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β-Galactosidase plays an important role in medicine and dairy industry. In this study, a new glycoside hydrolase family 42 (GH42) β-galactosidase-encoding gene, gal42, was cloned from a newly isolated marine bacterium Bacillus sp. BY02 and expressed in Escherichia coli. Structural characterization indicated that the encoding β-galactosidase, Gal42, is a homotrimer in solution, and homology modeling indicated that it retains the zinc binding sites of the Cys cluster. The reaction activity of Gal42 was significantly increased by Zn2+ (229.6%) and other divalent metal ions (Mn2+, Mg2+, and Co2+), while its activity was inhibited by EDTA (53.9%). Meanwhile, the thermo-stability of the Gal42 was also significantly enhanced by 5 and 10 mM of zinc ion supplement, which suggested that the “Cys-Zn” motif played important roles in both structural stability and catalytic function. Furthermore, Gal42 showed effective lactose hydrolysis activity, which makes the enzyme hydrolyze the lactose in milk effectively. These properties make Gal42 a potential candidate in food technology.

In order to control the release of mesalazine (MSZ) in the gastrointestinal tract to achieve better pharmacological effects in the colon, in this study, MSZ was added to hydroxypropyl-β-cyclodextrin (HP-β-CD) to form a water-soluble HP-β-CD/MSZ inclusion complex. Then, the inclusion compound was loaded into the structure of the bilayer polyelectrolyte complex microsphere formed by alginate (Alg), chitosan (Cs), and kappa carrageenan (κ-Car) as the hydrogel carrier, and the hydrogel beads with colon-specific release MSZ after oral administration were formed. The formed hydrogel beads have different swelling capabilities in different pH media and have the greatest swelling degree under pH 7.4. The encapsulation efficiency and drug loading of hydrogel beads can reach up to 83.23 and 18.31%, respectively, and the size of hydrogel beads can be reduced to less than 1 mm after drying, so that the size of oral administration can be reached. In vivo experiments also showed that the formed hydrogel beads had a better therapeutic effect on colitis than free drugs, and the microspheres were biodegradable, so the double-layer pH-sensitive microspheres could be effectively used in colon-targeting drug delivery.

β-Galacto-oligosaccharide (β-GOS) showed great potential in ulcerative colitis (UC) adjuvant therapy. Herein, the preventive and prebiotic effect of enzymatic-synthesized α-linked galacto-oligosaccharide (α-GOS) was investigated in dextran sodium sulfate-induced colitis and gut microbiota dysbiosis mice. Compared with β-GOS, the α-GOS supplement was more effective in improving preventive efficacy, promoting colonic epithelial barrier integrity, and alleviating inflammation cytokines. Moreover, the activation of the NOD-like receptor (NLR) family member NLRP3 inflammasome-mediated inflammation was significantly inhibited by both α-GOS and β-GOS. Gut microbiota analysis showed that α-GOS treatment reshaped the dysfunctional gut microbiota. The subsequent Spearman's correlation coefficient analysis indicated that these gut microbiota changes were significantly correlated with the inflammatory parameters. These results suggested that the enzymatic-synthesized α-GOS is a promising therapeutic agent in UC prevention and adjuvant treatment by maintaining intestinal homeostasis.

Alginate oligosaccharides (AOS) showed various biological activities. Traditional protocol for producing AOS was a multiple-step and high-pollution procedure. In this study, a rapid and efficient AOS producing method was developed directly from Laminaria japonica. Natural sun-dried L. japonica with a feed ratio of 1:7 (w/v) was pretreated using cellulase with a dry weight of 3%, for releasing the fermentable sugars (8.5 g/L glucose and 15.2 g/L mannitol). Then, the engineered yeast Yarrowia lipolytica strain with alginate lyase activity was grown using an algae-based medium. After fermentation for 72 h, glucose and mannitol were completely consumed, and 71.8 mM AOS was extracted from the fermentation supernatant. The degree of polymerization (DP) was ranging from 2 to 3. The recovery yield of AOS was about 91.7%. The purity of the extracted AOS was 92.6%. Overall, our work provided new insights for the development of green biotechnologies for oligosaccharide production from seaweed.

