目的:D-氨基葡萄糖(GlcN)是一种重要的氨基糖,在医学中具有多种应用,食品和饮料,营养补充剂,和乳制品。本研究的目的是从N-乙酰-D-氨基葡萄糖(GlcNAc)生产GlcN与有效的脱乙酰酶,并应用不同的策略来提高GlcN的产量。
结果:我们筛选了一系列涉及GlcNAc脱乙酰以形成GlcN的脱乙酰酶。来自柯达红球菌的二乙酰几丁糖脱乙酰酶(TKDac)对GlcNAc具有高效的脱乙酰活性,但大多是包涵体的形式。通过共表达分子伴侣(groEL)和TKDac提高了TKDac的可溶性表达,并插入编码异亮氨酸的稀有密码子ATA。因此,构建表达TKDac的重组菌株TKEL4,通过TKDac催化的脱乙酰反应获得48g/L的GlcN。为了克服副产物(乙酸盐)的抑制作用,进行固定化TKDac以从GlcNAc生产GlcN。将固定化的TKDac方便地重新用于几个批次(8个以上),转化率为90%。
结论:发现来自柯达红藻的TKDac是产生GlcN的有效脱乙酰酶。分子伴侣与靶蛋白共表达,插入稀有密码子可有效提高TKDac的可溶性表达。固定化的TKDac代表了未来GlcN生产的有希望的方法。
OBJECTIVE: D-Glucosamine (GlcN) is an important amino sugar with various applications in medicine, food & beverages, nutritional supplements, and dairy products. This study aimed to produce GlcN from N-acetyl-D-glucosamine (GlcNAc) with an efficient deacetylase, and apply different strategies to enhance GlcN production.
RESULTS: We screened a series of deacetylases that involved in the deacetylation of GlcNAc to form GlcN. A diacetylchitobiose deacetylase (TKDac) from Thermococcus kodakarensis exhibited high-efficient deacetylation activity for GlcNAc, yet mostly in the form of inclusion bodies. The soluble expression of TKDac was improved by a co-expressing molecular chaperone (groEL) and TKDac, and insertion of rare codon ATA encoding isoleucine. As such, the recombinant strain TKEL4 was constructed to express TKDac, and 48 g/L GlcN was achieved by TKDac-catalyzed deacetylation. To overcome the inhibition of byproduct (acetate), immobilized TKDac was carried out to produce GlcN from GlcNAc. The immobilized TKDac was conveniently re-used for several batches (above 8) with a 90% conversion rate.
CONCLUSIONS: TKDac from T. kodakarensis was found to be an efficient deacetylase to produce GlcN. Co-expression of molecular chaperone and target protein, and insertion of rare codons were effective to improve the soluble expression of TKDac. The immobilized TKDac represents a promising method for future GlcN production.