κ-Carrageenan is a linear polysaccharide with repeating disaccharide units of 3,6-anhydro-α-D-galactose and β-D-galactose-4-sulfate linked by alternating α-(1,3) and β-(1,4) glycosidic bonds [1]. The κ-carrageenases (EC 3.2.1.83) belong to the glycoside hydrolase (GH) family 16 (www.cazy.org) and can specifically hydrolyze the (1→4)-β-D-linkages between β-D-galactose-4-sulfate and 3,6-anhydro-α-D-galactose in κ-carrageenans, producing even-numbered κ-carrageenan oligosaccharides [2], [3]. κ-Carrageenase is mainly derived from marine microorganisms and marine animals. The microbial sources include Pseudoalteromonas sp. Q203, Pseudomonas aeruginosa ZSL-2, Shewanella sp. Kz7, Vibrio sp. CA-1004, Cellulophaga lytica strain N5-2, Cytophaga strains MCA-2 and lk-C783, Tamlana sp. HC4, Pseudoalteromonas carrageenovora HLX250, Pedobacter hainanensis 13-Q, and Zobellia galactanivorans [2], [3]. The hydrolysis of κ-carrageenan by κ-carrageenase is achieved by the coordination of acid/base and nucleophile catalysts consisting of glutamate and/or aspartate residues [3]. κ-Carrageenases degrade κ-carrageenan polysaccharides by a retaining mechanism via double replacement reactions, in which the catalytic carboxylate residues distributed at the opposite site of the sugar plane attack the glycosyl oxygen and anomeric carbon atoms, respectively, to complete the glycosylation and deglycosylation steps [3], [4]. In addition to κ-carrageenan oligosaccharides preparation, κ-carrageenases can also be used for algal protoplast preparation and providing substrates for bioethanol fuel production [4]. The biological activities of carrageenan oligosaccharides and their derivatives mainly include antioxidant, anti-inflammatory, anti-tumor, antiviral, antibacterial, and immunomodulatory activities [5]. The carrageenan oligosaccharides have potential utilization in food, agricultural, and pharmaceutical industries [5].

Thermostability and catalytic activity are essential features for an enzyme to be suitable for industrial application. One of the advantages of excellent thermostability of κ-carrageenase is to allow the enzymatic reaction to process a high concentration of κ-carrageenan at a high temperature with effectively reduced viscosity of the κ-carrageenan solution [6]. In addition, the tolerance to high temperature is greatly favored for the depolymerization process as the high temperature promotes the polysaccharide conformational change to be more accessible to carrageenase [7].

To improve the enzyme properties, attempts have been made by the enzyme structure modification through directed evolution, rational design, and semi-rational design [8]. Protein engineering based on rational design has successfully improved the thermostability of the engineered enzymes to strengthen the interactions by enhanced bonding through disulfide bridges, hydrogen bonds, and other non-covalent attractions [8]. These modifications can normally lead to reduced protein folding free energy to be thermodynamically favorable [9], [10]. The effects of mutations in the amino acid sequence on protein folding free energy can be predicted through some computer algorithms [11], [12]. Among them, the PoPMuSiC online server has been successfully used for the computer-aided design for enzyme thermostability improvement [13], [14]. The structural differences between enzymes complicate the relationship of enzyme structure and function. The characteristics of individual enzyme are needed to be studied. To the best of our knowledge, no study has been reported on the thermostability improvement of κ-carrageenase.

Most κ-carrageenases show the enzymatic activity and thermal stability in the range of 30−40 °C [4]. The κ-carrageenase from Pseudoalteromonas tetraodonis (GenBank accession number AB572925) demonstrates good enzyme activity towards κ-carrageenan at the alkaline conditions but displays low thermal stability [15]. After the enzyme is treated at 30 °C for 15 min, the residual activity is only about 60% of its original activity [15]. In this study, mutations were introduced to this κ-carrageenase gene by site-directed mutagenesis of some selected amino acid residues determined by the PoPMuSiC algorithm, and their thermostability and enzymatic activity were analyzed. In addition, the κ-carrageenan degradation products by the mutant κ-carrageenase were identified, and their pancreatic lipase inhibitory activities were evaluated.