Oral route colon-targeted drug delivery systems (CDDSs) are desirable for the treatment of ulcerative colitis (UC). However, CDDSs are challenging owing to the physiological and anatomical barriers associated with the gastrointestinal tract (GIT). In this study, we developed an effective enzyme-triggered controlled release system using curcumin–cyclodextrin (CD–Cur) inclusion complex as core and low molecular weight chitosan and unsaturated alginate resulting nanoparticles (CANPs) as shell. The formed CD–Cur–CANPs showed a narrow particle-size distribution and a compact structure. In vitro drug release determination indicated that CD–Cur–CANPs showed pH-sensitive and α-amylase-responsive release characteristics. Furthermore, in vivo experiments demonstrated that oral administration of CD–Cur–CANPs had an efficient therapeutic efficacy, strong colonic biodistribution and accumulation, rapid macrophage uptake, promoted colonic epithelial barrier integrity and modulated production of inflammatory cytokines, reshaped the gut microbiota in mice with dextran sodium sulfate (DSS)-induced colitis. Taken together, our synthetic CD–Cur–CANPs are a promising synergistic colon-targeted approach for UC treatment.

In this study, the two enzymatic low molecular weight chitosan oligosaccharides (LMW-COSs), LMW-COS-H and LMW-COS-L, were prepared with average MWs of 879.6 Da and 360.9 Da, respectively. Compared to LMW-COS-L, the LMW-COS-H was more effective in improving high-fat diet (HFD)-induced metabolic abnormalities, such as obesity, hyperlipidemia, low-grade inflammation and insulin resistance. The subsequent analysis of gut microbiota showed that the supplement of LMW-COSs caused overall structural and genus/species-specific changes in the gut microbiota, which were significantly correlated with the metabolic parameters. Specifically, both of the LMW-COSs significantly decreased the relative abundance of inflammatory bacteria such as Erysipelatoclostridium and Alistipes, whereas that of the beneficial intestinal bacteria (such as Akkermansia and Gammaproteobacteria) increased significantly. This study suggested that there were potential prebiotic functions of LMW-COSs in HFD fed mice, which regulated the dysfunctional gut microbiota, alleviated low-grade inflammation and maintained the intestinal epithelial barrier.

As prebiotics, galacto-oligosaccharides (GOSs) can improve the intestinal flora and have important applications in medicine. &beta, galactosidases could promote the synthesis of GOSs in lactose and catalyze the hydrolysis of lactose. In this study, a new &beta, galactosidase gene (gal2A), which belongs to the glycoside hydrolase family 2, was cloned from marine bacterium Alteromonas sp. QD01 and expressed in Escherichia coli. The molecular weight of Gal2A was 117.07 kDa. The optimal pH and temperature of Gal2A were 8.0 and 40 &deg, C, respectively. At the same time, Gal2A showed wide pH stability in the pH range of 6.0&ndash, 9.5, which is suitable for lactose hydrolysis in milk. Most metal ions promoted the activity of Gal2A, especially Mn2+ and Mg2+. Importantly, Gal2A exhibited high transglycosylation activity, which can catalyze the formation of GOS from milk and lactose. These characteristics indicated that Gal2A may be ideal for producing GOSs and lactose-reducing dairy products.

Chitooligosaccharides (COS) are derived from chitosan, which can be used as nutraceuticals and functional foods. Because of their various biological activities, COS are widely used in the food, medicine, agriculture, and other fields. COS were prepared by chitosanase from Pseudoalteromonas sp. SY39 and their anti-obesity activity was researched in mice in this study. The effects of hydrolysis time, temperature, the ratio of enzyme to chitosan, and pH on the productivity of COS were discussed. Preparation process of COS was established in a 5-L fermenter. COS were characterized and their anti-obesity activity was studied in animal experiments. The results showed that COS could effectively reduce serum lipid levels and obesity in mice, and have a good anti-obesity activity. The preparation technology and remarkable anti-obesity activity of COS further expand their applications in the food and pharmaceutical industries.

Oligoalginate lyases catalyze the degradation of alginate polymers and oligomers into monomers, a prerequisite for biotechnological utilizing alginate. In this study, we report the cloning, expression and biochemical characterization of a new polysaccharide lyase (PL) family 17 oligoalginate lyase, OalV17, from the marine bacterium Vibrio sp. SY01. The recombinant OalV17 showed metal ion independent and detergent resistant properties. Furthermore, OalV17 is an exo-type enzyme that yields alginate monomers as the main product and recognizes alginate disaccharides as the minimal substrate. Site-directed mutagenesis followed by kinetic analysis indicates that the residue Arg231 plays a key role in substrate specificity. Furthermore, a rapid and efficient alginate monomer-producing method was developed directly from Laminaria japonica. These results suggest that OalV17 is a potential candidate for saccharification of alginate.

