Efficient preparation of alginate oligosaccharides by using alginate lyases and evaluation of the development promoting effects on Brassica napus L. in saline-alkali environment.

Enzymatic degradation of alginate for the preparation of alginate oligosaccharides (AOS) is currently receiving significant attention in the field. AOS has been shown to promote crop growth and improve plant resistance to abiotic stresses. In this study, two PL6 family alginate lyases, AlyRmA and AlyRmB, were expressed and characterized. These enzymes demonstrate exceptional activity and stable thermophilicity compared to other known alginate lyases. AlyRmA (8855.34 U/mg) and AlyRmB (7879.44 U/mg) exhibited excellent degradation activity towards sodium alginate even at high temperatures (70 °C). The AlyRmA and AlyRmB were characterized and utilized to efficiently produce AOS. The study investigated the promotional effect of AOS on the growth of Brassica napus L. seedlings in a saline-alkaline environment. The results of this study demonstrate the high activity and thermal stability of AlyRmA and AlyRmB, highlighting their potential in the preparation of AOS. Moreover, the application of AOS prepared by AlyRmB could enhance the resistance of Brassica napus L. to saline-alkali environments, thereby broadening the potential applications of AOS.

Immobilization of Thermomyces lanuginosus lipase in a novel polysaccharide-based hydrogel by a two-step crosslinking method and its use in the lauroylation of α-arbutin.

The Thermomyces lanuginosus lipase (TLLs) was successfully immobilized within a novel hydrogel matrix through a two-step crosslinking method. TLLs were initially crosslinked through the Schiff base reaction by oxidized carboxymethyl cellulose (OCMC). The water-soluble OCMC@TLLs complex was subsequently crosslinked by carboxymethyl chitosan (CMCSH) in a microfluidic apparatus to form the CMCHS/OCMC@TLLs microspheres. The CD (Circular Dichroism, CD) and FT-IR (Fourier Transform infrared spectroscopy, FT-IR) spectra demonstrated that the crosslinking of TLLs with OCMC resulted in a less significant impact on their structure compared to that with glutaraldehyde. CMCHS/OCMC@TLLs showed decreased catalytic performance due to the mass transfer resistance, while its thermal stability was greatly improved. The CMCHS/OCMC@TLLs were used to catalyze the lauroylation of arbutin in tetrahydrofuran. After 12 h of reaction under optimal conditions, the yield of 6'-O-lauryl arbutin reached an impressive 92.12%. The prepared 6'-O-lauryl arbutin has high lipophilicity and exhibits similar tyrosinase inhibitory activity and higher antioxidant activity compared to its parent compound.

Separation, purification and structural characterization of marine oligosaccharides: A comprehensive and systematic review of chromatographic methods.

Marine oligosaccharides have now been applied in a wide range of industry due to various kinds of physiological activities. However, the oligosaccharides with different polymeric degrees (Dps) differed in physiological activities and applicable fields. So it is promising and essential to separate, purify and structurally characterize these oligosaccharides for understanding their structure-function relationship. This review will summarize the lasted developments in the separation, purification and structural characterization of marine oligosaccharides, including the alginate oligosaccharides, carrageenan oligosaccharides, agar oligosaccharides, chitin oligosaccharides and chitosan oligosaccharides, emphasizing the successful examples of methods for separation and purification. Furthermore, an outlook for preparation of functional oligosaccharides in food biotechnology and agriculture fields is also included. This comprehensive review could definitely promote the utilization of marine functional polysaccharides for food and agriculture.

Biochemical characterization of a new ulvan lyase and its applicability in utilization of ulvan and preparation of ulva oligosaccharides.

Ulva is globally distributed specie and has a high economic value. Ulvan is one of the main active substances in Ulva, which has a variety of biological properties. Ulvan lyase degrades ulvan through a ß-elimination mechanism which cleaves the ß-glycosidic bond between Rha3S and GlcA or IdoA. The complex monosaccharide composition of ulvan makes it promising for use in food and pharmaceutical applications. This thesis explores a putative ulvan lyase from Alteromonas sp. KUL_42. We expressed and purified the protein, performed a series of characterizations and signal peptide had been removed. The results showed that the protein molecular weight of ULA-2 was 53.97 kDa, and it had the highest catalytic activity at 45 °C and pH 8.0 in Tris-HCl buffer. The Km and Vmax values were 2.24 mg · mL-1 and 2.048 µmol · min-1 · mL-1, respectively. The activity of ULA-2 was able to maintain more than 80% at 20 ~ 30 °C. ESI-MS analysis showed that the primary end-products were mainly disaccharides to tetrasaccharides. The study of ULA-2 enriches the ulvan lyase library, promotes the development and high-value utilization of Ulva resources, and facilitates further research applications of ulvan lyase in ulva oligosaccharides.

