Research Progress on Preparation and Functions of Alginate Oligosaccharides with Different Structures

Alginate is one of the most abundant marine polysaccharides, which can be degraded into alginate oligosaccharides (AOS) by chemical, physical and biological methods. AOS, linear oligosaccharide composed of guluronic acid and mannuronic acid, have a variety of biological activities such as antimicrobial, anti-inflammatory, and immunoregulatory activities, and these biological activities are closely related to their structural diversity. AOS prepared by different degradation methods have different uronic acid compositions, polymerization degrees and special structures. Therefore, exploring the structure-function relationship of AOS will help to fully understand the activity of AOS and improve its application value. In this paper, the reaction mechanisms of the various methods to prepare AOS, the structures of the resulting products, and the structure-function relationship of AOS are reviewed in order to provide a reference for further research and application of AOS

Expression and Characterization of a New PolyG-Specific Alginate Lyase From Marine Bacterium Microbulbifer sp. Q7

Alginate lyases play an important role in preparation of alginate oligosaccharides. Although a large number of alginate lyases have been characterized, reports on directional preparation of alginate oligosaccharides by alginate lyases are still rather less. Here, a gene alyM encoding a new alginate lyase AlyM was cloned from Microbulbifer sp. Q7 and expressed in Escherichia coli. AlyM exhibited the maximumactivity at pH 7.0 and 55°C and showed special preference to poly-guluronic acid (polyG). Glycine promoted the extracellular secretion of AlyM by 3.6 times. PBS and glycerol significantly improved the thermal stability of AlyM, the enzyme activity remained 75 and 78% after heat-treatment at 45°C for 2 h, respectively. ESI-MS analysis suggested that AlyM mainly produced oligosaccharides with degrees of polymerization (DP) of 2–5. The results of 1H-NMR showed that guluronic acid (G) occupied the reducing end of the end products, indicating that AlyM preferred to degrade the glycosidic bond at the G-X linkage. HPLC analysis showed that the hydrolysis products with a lower degree of polymerization contained more G. Therefore, AlyM shows good potential to produce alginate oligosaccharides with specific M/G ratio and molecular weights

optimizationofkcarrageenaseproductionbypseudoalteromonasspaj5

【目的】本研究的目的是优化Pseudoalteromonas sp．AJ5菌株的培养条件使之产生高活性的胞外κ-卡拉胶酶。【方法】通过富集培养技术从刺参肠道分离出一株卡拉胶降解菌AJ5，该菌株能利用卡拉胶作为惟一碳源和能源。依据形态学和生理学特征及16S rRNA基因序列分析，将该菌株鉴定为假交替单胞菌属（Pseudoalteromonas）。通过单因素试验和正交试验对Pseudoalteromonas sp．AJ5菌株产胞外κ-卡拉胶酶的培养条件进行了优化。【结果】单因素试验结果表明，Pseudoalteromonas sp．AJ5菌株的最佳培养条件为250mL三角瓶装入75mL发酵培养基、摇床转速150r／min、接种量7％、pH8．0。单因素试验和正交试验结果显示该菌株的最佳培养基组成为κ-卡拉胶1g／L、牛肉膏2g／L、NaCl 20g／L、K2HPO4·3H2O 1g／L、MgSO4·7H2O 0．5g／L、MnCl2·4H2O 0．2g／L、FePO4·4H2O 0．01g／L；培养温度为28℃，培养时间为28h。【结论】Pseudoalteromonas sp．AJ5菌株分泌胞外κ-卡拉胶酶，在最佳培养条件下，该菌株的κ-卡拉胶酶活力比优化前提高了4倍

Extracellular expression of a novel β-agarase from Microbulbifer sp. Q7, isolated from the gut of sea cucumber