The recent emergence of antibiotic-resistant bacteria requires the development of new antibiotics or new agents capable of enhancing antibiotic activity. Lysozyme degrades bacterial cell wall without involving antibiotic resistance and has become a new antibacterial strategy. However, direct use of native, active proteins in clinical settings is not practical as it is fragile under various conditions. In this study, lysozyme was integrated into chitosan nanoparticles (CS-NPs) by the ionic gelation technique to obtain lysozyme immobilized chitosan nanoparticles (Lys-CS-NPs) and then characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM), which showed a small particle size (243.1 &plusmn, 2.1 nm) and positive zeta potential (22.8 &plusmn, 0.2 mV). The immobilization significantly enhanced the thermal stability and reusability of lysozyme. In addition, compared with free lysozyme, Lys-CS-NPs exhibited superb antibacterial properties according to the results of killing kinetics in vitro and measurement of the minimum inhibitory concentration (MIC) of CS-NPs and Lys-CS-NPs against Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), Escherichia coli (E. coli), and Staphylococcus aureus (S. aureus). These results suggest that the integration of lysozyme into CS-NPs will create opportunities for the further potential applications of lysozyme as an anti-bacterium agent.

Chitooligosaccharide (COS) has been recognized to exhibit efficient anti-oxidant activity. Enzymatic hydrolysis using chitosanases can retain all the amino and hydroxyl groups of chitosan, which are necessary for its activity. In this study, a new chitosanase encoding gene, csnQ, was cloned from the marine Bacillus sp. Q1098 and expressed in Escherichia coli. The recombinant chitosanase, CsnQ, showed maximal activity at pH 5.31 and 60 &deg, C. Determination of CsnQ pH-stability showed that CsnQ could retain more than 50% of its activity over a wide pH, from 3.60 to 9.80. CsnQ is an endo-type chitosanase, yielding chitodisaccharide as the main product. Additionally, in vitro and in vivo analyses indicated that chitodisaccharide possesses much more effective anti-oxidant activity than glucosamine and low molecular weight chitosan (LMW-CS) (~5 kDa). Notably, to our knowledge, this is the first evidence that chitodisaccharide is the minimal COS fragment required for free radical scavenging.

Alginate, the most abundant carbohydrate presents in brown macroalgae, has recently gained increasing attention as an alternative biomass for the production of biofuel. Oligoalginate lyases catalyze the degradation of alginate oligomers into monomers, a prerequisite for bioethanol production. In this study, two new oligoalginate lyase genes, oalC6 and oalC17, were cloned from Cellulophaga sp. SY116, and expressed them in Escherichia coli. The deduced oligoalginate lyases, OalC6 and OalC17, belonged to the polysaccharide lyase (PL) family 6 and 17, respectively. Both showed less than 50% amino acid identity with all of the characterized oligoalginate lyases. Moreover, OalC6 and OalC17 could degrade both alginate polymers and oligomers into monomers in an exolytic mode. Substrate specificity studies demonstrated that OalC6 preferred α-L-guluronate (polyG) blocks, while OalC17 preferred poly β-D-mannuronate (polyM) blocks. The combination of OalC6 and OalC17 showed synergistic degradation ability toward both alginate polymers and oligomers. Finally, an efficient process for the production of alginate monomers was established by combining the new-isolated exotype alginate lyases (i.e., OalC6 and OalC17) and the endotype alginate lyase AlySY08. Overall, our work provides new insights for the development of novel biotechnologies for biofuel production from seaweed.