Biochemical characterization and elucidation of the action mode of a GH16 family κ-carrageenase for efficient preparation of carrageenan oligosaccharides.

κ-Carrageenan oligosaccharides have a variety of biological activities. Degradation of κ-carrageenan by κ-carrageenase leads to degradation products with different degrees of polymerization (DPs). A novel gene (CecgkA) encoding a new κ-carrageenase was cloned from Colwellia echini and heterologously expressed in Escherichia coli BL21 (DE3). The enzyme is 1104 bp in length, encodes 367 amino acid residues and has a molecular weight of 41.30 kDa. Multiple alignment analysis showed that CeCgkA belongs to the glycoside hydrolase (GH16) family and has the highest homology with the κ-carrageenase of Rhodopirellula maiorica SM1, with 58% homology. The CeCgkA showed maximum activity (453.15 U/mg) at pH 8.0 and 35 °C. Determination of biochemical properties showed that CeCgkA was a thermal recovery enzyme, and 51.6% of the initial enzyme activity was recovered by immediately placing the sample at 35 °C for 60 min after enzymatic inactivation by boiling for 10 min. K+, Na+, and EDTA had an activating effect on the enzyme activity, while Ni2+, Cu2+, and Zn2+ inhibited the activity of the enzyme. In addition, TLC and ESI-MS analysis showed that the maximum recognition unit of CecgkA was decasaccharide and that the main degradation products were disaccharides, tetrasaccharides and hexasaccharides, indicating that the enzyme is an endo-type carrageenase.

Directed preparation, structure-activity relationship and applications of alginate oligosaccharides with specific structures: A systematic review.

The alginate oligosaccharides (AOS) possess versatile activities (such as antioxidant, anti-inflammatory, antitumor, and immune-regulatory activities) and have been the research topic in marine bioresource utilization fields. The degree of polymerization (DP) and the ß-D-mannuronic acid (M)/α-L-guluronic acid (G)-units ratio strongly affect the functionality of AOS. Therefore, directed preparation of AOS with specific structures is essential for expanding the applications of alginate polysaccharides and has been the research topic in the marine bioresource field. Alginate lyases could efficiently degrade alginate and specifically produce AOS with specific structures. Therefore, enzymatic preparation of AOS with specific structures has drawn increasing attention. Herein, we systematically summarized the current research progress on the structure-function relation of AOS and focuses on the application of the enzymatic properties of alginate lyase to the specific preparation of various types of AOS. At the same time, current challenges and opportunities for AOS applications are presented to guide and improve the preparation and application of AOS in the future.

Recent advances in the production, properties and applications of alginate oligosaccharides - a mini review.

Alginate oligosaccharides (AOS) made from the degradation of alginate, to some extent, makes up for the poor solubility and bioavailability of alginate as a macromolecular substance and possess several beneficial biological activities that are absent in alginate. These properties include prebiotic, glycolipid regulatory, immunomodulatory, antimicrobial, antioxidant, anti-tumor, promoting plant growth and other activities. Consequently, AOS has significant potential for use in the agricultural, biomedical, and food industries, and has been the focus of research in the field of marine biological resources. This review comprehensively covers methods (physical, chemical, and enzymatic methods) for the production of AOS from alginate. More importantly, this paper reviews recent advances in the biological activity and potentially industrial and therapeutic applications of AOS, providing a reference for future research and applications of AOS.

Fabrication of carboxymethyl chitosan/oxidized carboxymethyl cellulose composite film and its assessment for coating preservation of strawberry.

In the current study, a novel chitosan-based composite, carboxymethyl chitosan (CMCHS)/oxidized carboxymethyl cellulose (OCMC) was fabricated and characterized. The composite film (CMCHS 1.5%w/v + OCMC 0.8%w/v) was more uniform and had better tensile properties, UV blocking, water vapor permeability, and antifungal properties than pure CMCHS film. Preservation experiments showed that the CMCHS/OCMC film was more efficient for retaining the quality decrease of strawberry during storage. By the end of 7 days' storage, the hardness, contents of organic acid, soluble solids, and reducing sugar of coated strawberries were increased by 35.1%, 38.5%, 14.1%, and 3.5%, respectively, compared to the control group; and the decay rate of strawberries in CMCHS/OCMC group also dropped to 36%, about 42% decrease than that in control, suggesting the promising application of CMCHS/OCMC composite in coating preservation.

