61 ± 0.13 U/mL to 14.37 ± 0.22 U/mL in shaking flask culture (a 5.5-fold increase). Results of N-terminal sequencing showed that secreted active ARA of recombinant strain p-oARA had theoretical initial five amino acids (GPCDI) comparable to the mature sequences of α-oARA (EAEAG) and αp-oARA (NLVAL). The kinetic values have been determined for ARA of recombinant strain p-oARA ( = 747.55 μmol/min/mg, = 5.36 mmol/L), optimal activity temperature 60°C and optimal pH 4.0. Scaling up of ARA production by p-oARA in a 7.5-L fermentor resulted in remarkably high extracellular ARA specific activity (479.50 ± 12.83 U/mg) at 168 h, and maximal production rate 164.47 ± 4.40 U/mL. In studies of corn stover degradation activity, degree of synergism for ARA and xylanase was 32.4% and enzymatic hydrolysis yield for ARA + xylanase addition was 15.9% higher than that of commercial cellulase, indicating significant potential of ARA for catalytic conversion of corn stover to fermentable sugars for biofuel production.
多种工业应用需要具有更高比活性且大量的α-L-阿拉伯呋喃糖苷酶（ARA）。为了利用工程化甲基营养酵母提高ARA产量，对来自ND-1的一个基因进行了基因改造研究。通过密码子优化并用天然前肽（MFSRRNLVALGLAATVSA）合理替换α-因子信号肽，摇瓶培养中ARA产量从2.61±0.13 U/mL提高到14.37±0.22 U/mL（增加了5.5倍）。N端测序结果表明，重组菌株p-oARA分泌的活性ARA理论上最初的五个氨基酸（GPCDI）与α-oARA（EAEAG）和αp-oARA（NLVAL）的成熟序列相当。已测定重组菌株p-oARA的ARA的动力学值（ = 747.55 μmol/min/mg， = 5.36 mmol/L）、最佳活性温度60°C和最佳pH 4.0。在7.5-L发酵罐中扩大p-oARA的ARA生产，在168 h时细胞外ARA比活性显著提高（479.50±12.83 U/mg），最大生产率为164.47±4.40 U/mL。在玉米秸秆降解活性研究中，ARA与木聚糖酶协同作用程度为32.4%，添加ARA +木聚糖酶的酶解产率比商业纤维素酶高15.9%，表明ARA在将玉米秸秆催化转化为可发酵糖用于生物燃料生产方面具有巨大潜力。