(1) Background: In the treatment of ulcerative colitis (UC), accurate delivery and release of anti-inflammatory drugs to the site of inflammation can reduce systemic side effects. (2) Methods: We took advantage of this goal to prepare resveratrol-loaded PLGA nanoparticles (RES-PCAC-NPs) by emulsification solvent volatilization. After layer-by-layer self-assembly technology, we deposited chitosan and alginate to form a three-layer polyelectrolyte film. (3) Results: It can transport nanoparticles through the gastric environment to target inflammation sites and slowly release drugs at a specific pH. The resulting RES-PCAC-NPs have an ideal average diameter (~255 nm), a narrow particle size distribution and a positively charged surface charge (~13.5 mV). The Fourier transform infrared spectroscopy showed that resveratrol was successfully encapsulated into PCAC nanoparticles, and the encapsulation efficiency reached 87.26%. In addition, fluorescence imaging showed that RES-PCAC-NPs with positive charges on the surface can effectively target and accumulate in the inflammation site while continuing to penetrate downward to promote mucosal healing. Importantly, oral RES-PCAC-NPs treatment in DSS-induced mice was superior to other results in significantly improved inflammatory markers of UC. (4) Conclusions: Our results strongly prove that RES-PCAC-NPs can target the inflamed colon for maximum efficacy, and this oral pharmaceutical formulation can represent a promising formulation in the treatment of UC.

ι-Carrageenases play a role in marine ι-carrageenan degradation, and their enzymatic hydrolysates are thought to be excellent antioxidants. In this study, we identified a new ι-carrageenase, encoded by cgiF, in psychrophilic bacterium Flavobacterium sp. YS-80-122. The deduced ι-carrageenase, CgiF, belongs to glycoside hydrolase family 82 and shows less than 40% amino acid identity with characterized ι-carrageenases. The activity of recombinant CgiF peaked at 30°C (1,207.8U/mg). Notably, CgiF is a cold-adapted ι-carrageenase, which showed 36.5% and 57% of the maximum activity at 10°C and 15°C, respectively. In addition, it is a thermo-tolerant enzyme that recovered 58.2% of its initial activity after heat shock. Furthermore, although the activity of CgiF was enhanced by NaCl, the enzyme is active in absence of NaCl. This study also shows that CgiF is an endo-type ι-carrageenase that hydrolyzes β-1,4-linkages of ι-carrageenan, yielding neo-ι-carratetraose as the main product. Its cold-adaptation, thermo-tolerance, NaCl independence and high neo-ι-carratetraose yield make CgiF an excellent candidate for industrial applications in production of ι-carrageen oligosaccharides from seaweed polysaccharides.

Serralysin inhibitors have been proposed as potent drugs against many diseases and may help to prevent further development of antibiotic-resistant pathogenic bacteria. In this study, a novel serralysin inhibitor gene, lupI, was cloned from the marine bacterium Flavobacterium sp. YS-80-122 and expressed in Escherichia coli. The deduced serralysin inhibitor, LupI, shows

The metalloproteinase MP belongs to the serralysin family, which is involved in important functions such as nutrient acquisition and infection pathogenesis. Serralysin proteases in highly purified form are commonly used at the industrial level with several purposes. In this study, we set up an efficient and rapid purification protocol for MP using a p-aminobenzamidine-modified affinity chromatography. The affinity medium was synthesized by using p-aminobenzamidine as affinity ligand immobilized via cyanuric chloride spacer to Sepharose 6B sorbent carrier. According to the adsorption analysis, the dissociation constant Kd and theoretical maximum adsorption Qmax of this medium were 24.2 μg/mL and 24.1 mg/g wet sorbent, respectively. The purity of MP was assessed by a high-performance liquid chromatography on a TSK3000SW column and sodium dodecyl sulfate polyacrylamide gel electrophoresis, revealing values of 98.7 and ∼98%, respectively. The specific activity of purified MP was 95.6 U/mg, which is similar to values obtained through traditional purification protocols. In conclusion, our protocol could be easily employed for the rapid isolation of MP with high purity, and could be implemented for other serralysin family proteases.

As an important medical enzyme, &beta, galactosidases catalyze transgalactosylation to form prebiotic Galacto-Oligosaccharides (GOS) that assist in improving the effect of intestinal flora on human health. In this study, a new glycoside hydrolase family 2 (GH2) &beta, galactosidase-encoding gene, galA, was cloned from the Antarctic bacterium Alteromonas sp. ANT48 and expressed in Escherichia coli. The recombinant &beta, galactosidase GalA was optimal at pH 7.0 and stable at pH 6.6&ndash, 7.0, which are conditions suitable for the dairy environment. Meanwhile, GalA showed most activity at 50 &deg, C and retained more than 80% of its initial activity below 40 &deg, C, which makes this enzyme stable in normal conditions. Molecular docking with lactose suggested that GalA could efficiently recognize and catalyze lactose substrates. Furthermore, GalA efficiently catalyzed lactose degradation and transgalactosylation of GOS in milk. A total of 90.6% of the lactose in milk could be hydrolyzed within 15 min at 40 &deg, C, and the GOS yield reached 30.9%. These properties make GalA a good candidate for further applications.