Purification and biochemical characterization of a novel chitosanase cloned from the gene of Kitasatospora setae KM-6054 and its application in the production of chitooligosaccharides.

Chitosanase could degrade chitosan efficiently under mild conditions to prepare chitosan oligosaccharides (COSs). COS possesses versatile physiological activities and has wide application prospects in food, pharmaceutical and cosmetic fields. Herein, a new glycoside hydrolase (GH) family 46 chitosanase (CscB) was cloned from Kitasatospora setae KM-6054 and heterologously expressed in Escherichia coli. The recombinant chitosanase CscB was purified by Ni-charged magnetic beads and showed a relative molecular weight of 29.19 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). CscB showed the maximal activity (1094.21 U/mg) at pH 6.0 and 30 °C. It was revealed that CscB is a cold-adapted enzyme. CscB was determined to be an endo-type chitosanase with a polymerization degree of the final product mainly in the range of 2-4. This new cold-adapted chitosanase provides an efficient enzyme tool for clean production of COSs.

Efficient Degradation of Alginate and Preparation of Alginate Oligosaccharides by a Novel Biofunctional Alginate Lyase with High Activity and Excellent Thermophilic Features.

The enzymatic degradation of seaweed polysaccharides is gaining interest for its potential in the production of functional oligosaccharides and fermentable sugars. Herein, a novel alginate lyase, AlyRm3, was cloned from a marine strain, Rhodothermus marinus DSM 4252. The AlyRm3 showed optimal activity (37,315.08 U/mg) at 70 °C and pH 8.0, with the sodium alginate used as a substrate. Noticeably, AlyRm3 was stable at 65 °C and also exhibited 30% of maximal activity at 90 °C. These results indicated that AlyRm3 is a thermophilic alginate lyase that efficiently degrades alginate at high industrial temperatures (>60 °C). The FPLC and ESI-MS analyses suggested that AlyRm3 primarily released disaccharides and trisaccharides from the alginate, polyM, and polyG in an endolytic manner. In the saccharification process of sodium alginate (0.5%, w/v), the AlyRm3 yielded numerous reducing sugars (1.73 g/L) after 2 h of reaction. These results indicated that AlyRm3 has a high enzymatic capacity for saccharifying the alginate, and could be used to saccharify the alginate biomass before the main fermentation process for biofuels. These properties make AlyRm3 a valuable candidate for both fundamental research and industrial applications.

A novel alginate lyase and its domain functions for the preparation of unsaturated monosaccharides.

Brown algae are considered promising crops for the production of sustainable biofuels. However, the commercial application has been limited by lack of efficient methods for converting alginate into fermentable sugars. Herein, we cloned and characterized a novel alginate lyase AlyPL17 from Pedobacter hainanensis NJ-02. It possessed outstanding catalytic efficiency toward polymannuronic acid (polyM), polyguluronic acid (polyG), and alginate sodium, with kcat of 39.42 ± 1.9 s-1, 32.53 ± 0.88 s-1, and 38.30 ± 2.12 s-1, respectively. AlyPL17 showed maximum activity at 45 °C and pH 9.0. The domain truncation did not change the optimal temperature and optimal pH, but greatly reduced the activity. In addition, AlyPL17 degrades alginate through the cooperative action of two structural domains in an exolytic mode. The minimal degradation substrate of AlyPL17 is a disaccharide. Furthermore, AlyPL17 and AlyPL6 can synergistically degrade alginate to prepare unsaturated monosaccharides that can be converted to 4-deoxy-L-erythron-5-hexoseuloseuronate acid (DEH). DEH is reduced to KDG by DEH reductase (Sdr), which enters the Entner-Doudoroff (ED) pathway as a common metabolite and is converted to bioethanol. KEY POINTS: • Biochemical characterization of alginate lyase from Pedobacter hainanensis NJ-02 and its truncated form. • Degradation patterns of AlyPL17 and the role of its domains in product distribution and mode of action. • Potential of synergistic degradation system for efficient preparation of unsaturated monosaccharides.

Biochemical characterization and elucidation the degradation pattern of a new cold-adapted and Ca2+ activated alginate lyase for efficient preparation of alginate oligosaccharides.

