Hyaluronic acid (hyaluronan; HA) is a class of non-sulfated glucosaminoglycan (GAG) composed of repeating disaccharide units of D-gluconic acid and N-acetylglucosamine, and can be degraded by hyaluronidase (HAase). Hyaluronate lyase (HA lyase) is a group of HAase that cleave β-1,4 bond of HA through β-elimination, generating products with unsaturated D-gluconic acid on the non-reducing end (Stern and Jedrzejas, 2006). Unlike HA hydrolases that degrade HA into HA tetrasaccharides as minimum product, final product of HA lyases digestion is unsaturated HA disaccharide (ΔHA2) (El-Safory et al., 2010, Guo et al., 2009).

In recent years, significant application values of HA lyase have been elucidated in fields of biotechnology. One is its application in preparing HA oligomers (o-HA). Unsaturated o-HAs prepared by HA-lyase was proved to have antioxidant effect and inhibit tumor cell growth (El-Safory and Lee, 2010), and the purified ΔHA2 had shown significant anti-inflammatory effect on both cell and animal experiments (Han et al., 2022). Meanwhile, applying HA lyase in medical treatments have also been investigated. A recombinant HA lyase from Streptomyces koganeiensis has been developed by Fidia Farmaceutici S.p.A. (www.fidiapharma.com), and has been proved an effective “spreading factor” that degrade HA in the extracellular matrix (ECM) to accelerate drug delivery(Ebelt et al., 2020; Pavan et al., 2016). The advantage of such bacterial-originated HA lyases in medical treatments is that they possess higher activities and stabilities comparing to commercialized animal hyaluronidases. Also, they do not need glycosylation to maintain their activities, which implicates less risks of calling immune reactions (Santaella-Sosa, 2020). In this case, HA lyases might also be preferable to be applied in cell pretreatments to separate single cells from tissues for applications such as assisted reproduction technology (Moura et al., 2017).

HA lyase-producing microorganisms distribute widely in nature (Girish and Kemparaju, 2007). HA lyase from species including Streptococcus sp., Streptomyces sp., Clostridium sp., Arthrobacter sp., Bacillus sp. have been characterized by researchers (Girish and Kemparaju, 2007, Guo et al., 2014, Pavan et al., 2016, Zhu et al., 2017). However, a large number of microbial HA lyases to date, still remain uncharacterized (Zhang et al., 2022). Also, few studies have explored the potential of optimizing recombinant expression to facilitate high level production of specific HA lyase.

In this work, we first isolate a HA lyase producing strain Cf1 and identify it as Citrobacter freundii. The whole-length gene of a highly active HA lyase, HylC is then cloned from Cf1 and its amino acid sequence is analyzed. Then, encoding gene of HA lyase HylC with a His6 tag is amplified from Cf1, recombinantly expressed in the host E.coli BL21(DE3), and the recombinant enzyme is purified to characterize its biochemical properties. We also analyze the degradation pattern and final product of HylC. In order to facilitate a higher expression level, promoter optimization is carried out on the basis of original expression vector. Finally, scale-up batch fermentation is performed to further enhance the productivity of recombinant strain.