Abstract A novel endo-type β-agarase was cloned from an agar-degrading bacterium, Microbulbifer sp. Q7 (CGMCC No. 14061), that was isolated from sea cucumber gut. The agarase-encoding gene, ID2563, consisted of 1800 bp that encoded a 599-residue protein with a signal peptide of 19 amino acids. Sequence analysis suggested that the agarase belongs to the GH16 family. The agarase was expressed in Escherichia coli with a total activity of 4.99 U/mL in fermentation medium. The extracellular enzyme activity accounted for 65.73% of the total activity, which indicated that the agarase can be extracellularly secreted using the wild-type signal peptide from Microbulbifer sp. Q7. The agarase exhibited maximal activity at approximately 40 °C and pH 6.0. It was stable between pH 6.0 and pH 9.0, which was a much wider range than most of the reported agarases. The agarase was sensitive to some metal ions (Cu2+, Zn2+ and Fe3+), but was resistant to urea and SDS. The agarase hydrolyzed β-1,4-glycosidic linkages of agarose, primarily yielding neoagarotetraose and neoagarohexaose as the final products. These indicate that this recombinant agarase can be an effective tool for the preparing functional neoagaro-oligosaccharides

Microalgae-Derived Pigments for the Food Industry

In the food industry, manufacturers and customers have paid more attention to natural pigments instead of the synthetic counterparts for their excellent coloring ability and healthy properties. Microalgae are proven as one of the major photosynthesizers of naturally derived commercial pigments, gaining higher value in the global food pigment market. Microalgae-derived pigments, especially chlorophylls, carotenoids and phycobiliproteins, have unique colors and molecular structures, respectively, and show different physiological activities and health effects in the human body. This review provides recent updates on characteristics, application fields, stability in production and extraction processes of chlorophylls, carotenoids and phycobiliproteins to standardize and analyze their commercial production from microalgae. Potential food commodities for the pigment as eco-friendly colorants, nutraceuticals, and antioxidants are summarized for the target products. Then, recent cultivation strategies, metabolic and genomic designs are presented for high pigment productivity. Technical bottlenecks of downstream processing are discussed for improved stability and bioaccessibility during production. The production strategies of microalgal pigments have been exploited to varying degrees, with some already being applied at scale while others remain at the laboratory level. Finally, some factors affecting their global market value and future prospects are proposed. The microalgae-derived pigments have great potential in the food industry due to their high nutritional value and competitive production cost

Characterization of Recombinant Antimicrobial Peptide BMGlv2 Heterologously Expressed in Trichoderma reesei

Antimicrobial peptides (AMPs) serve as alternative candidates for antibiotics and have attracted the attention of a wide range of industries for various purposes, including the prevention and treatment of piglet diarrhea in the swine industry. Escherichia coli, Salmonella, and Clostridium perfringens are the most common pathogens causing piglet diarrhea. In this study, the antimicrobial peptide gloverin2 (BMGlv2), derived from Bombyx mandarina, was explored to determine the efficient prevention effect on bacterial piglet diarrhea. BMGlv2 was heterologously expressed in Trichoderma reesei Tu6, and its antimicrobial properties against the three bacteria were characterized. The results showed that the minimum inhibitory concentrations of the peptide against E. coli ATCC 25922, S. derby ATCC 13076, and C. perfringens CVCC 2032 were 43.75, 43.75, and 21.86 μg/mL, respectively. The antimicrobial activity of BMGlv2 was not severely affected by high temperature, salt ions, and digestive enzymes. It had low hemolytic activity against rabbit red blood cells, indicating its safety for use as a feed additive. Furthermore, the measurements of the leakage of bacterial cell contents and scanning electron microscopy of C. perfringens CVCC 2032 indicated that BMGlv2 exerted antimicrobial activity by destroying the cell membrane. Overall, this study showed the heterologous expression of the antimicrobial peptide BMGlv2 in T. reesei and verified its antimicrobial properties against three common pathogenic bacteria associated with piglet diarrhea, which can provide a reference for the applications of AMPs as an alternative product in industrial agriculture

Characterization of Lipopeptide Biosurfactants Produced by Bacillus licheniformis MB01 from Marine Sediments