Alginate lyase as the excellent tool enzyme could produce alginate oligosaccharides under mild reaction conditions and good specificity. Herein, the new Ca2+ activated alginate lyase gene (TAPL7B) with metal ions stability and cold-adapted was cloned and expressed heterologously from Thalassotalea algicola. TAPL7B was identified as a polysaccharide lyases family 7 (PL7) alginate lyase with endolytic activity. TAPL7B is a polyM-preferred and cold-adapted enzyme with an optimal temperature of 20 °C and pH 11.0. The pH stability analysis indicated that TAPL7B possessed broad pH tolerant range (4.0-12.0) and preferred alkaline environments. Interestingly, the enzymatic activity of TAPL7B was greatly activated by Ca2+ and it increased 9-fold in the presence of 1.2 mM Ca2+, which is rare in other alginate lyases. The activity of TAPL7B is not only activated by Ca2+, but also by some other metal ions (such as K+, Na+, Ni+, Cu2+, Mg2+, Zn2+, Co2+, Fe3+). The results of fast protein liquid chromatography (FPLC) and electrospray ionization mass spectrometry (ESI-MS) indicate that TAPL7B adopts an endo-action mode on all three substrates, and the products are all oligosaccharides with a degree of polymerization of 2-6. Therefore, the cold adaptability, broad pH stability, excellent metal ions stability, and Ca2+ activation of TAPL7B make it a potential industrial biocatalyst for the preparation of alginate oligosaccharides.

Ulvan and Ulva oligosaccharides: a systematic review of structure, preparation, biological activities and applications.

Ulva is one of the main green algae causing green tide disasters. Ulvan is the primarily component polysaccharide of the cell wall of Ulva and its complex structure and monosaccharide composition resulted in various biological activities. However, the high-value and effective utilization of extracted ulvan have been obstructed by limitations ranging from large molecular weight and low solubility to poor bioavailability. Ulva oligosaccharide obtained by degrading ulvan can not only ideally retain the various biological activities of ulvan very well but also effectively solve the problems of low solubility and poor bioavailability. The preparation and biological activity studies of ulvan and Ulva oligosaccharides have become a hot spot in the field of marine biological resources development research. At present, the comprehensive reviews of ulvan and Ulva oligosaccharides are still scarce. What are overviewed in this paper are the chemical composition, structure, extraction, and purification of ulvan and Ulva oligosaccharides, where research progress on the biological activities of ulvan and Ulva oligosaccharides is summarized and prospected. A theoretical and practical basis has been provided for further research on ulvan and Ulva oligosaccharides, as well as the high-value development and effective utilization of marine algae resources.

Biochemical Characterization and Elucidation of the Hybrid Action Mode of a New Psychrophilic and Cold-Tolerant Alginate Lyase for Efficient Preparation of Alginate Oligosaccharides.

Alginate lyases with unique biochemical properties have irreplaceable value in food and biotechnology industries. Herein, the first new hybrid action mode Thalassotalea algicola-derived alginate lyase gene (TAPL7A) with both psychrophilic and cold-tolerance was cloned and expressed heterologously in E. coli. With the highest sequence identity (43%) to the exolytic alginate lyase AlyA5 obtained from Zobellia galactanivorans, TAPL7A was identified as a new polysaccharide lyases family 7 (PL7) alginate lyase. TAPL7A has broad substrate tolerance with specific activities of 4186.1 U/mg, 2494.8 U/mg, 2314.9 U/mg for polyM, polyG, and sodium alginate, respectively. Biochemical characterization of TAPL7A showed optimal activity at 15 °C, pH 8.0. Interestingly, TAPL7A exhibits both extreme psychrophilic and cold tolerance, which other cold-adapted alginate lyase do not possess. In a wide range of 5-30 °C, the activity can reach 80-100%, and the residual activity of more than 70% can still be maintained after 1 h of incubation. Product analysis showed that TAPL7A adopts a hybrid endo/exo-mode on all three substrates. FPLC and ESI-MS confirmed that the final products of TAPL7A are oligosaccharides with degrees of polymerization (Dps) of 1-2. This study provides excellent alginate lyase candidates for low-temperature environmental applications in food, agriculture, medicine and other industries.

Ulva (Enteromorpha) Polysaccharides and Oligosaccharides: A Potential Functional Food Source from Green-Tide-Forming Macroalgae.

The high-valued utilization of Ulva (previously known as Enteromorpha) bioresources has drawn increasing attention due to the periodic blooms of world-wide green tide. The polysaccharide is the main functional component of Ulva and exhibits various physiological activities. The Ulva oligosaccharide as the degradation product of polysaccharide not only possesses some obvious activities, but also possesses excellent solubility and bioavailability. Both Ulva polysaccharides and oligosaccharides hold promising potential in the food industry as new functional foods or food additives. Studies on Ulva polysaccharides and oligosaccharides are increasing and have been the focus of the marine bioresources field. However, the comprehensive review of this topic is still rare and do not cover the recent advances of the structure, isolation, preparation, activity and applications of Ulva polysaccharides and oligosaccharides. This review systematically summarizes and discusses the recent advances of chemical composition, extraction, purification, structure, and activity of Ulva polysaccharides as well as oligosaccharides. In addition, the potential applications as new functional food and food additives have also been considered, and these will definitely expand the applications of Ulva oligosaccharides in the food and medical fields.