Chitosanase plays an important role in the production of chitooligosaccharides (CHOS), which possess various biological activities. Herein, a glycoside hydrolase (GH) family 46 chitosanase-encoding gene, csnB, was cloned from marine bacterium Bacillus sp. BY01 and heterologously expressed in Escherichia coli. The recombinant chitosanase, CsnB, was optimally active at 35 &deg, C and pH 5.0. It was also revealed to be a cold-adapted enzyme, maintaining 39.5% and 40.4% of its maximum activity at 0 and 10 &deg, C, respectively. Meanwhile, CsnB showed wide pH-stability within the range of pH 3.0 to 7.0. Then, an improved reaction condition was built to enhance its thermostability with a final glycerol volume concentration of 20%. Moreover, CsnB was determined to be an endo-type chitosanase, yielding chitosan disaccharides and trisaccharides as the main products. Overall, CsnB provides a new choice for enzymatic CHOS production.

Alginate oligosaccharides (AOS) show versatile bioactivities. Although various alginate lyases have been characterized, enzymes with special characteristics are still rare. In this study, a polysaccharide lyase family 7 (PL7) alginate lyase-encoding gene, aly08, was cloned from the marine bacterium Vibrio sp. SY01 and expressed in Escherichia coli. The purified alginate lyase Aly08, with a molecular weight of 35 kDa, showed a specific activity of 841 U/mg at its optimal pH (pH 8.35) and temperature (45 &deg, C). Aly08 showed good pH-stability, as it remained more than 80% of its initial activity in a wide pH range (4.0&ndash, 10.0). Aly08 was also a thermo-tolerant enzyme that recovered 70.8% of its initial activity following heat shock treatment for 5 min. This study also demonstrated that Aly08 is a polyG-preferred enzyme. Furthermore, Aly08 degraded alginates into disaccharides and trisaccharides in an endo-manner. Its thermo-tolerance and pH-stable properties make Aly08 a good candidate for further applications.

Chitooligosaccharides (CHOS) have gained increasing attention because of their important biological activities. Enhancing the efficiency of CHOS production essentially requires screening of novel chitosanase with unique characteristics. Therefore, a rapid and efficient one-step affinity purification procedure plays important roles in screening native chitosanases. In this study, we report the design and synthesis of affinity resin for efficient purification of native chitosanases without any tags, using chitodisaccharides (CHDS) as an affinity ligand, to couple with Sepharose 6B via a spacer, cyanuric chloride. Based on the CHDS-modified affinity resin, a one-step affinity purification method was developed and optimized, and then applied to purify three typical glycoside hydrolase (GH) families: 46, 75, and 80 chitosanase. The three purified chitosanases were homogeneous with purities of greater than 95% and bioactivity recovery of more than 40%. Moreover, we also developed a rapid and efficient affinity purification procedure, in which tag-free chitosanase could be directly purified from supernatant of bacterial culture. The purified chitosanases samples using such a procedure had apparent homogeneity, with more than 90% purity and 10&ndash, 50% yield. The novel purification methods established in this work can be applied to purify native chitosanases in various scales, such as laboratory and industrial scales.

κ-Carrageenase cleaves the β-(1–4) linkages of κ-carrageenan into κ-carrageenan oligosaccharides (κ-COS), which exhibit various biological activities. In this study, a glycoside hydrolase (GH) family 16 κ-carrageenase gene, cgkA, was cloned from the marine bacterium Vibrio sp. SY01 and secretory expressed in a food-grade host, Yarrowia lipolytica. The specific activity of the purified CgkA was 12.5 U/mg. Determination of biochemical properties showed that CgkA was a thermo-tolerant enzyme, and 59.9% of the initial enzyme activity was recovered by immediately placing the sample at 20 °C for 30 min after enzymatic inactivation by boiling for 5 min. The recombinant CgkA was an endo-type enzyme, the main enzymatic product was κ-carradiaose (accounting for 87.6% of total products), and κ-carratetraose was the minimum substrate. Additionally, in vitro and in vivo analyses indicated that enzymatic κ-carradiaose possesses anti-oxidant activity. These features make CgkA as a promising candidate for biotechnological applications in the production of anti-oxidant κ-COS.