Antibiotic resistance has become one of the world's most severe problems because of the overuse of antibiotics. Antibiotic-resistant bacteria are more difficult to kill and more expensive to treat. Researchers have been studied on antibiotic alternatives such as antimicrobial peptides and lipopeptides. A functional bacteria MB01 producing lipopeptides which can be used as bacteriostat was isolated from the Bohai Sea sediments, which had been identified as Bacillus licheniformis by the morphological, physiological, and biochemical identification and 16s rDNA sequence. The lipopeptides produced by MB01 were determined to be cyclic surfactin homologs by LC-ESI-MS structural identification after crude extraction and LH-20 chromatography. [M + H](+) m/z 994, 1008, 1022, and 1036 were all the characteristic molecular weight of surfactin homologs. CID analysis revealed that the molecular structure of the lipopeptides was R-n-Glu(1)-Leu/lle(2)-Leu(3)-Val(4)-Asp(5)-Leu(6)-Leu/Ile(7). The lipopeptides showed well resistance to UV light and the change of pH and temperature

Characterization of Full-Length and Truncated Recombinant κ-Carrageenase Expressed in Pichia pastoris

κ-Carrageenase belongs to glycoside hydrolase family 16 and cleaves the β-(1→4) linkages of κ-carrageenan. In this study, genes encoding the full-length (cgkZ), Por secretion tail-truncated (cgkZΔPst) and carbohydrate binding domain-truncated (cgkZΔCBM) κ-carrageenase proteins were expressed in Pichia pastoris. The copy numbers of gene cgkZ, cgkZΔPst and cgkZΔCBM were 7, 7 and 6, respectively. The enzymatic activities of recombinant enzymes cgkZ, cgkZΔPst and cgkZΔCBM reached 4.68, 5.70, and 3.02 U/mL, respectively, after 120 h of shake flask fermentation at 22°C and pH 6 in the presence of 1 % (v/v) methanol. The molecular weights of recombinant cgkZ, cgkZΔPst, and cgkZΔCBM were approximately 65, 45, and 40 kDa; their Km values were 2.07, 1.85, and 1.04 mg/mL; and they exhibited optimal activity at 45–50°C and pH 6–7. All the recombinant enzymes were stimulated by Na+, Mg2+, Ca2+, and dithiothreitol. The end-products of enzymatic hydrolysis were mainly composed of κ-carrageenan tetrasaccharide and hexasaccharide. The removal of the Por secretion tail of κ-carrageenase promoted the transcription of κ-carrageenase gene, enhancing the specific activity of κ-carrageenase without significantly changing its catalytic properties. Although the transcription level of κ-carrageenase gene after the removal of the carbohydrate binding domain was relatively high, the specific activity of the recombinant enzyme significantly decreased. The comprehensive application of the P. pastoris expression system combined with the rational modification of genes may provide a novel approach for the heterologous expression of various marine enzymes with high activities

Expression, Purification and Characterization of Chondroitinase AC II from Marine Bacterium <i>Arthrobacter</i> sp. CS01

Chondroitinase (ChSase), a type of glycosaminoglycan (GAG) lyase, can degrade chondroitin sulfate (CS) to unsaturate oligosaccharides, with various functional activities. In this study, ChSase AC II from a newly isolated marine bacterium Arthrobacter sp. CS01 was cloned, expressed in Pichia pastoris X33, purified, and characterized. ChSase AC II, with a molecular weight of approximately 100 kDa and a specific activity of 18.7 U/mg, showed the highest activity at 37 &#176;C and pH 6.5 and maintained stability at a broad range of pH (5&#8211;7.5) and temperature (below 35 &#176;C). The enzyme activity was increased in the presence of Mn2+ and was strongly inhibited by Hg2+. Moreover, the kinetic parameters of ChSase AC II against CS-A, CS-C, and HA were determined. TLC and ESI-MS analysis of the degradation products indicated that ChSase AC II displayed an exolytic action mode and completely hydrolyzed three substrates into oligosaccharides with low degrees of polymerization (DPs). All these features make ChSase AC II a promising candidate for the full use of GAG to produce oligosaccharides