Biochemical characterization and immobilization of a novel pectate lyase ErPL2 for efficient preparation of pectin oligosaccharides.

Pectate lyase (ErPL2) from Echinicola rosea JL3085 showed maximal activity at 45 °C and pH 9.0 with 0.6 mM CaCl2. The Km and Vmax values of ErPL2 for polygalacturonic sodium were 2.098 mmol/L and 0.955 mmol/s, respectively. ErPL2 endolytically degraded pectic substances into oligosaccharides with degree of polymerization (DP) 1-5. To improve the thermostability and pH operation range, recombinant ErPL2 was immobilized onto mesoporous titanium oxide particles (MTOPs). MTOPs have abundant hydroxyl groups on the surface, which is a non-toxicity and good biocompatibility carrier. The residual enzyme activity of immobilized ErPL2 at 40 °C increased remarkably from 11% to 91% compared with free enzyme. The operable pH range was extended from 8-9 to 9-11. Surprisingly, the catalytic efficiency of immobilized ErPL2 was about 19 times higher than free enzyme. To our knowledge this is the first attempt of pectate lyase immobilized on MTOPs and it provides a new option for improving the catalytic performance.

Evolving strategies for marine enzyme engineering: recent advances on the molecular modification of alginate lyase.

Alginate, an acidic polysaccharide, is formed by ß-d-mannuronate (M) and α-l-guluronate (G). As a type of polysaccharide lyase, alginate lyase can efficiently degrade alginate into alginate oligosaccharides, having potential applications in the food, medicine, and agriculture fields. However, the application of alginate lyase has been limited due to its low catalytic efficiency and poor temperature stability. In recent years, various structural features of alginate lyase have been determined, resulting in modification strategies that can increase the applicability of alginate lyase, making it important to summarize and discuss the current evidence. In this review, we summarized the structural features and catalytic mechanisms of alginate lyase. Molecular modification strategies, such as rational design, directed evolution, conserved domain recombination, and non-catalytic domain truncation, are also described in detail. Lastly, the application of alginate lyase is discussed. This comprehensive summary can inform future applications of alginate lyases.

[Innovation and exploration of diversified teaching patterns for Biochemical Reaction Engineering under the new engineering education background].

Biochemical Reaction Engineering is a professional discipline with emphasis on both theory and practice. It is the core course in the curriculum provision for students majoring in bioengineering and plays an important role in the cultivation of professional talents. However, there are so much theory knowledge, such as formulas and deduction, that students showed poor efficiency in their study. Herein, to cater the objective command of innovative talents under new education background, and to improve the practical teaching efficiency and the students' enthusiasm as well as the practical innovation capability, the teaching team innovated and explored the teaching pattern, the teaching method as well as the teaching technologies. The teaching efficiency has been remarkably improved by introducing the virtual simulate technology, the micro-lecture, the case teaching pattern attempts and the scientific platform, which can be used as reference by other peers.

Alginate degrading enzymes: an updated comprehensive review of the structure, catalytic mechanism, modification method and applications of alginate lyases.

Alginate, a kind of linear acidic polysaccharide, consists of α-L-guluronate (G) and ß-D-mannuronate (M). Both alginate and its degradation products (alginate oligosaccharides) possess abundant biological activities such as antioxidant activity, antitumor activity, and antimicrobial activity. Therefore, alginate and alginate oligosaccharides have great value in food, pharmaceutical, and agricultural fields. Alginate lyase can degrade alginate into alginate oligosaccharides via the ß-elimination reaction. It plays an important role in marine carbon recycling and the deep utilization of brown algae. Elucidating the structural features of alginate lyase can improve our knowledge of its catalytic mechanisms. With the development of structural analysis techniques, increasing numbers of alginate lyases have been characterized at the structural level. Hence, it is essential and helpful to summarize and discuss the up-to-date findings. In this review, we have summarized progress on the structural features and the catalytic mechanisms of alginate lyases. Furthermore, the molecular modification strategies and the applications of alginate lyases have also been discussed. This comprehensive information should be helpful to expand the applications of alginate lyases.