In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu-iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14-atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high-performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases.

κ-carrageenan oligosaccharides exhibit various biological activities. Enzymatic degradation by κ-carrageenase is safe and controllable. Therefore, κ-carrageenases have captured more and more attentions. In this study, a κ-carrageenase encoding gene, cgkX, was cloned from Pseudoalteromonas sp. QY203 with degenerate and inverse PCR. It comprised an ORF of 1194 bp in length, encoding a protein with 397 amino acid residues. CgkX is a new member of glycoside hydrolase family 16. The deduced amino acid sequence shared a high similarity with CgkX of Pseudoalteromonas κ-carrageenase; however, the recombinant CgkX showed different biochemical characteristics. The recombinant enzyme was most active at pH 7.0 and 55°C in the presence of 300 mmol L−1 NaCl. It was stable in a broad range of acidity ranging from pH 3.0 to pH 10.0 when temperature was below 40°C. More than 80% of its activity was maintained after being incubated at pH 3.6–10.0 and 4°C for 24 h. CgkX retained more than 90% of activity after being incubated at 40°C for 1 h. EDTA and SDS (1 mmol L−1) did not inhibit its activity. CgkX hydrolyzed κ-carrageenan into disaccharide and tetrasaccharide as an endo-cleaver. All these characteristics demonstrated that CgkX is applicable to both κ-carrageenan oligosaccharide production and κ-carrageenase structure-function research.

Alginate lyase played an important role in brown algae degradation, and its enzymatic degradation products showed various biological activities. Although many alginate lyases have been characterized, the enzymes with special characterizations are still rather rare. In this study, a new alginate lyase gene, tsaly6A, has been cloned from marine bacterium Thalassomonas sp. LD5, and expressed in Escherichia coli. The deduced alginate lyase, TsAly6A, belonged to the polysaccharide lyase (PL) family 6 and showed the highest amino acid identity (63%) with an exo-type oligoalginate lyase AlyGC. However, this study showed that TsAly6A was an endo-type enzyme yielding alginate trisaccharides (64.5%) as the main products. Compared with other alginate lyases, TsAly6A showed high trisaccharide-yielding levels. Meanwhile, TsAly6A showed the specific activity of 15,960 U/μmol at its optimal pH (pH 8.0) and temperature (35 °C). In addition, TsAly6A was a cold-adapted, salt-activated and metal ions-resisted alginate lyase, which will enable it to perform high activity in the solution containing various ions. Its cold-adaptation, metal ions-tolerance and high trisaccharides yields make TsAly6A an excellent candidate for industrial applications.

A new κ-carrageenase gene cgkS was cloned from marine bacterium Shewanella sp. Kz7 by using degenerate and site-finding PCR. The gene was comprised of an open reading frame of 1224 bp, encoding 407 amino acid residues, with a signal peptide of 24 residues. Based on the deduced amino acid sequence, the κ-carrageenase CgkS was classified into the Glycoside Hydrolase family 16. The cgkS gene was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity with a specific activity of 716.8 U mg−1 and a yield of 69%. Recombinant CgkS was most active at 45°C and pH 8.0. It was stable at pH 6.0–9.0 and below 30°C. The enzyme did not require NaCl for activity, although its activity was enhanced by NaCl. CgkS degraded κ-carrageenan in an endo-fashion releasing tetrasaccharides and disaccharides as main hydrolysis products.

The purpose of this study was to find a cold-adapted and surfactant-stable alginate lyase as a candidate for biotechnological and industrial applications. The gene for a new alginate lyase, AlyL1, from Agarivorans sp. L11 was cloned and expressed in Escherichia coli. The recombinant AlyL1 was most active at 40°C (1,370 U/mg). It was a cold-adapted alginate lyase, which showed 54.5% and 72.1% of maximum activity at 15°C and 20°C, respectively. AlyL1 was an alkaliphilic enzyme and most active at pH 8.6. In addition, it showed high stability in the presence of various surfactants at a high concentration (from 0.1% to 1% (w/v)). AlyL1 was an endo-type alginate lyase that degraded both polyM and polyG blocks, yielding disaccharides and trisaccharides as the main products. This is the first report of the cloning and functional expression of a cold-adapted and surfactant-stable alginate lyase. AlyL1 might be an interesting candidate for biotechnological and industrial applications.

