河北省重点研发计划(21372802D 和 19222906D)
College of Food Science and Biology,Hebei University of Science and Technology
为了提高β-甘露聚糖酶的活性,本研究采用易错PCR将来源于地衣芽孢杆菌(Bacillus licheniformis)KD-1的β-甘露聚糖酶基因manBl进行分子进化,并在枯草芽孢杆菌(B. subtilis)中进行表达,以定向筛选酶活性提高的β-甘露聚糖酶突变体。筛选得到的突变体ManBl (I91N/L211I),其比酶活性为 15554.7 U/mg,是野生型ManBl的4.2倍,食品级表达的胞外酶产量达17601.3 U/mL。应用AlphaFold2对该酶的三维结构进行预测,结果表明,尽管β-甘露聚糖酶的2个位点(I91N 和 L211I)位于催化中心之外,但在很大程度上影响酶活性。β-甘露聚糖酶ManBl (I91N/L211I)水解魔芋胶产物主要由甘露六糖、甘露三糖和甘露二糖组成。该研究首次报道I91N/L211I 2个位点联合突变能够提高β-甘露聚糖酶活性；ManBl (I91N/L211I)食品级表达,为该酶绿色安全地应用奠定了基础。
To improve β-mannanase activity, the molecular evolution of β-mannanase gene from Bacillus licheniformis KD-1 was carried out by error-prone PCR. The mutated genes were expressed in B. subtilis to directed screen the mutants with higher β-mannanase activity than the wildtype. Among mutants, the specific activity of β-mannanase ManBl (I91N/L211I) was 15554.7 U/mg, which was 4.2 times of the wild-type ManBl. The extracellular yield of ManBl (I91N/L211I) expressed in B. subtilis at food-grade level reached 17601.3 U/mL. The β-mannanase structure predicted by AlphaFold2 demonstrates that the two loci (I91N and L211I), which are not the β-mannanase catalytic center, influence the enzyme activity at a great degree. Furthermore, the β-mannanase ManBl (I91N/L211I) can hydrolyze konjac glucomannan (KGM) into mannooligosaccharides (MOS) with different degree of polymerization (DP), which were mainly composed of mannohexaose, mannotriose, and mannobiose. This is the first report that the combined mutation of I91N/L211I can increase β-mannanase activity. The food-grade expression of ManBl (I91N/L211I) opens up a way to its application with biosafety.