Biochemical characterization and cleavage pattern analysis of a novel chitosanase with cellulase activity.

Chitosanases are critical tools for the preparation of active oligosaccharides, whose composition is related to the cleavage pattern of the enzyme. Although numerous chitosanases have been characterized, the glycoside hydrolase (GH) family 5 chitosanases with other activities have rarely been investigated. Herein, a novel and second GH5 chitosanase OUC-Csngly from Streptomyces bacillaris was cloned and further characterized by expression in Escherichia coli BL21 (DE3). Interestingly, OUC-Csngly possessed dual chitosanase and cellulase activities. Molecular docking analysis showed that the C-2 group of sugar units affected the binding of the enzyme to oligosaccharides, which could result in different cleavage patterns toward chito-oligosaccharides (COSs) and cello-oligosaccharides. Further, we characterized OUC-Csngly's distinctive cleavage patterns toward two different types of oligosaccharides. Meanwhile, endo-type chitosanase OUC-Csngly generated (GlcN) - (GlcN)4 from chitosan, was significantly different from other chitosanases. To our knowledge, this is the first report to investigate the different cleavage patterns of chitosanase for COSs and cello-oligosaccharides.Key points• The molecular docking showed C-2 group of sugar units in substrate affecting the cleavage pattern.• The first chitosanase exhibited different cleavage patterns towards chito- and cello-oligosaccharides.• The groups at C-2 influence the subsite composition of the enzyme's active cleft.

A novel autolysis system for extracellular production and direct immobilization of a phospholipase D fused with cellulose binding domain.

BACKGROUND: Several types of phospholipases have been described in phospholipids modification. The majority of phospholipase D (PLD) superfamily members can catalyze two separate reactions: the hydrolysis of phospholipids to produce phosphatidic acid (PA) and the transphosphatidylation of phosphatidyl groups into various phosphatidyl alcohols to produce modified phospholipids. Transphosphatidylation is a useful biocatalytic method for the synthesis of functional phospholipids from lecithin or phosphatidylcholine (PC), which are both easily accessible. Different PLD coding genes have been cloned from various sources from viral, prokaryotic, and eukaryotic organisms. Despite the catalytic potential of PLD, their low productivity has hampered their practical applications, probably because PLD, which is highly toxic to the host cells, when transformation of the PLD genes into the host cells, degrade PLs in the cell membrane. In this study, we designed a novel two-step expression system to produce and secrete recombinant PLD in extracellular medium, cellulose-binding domains as an affinity fused with PLD for immobilization and purification proteins. RESULTS: The engineered BL21 (DE3) host strain, which harbored the final expression vector pET28a-PLD-CBD-araC-ESN, was induced by IPTG and L-arabinose, the cell density decreased rapidly over a 2 h period and the enzymes released into the extracellular medium accounts owned 81.75% hydrolytic activity. Scanning electron microscopy results showed that there were obvious structural changes on the cell surface. The extracellularly secreted PLD-CBD powder was used to catalyze the transphosphatidylation reaction synthesis of phosphatidylserine, 2.3 U enzymes reacted for 12 h, during which the conversion rate reached 99% with very few by-products being produced. When the fused protein PLD-CBD immobilized on microcrystalline cellulose, the enzymes can be cycle used five times with 26% conversion rate was preserved. CONCLUSIONS: This study introduced an effective method for use in the expression of recombinant proteins and their extracellular secretion that simplifies the steps of sonication and purification and demonstrates great potential in the industrial application of enzymes. Cellulose as the most abundant renewable biomass resources in nature, and the cost is low, used for PLD immobilization make it more simple, effective and sustainable.

Identification of an alkaline lipase capable of better enrichment of EPA than DHA due to fatty acids selectivity and regioselectivity.

The whole genome of Streptomyces violascens (=ATCC 27968) was sequenced and the cloning and expression of OUC-Lipase 6 were conducted in Bacillus subtilis WB800. The recombinant enzyme belongs to the lipolytic enzymes family V. OUC-Lipase 6 showed optimal activity at 30 °C and pH 9.0, and retained 90.2% of its activity in an alkaline buffer (pH 8.0, 30 °C and 96 h). OUC-Lipase 6 showed good stability under medium temperature conditions (residual activity of 68.8%, pH 8.0, 45 °C and 96 h). OUC-Lipase 6 could selectively hydrolyze fatty acids on the glyceride backbone, thus improving the contents of DHA and EPA in codfish oil. OUC-Lipase 6 also showed regioselectivity, resulting in a better enrichment efficiency for EPA than DHA. After hydrolyzing for 36 h via OUC-Lipase 6, the contents of EPA and DHA were improved to 3.24-fold and 1.98-fold, respectively.

Combining Cell Surface Display and DNA-Shuffling Technology for Directed Evolution of Streptomyces Phospholipase D and Synthesis of Phosphatidylserine.

Phospholipids have been widely used in food, medicine, cosmetics, and other fields because of their unique chemical structure and healthcare functions. Phospholipase D (PLD) is a key biocatalyst for the biotransformation of phospholipids. Here, an autodisplay expression system was constructed for rapid screening of mutants, and PLD variants were recombined using DNA shuffling technology and three beneficial mutations were obtained. The results of enzymatic performance and sequence information comparison indicated that C-terminal amino acids exerted a greater impact on the correct folding of PLDs, and N-terminal amino acids are more important for catalytic reaction. The best-performing recombinant enzyme in transphosphatidylation reactions was Recom-34, with a phosphatidylserine content accounting for 80.3% of total phospholipids and a 3.24-fold increased conversion rate compared to the parent enzyme. This study demonstrates great significance for screening ideal biocatalysts, facilitating soluble expression of inclusion body proteins, and identifying key amino acids.