Unsaturated alginate disaccharides (UADs), enzymatically derived from the degradation of alginate polymers, are considered powerful antioxidants. In this study, a new high UAD-producing alginate lyase, AlySY08, has been purified from the marine bacterium Vibrio sp. SY08. AlySY08, with a molecular weight of about 33 kDa and a specific activity of 1070.2 U/mg, showed the highest activity at 40 °C in phosphate buffer at pH 7.6. The enzyme was stable over a broad pH range (6.0–9.0) and retained about 75% activity after incubation at 40 °C for 2 h. Moreover, the enzyme was active in the absence of salt ions and its activity was enhanced by the addition of NaCl and KCl. AlySY08 resulted in an endo-type alginate lyase that degrades both polyM and polyG blocks, yielding UADs as the main product (81.4% of total products). All these features made AlySY08 a promising candidate for industrial applications in the production of antioxidants from alginate polysaccharides.

Obesity and its related complications have become one of the leading problems affecting human health. However, current anti-obesity treatments are limited by high cost and numerous adverse effects. In this study, we investigated the use of a non-toxic green food additive, known as unsaturated alginate oligosaccharides (UAOS) from the enzymatic degradation of Laminaria japonicais, which showed effective anti-obesity effects in a high-fat diet (HFD) mouse model. Compared with acid hydrolyzed saturated alginate oligosaccharides (SAOS), UAOS significantly reduced body weight, serum lipid, including triacylglycerol (TG), total cholesterol (TC) and free fatty acids (FFA), liver weight, liver TG and TC, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels, adipose mass, reactive oxygen species (ROS) formation, and accumulation induced in HFD mice. Moreover, the structural differences in &beta, d-mannuronate (M) and its C5 epimer &alpha, l-guluronate (G) did not cause significant functional differences. Meanwhile, UAOS significantly increased both AMP-activated protein kinase &alpha, (AMPK&alpha, ) and acetyl-CoA carboxylase (ACC) phosphorylation in adipocytes, which indicated that UAOS had an anti-obesity effect mainly through AMPK signaling. Our results indicate that UAOS has the potential for further development as an adjuvant treatment for many metabolic diseases such as fatty liver, hypertriglyceridemia, and possibly diabetes.

A new extracellular κ-carrageenase, namely CgkP, 34.0 kDa in molecular weight, was purified from Pseudoalteromonas sp. QY203. CgkP showed relatively high activity at acidities ranging from pH6.0 to pH9.0 and temperatures ranging from 30°C to 50°C with the highest activity at 45°C and pH7.2. Sodium chloride increased its activity markedly, and KCl increased its activity slightly. The divalent and trivalent metal ions including Cu2+, Ni2+, Zn2+, Mn2+, Al3+ and Fe3+ significantly inhibited its activity, while Mg2+ did not. CgkP remained 70% of original activity after being incubated at 40°C for 48 h, and remained 80% of the activity after being incubated at 45°C for 1 h. It exhibited endo-κ-carrageenase activity, mainly depolymerizing the κ-carrageenan into disaccharide and tetrasaccharide. CgkP was more thermostable than most of previously reported κ-carrageenases with a potential of being used in industry.

Two new alginate lyase genes, oalY1 and oalY2 , have been cloned from the newly isolated marine bacterium Halomonas sp. QY114 and expressed in Escherichia coli . The deduced alginate lyases, OalY1 and OalY2, belonged to polysaccharide lyase (PL) family 17 and showed less than 45% amino acid identity with all of the characterized oligoalginate lyases. OalY1 and OalY2 exhibited the highest activities at 45°C and 50°C respectively. Both of them showed more than 50% of the highest activity at 60°C, and 20% at 80°C. In addition, they were salt-dependent and salt-tolerant since both of them showed the highest activity in the presence of 0.5 M NaCl and preserved 63% and 68% of activity in the presence of 3 M NaCl. Significantly, OalY1 and OalY2 could degrade both polyM and polyG blocks into alginate monosaccharides in an exo-lytic type, indicating that they are bifunctional alginate lyases. In conclusion, our study indicated that OalY1 and OalY2 are good candidates for alginate saccharification application, and the salt-tolerance may present an exciting new concept for biofuel production from native brown seaweeds.

