Yan-Feng Guo

Enzymatic alginate oligosaccharides were proved with various biological activities. Developing robust alginate lyase with high production is essential for its industrial application. In this study, a novel alginate lyase gene, AL07, was successfully screened and expressed in Pichia pastoris, and its characteristics were characterized. The secreted alginate lyase has a molecular weight of approximately 40.0 kDa and an activity of 45.4 U/mL. AL07 exhibits superior biological activity at pH 7.0 and 40 °C, with a preference for polyG substrates. Notably, the enzyme exhibits more than 60% relative activity over a pH range of 5.0 to 10.0. AL07 is also independent of ions and exhibits salt tolerance properties. The main degradation product of AL07 is DP2 oligosaccharide. Using AL07-based enzyme cocktail, Laminaria japonica can be degraded within 120 min. Therefore, AL07 has been demonstrated to be an efficient tool for the preparation of alginate oligosaccharides and the degradation of L. japonica.

Purpose: L-Leucine is an essential amino acid for humans since it is only synthesized in fungi, bacteria, and plants. Its production has not been well documented as L-lysine and L-glutamic acid due to multivalent regulation and longer synthesis pathways. In Corynebacterium glutamicum, L-leucine synthesis is strongly regulated at α-isopropylmalate synthase (α-IMPS) node by the end product. Amino acid substitutions of the α-IMPS variant from strain ARTP-L04 were analyzed, and the α-IMPS variant was further characterized. To enhance the L-leucine production, the effect of α-IMPS variant was also evaluated.Methods: In the present study, construction of plasmid and recombination strain, protein expression and purification, enzymatic properties, fermentation, and amino acid analysis were performed.Results: Compared with leuA from wild-type strain ATCC 13032, 10 nucleotide exchanges were identified in the α-IMPS variant, leading to amino acid exchanges of Gly92Asp, Ile162Val, Gln494Arg, and Gly526Asp. Furthermore, the α-IMPS variant was expressed and characterized. It was found that the α-IPMS variant exhibited higher resistance to feedback inhibition by L-leucine. In the presence of 20 mM L-leucine, over 50% of α-IPMS variant activity was still observed. However, there was no significant difference in specific activity between the α-IPMS variant and wild-type α-IPMS. Therefore, the α-IPMS variant almost removed inhibition by L-leucine. Interestingly, co-expression of α-IPMS and AHAS both from strain ARTP-L04 could significantly enhance L-leucine production, accumulating 7.79 g/L L-leucine.Conclusions: Mutation that occurred in the α-IPMS variant played an important role in feedback resistance, almost removing feedback inhibition by L-leucine. In C. glutamicum, the expression of the α-IPMS variant could significantly enhance L-leucine production, especially when it was co-expressed with AHAS. Collectively, our findings provided valuable insights into further development in gene engineering.