Alginate, the most abundant carbohydrate in brown macroalgae, is widely used in the food and pharmaceutical industries. Recently, alginate has attracted increasing attention, as it may serve as an alternative biomass for the production of biofuel. The degradation of alginate into monomeric units is the prerequisite for bioethanol production. All known oligoalginate lyases belong to the polysaccharide lyase (PL) family 7, 14, 15 and 17, and most of them preferred to degrade the polyM blocks to yield 4-deoxy-l-erythro-5-hexoseulose uronic acid as the primary product. In this study, we cloned an oligoalginate lyase gene, oalS6, from Shewanella sp. Kz7 and expressed it in Escherichia coli. The PL family 6 oligoalginate lyase (OalS6) has no significant sequence similarity with other known oligoalginate lyases. OalS6 contains a chondroitinase-like domain and was assigned to the PL family 6. This lyase is an exo-type oligoalginate lyase and prefer to depolymerize polyG block into 2, 4, 5, 6-tetrahydroxytetrahydro-2H-pyran-2-carboxylic acid. All of these results indicate that OalS6 is a novel oligoalginate lyase that is structurally and functionally different from other known oligoalginate lyases. This finding provides new insights into the development of biofuel processing biotechnologies from seaweed.

The carbohydrate-binding module (CBM) in polysaccharide hydrolases plays a key role in the hydrolysis of cellulose, xylan and chitin. However, the function of CBM in alginate lyases has not been elucidated. A new alginate lyase gene, alyL2, was cloned from the marine bacterium Agarivorans sp. L11 by using degenerate and site-finding PCR. The alginate lyase, AlyL2, contained an N-terminal CBM13 and a C-terminal catalytic family 7 polysaccharide lyase (PL7) module. To better understand the function of CBM13 in alginate lyase AlyL2, the full-length enzyme (AlyL2-FL) and its catalytic module (AlyL2-CM) were expressed in Escherichia coli and characterized. The specific activity and catalytic efficiency of AlyL2-FL were approximately twice those of AlyL2-CM. The half-lives of AlyL2-FL were 4.7-6.6 times those of AlyL2-CM at 30-50°C. In addition, the presence of CBM13 in AlyL2 changed its substrate preference and increased the percentage of disaccharides from 50.5% to 64.6% in the total products. This first report of the function of CBM13 in alginate lyase provides new insights into the degradation of alginate by marine microorganisms.

Is to report an oligoalginate lyase with high enzymatic activity and high-level expression. Using site-finding PCR and degenerate PCR, a gene (designated oalS17) encoding a new oligoalginate lyase was cloned from Shewanella sp. Kz7 and expressed in Escherichia coli. The gene consisted of 2,292 bp with deduced amino acid size of 763 including a putative signal peptide of 44 amino acid residues belonging to polysaccharide lyase (PL) family 17. The recombinant protein was most active at 50 °C and pH 6.2 in 50 mM phosphate buffer. It degraded alginate more efficiently than polyM and polyG block into a monomeric sugar acid, with a specific activity of 32 U mg(-1) toward alginate, 24 U mg(-1) toward polyM and 5 U mg(-1) toward polyG. With the high-level expression and high enzymatic activity, the recombinant oligoalginate lyase OalS17 could be a potential enzyme for further research on alginate saccharification and biofuels production.

Metalloproteases are emerging as useful agents in the treatment of many diseases including arthritis, cancer, cardiovascular diseases, and fibrosis. Studies that could shed light on the metalloprotease pharmaceutical applications require the pure enzyme. Here, we reported the structure-based design and synthesis of the affinity medium for the efficient purification of metalloprotease using the 4-aminophenylboronic acid (4-APBA) as affinity ligand, which was coupled with Sepharose 6B via cyanuric chloride as spacer. The molecular docking analysis showed that the boron atom was interacting with the hydroxyl group of Ser176 residue, whereas the hydroxyl group of the boronic moiety is oriented toward Leu175 and His177 residues. In addition to the covalent bond between the boron atom and hydroxyl group of Ser176, the spacer between boronic acid derivatives and medium beads contributes to the formation of an enzyme-medium complex. With this synthesized medium, we developed and optimized a one-step purification procedure and applied it for the affinity purification of metalloproteases from three commercial enzyme products. The native metalloproteases were purified to high homogeneity with more than 95% purity. The novel purification method developed in this work provides new opportunities for scientific, industrial and pharmaceutical projects.