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Journal of Oceanology and Limnology 2018年第4期36卷 1304-1314页

作者： XIE Maisheng LI Jiang HE Peiqing LIN Xuezheng Key Laboratory of Marine Bioactive Substances The First Institute of Oceanography State Oceanic Administration (SOA) Qingdao 266061 China Key Laboratory of Marine Natural Products of Qingdao Qingdao 266061 China

In this study, an endolytic alginate lyase, named Al163, was identified, cloned, and characterized from the Antarctic bacterium Pseudoalteromonas sp. NJ-21. Comparative sequence analysis showed that the predicted amino acid sequence encoded by al163 belongs to the polysaccharide lyase 6(PL-6) family and has a molecular mass of about 80 kDa. Recombinant enzyme was purified by Ni-Sepharose affinity chromatography. Recombinant Al163 exhibited maximum activity(258 U/mg) at pH 7.0 and 40℃, and thermal stability assays showed retention of almost 90% activity after incubation at 30℃ for 30 min. Al163 activity was stimulated by Cd^(2+), Ca^(2+), Fe^(3+), and Mn^(2+), but inhibited by Cu^(2+), Si^(2+), Fe^(2+), and Ni^(2+). Thin-layer chromatographic analysis indicated that Al163 degraded sodium alginate, poly M, and poly G, generating disaccharides and trisaccharides as the final products. Only a few bacterial strains that produce a bifunctional alginate lyase have been reported. Our results indicate that recombinant Al163 exhibits broad substrate specificity and its products exhibit low degrees of polymerization. Both properties imply high potential for use of the enzyme in several industrial fields, including cosmetics and pharmaceuticals, based on the high demand for biologically active oligosaccharides.

关键词： Pseudoalteromonas sp. alginate lyase PL-6 family

Cloning,expression and improvement of catalytic activity of alginate lyase by site-directed mutation

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Systems Microbiology and Biomanufacturing 2022年第3期2卷 555-567页

作者： Yu Lu Jianli Zhou Qiuya Gu Wenhua Yang Liu Yang Xiaobin Yu The Key Laboratory of Industrial Biotechnology Ministry of EducationSchool of BiotechnologyJiangnan University1800 Lihu RoadWuxi 214122China

alginate lyase mainly produces active alginate oligosaccharides(AOS)by degrading alginate viaβ-elimination *** this study,the Pseudoalteromonas ***6B alginate lyase-encoding gene alg6B-7 from polysaccharide lyase(PL)-7 family was successfully cloned,sequenced,expressed in Escherichia *** on rational design and amino acid sequence alignment of the alginate lyase from various sources,four positive mutants were *** specific enzyme activities of four mutants I62A,A99K,V132S,and L157T were 38.84%,42.85%,75.8%and 51.83%higher than that of the wild enzyme,*** K_(cat)/K_(m) values of the four mutants were both increased,and the catalytic efficiency of V132S was 1.92-fold higher than that of the wild enzyme,*** rational design that was employed in this study achieved the dramatic improvement of catalytic activity,which may provide the application potential in industrial production.

关键词： alginate lyase Rational design Active center Site-directed mutation Catalytic efficiency

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

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Marine Life Science & Technology 2022年第1期4卷 106-116页

作者： Shengsheng Cao Qian Li Yinxiao Xu Tiancheng Tang Limin Ning Benwei Zhu College of Food Science and Light Industry Nanjing Tech UniversityNanjing211816China School of Medicine and Holistic Integrative Medicine Nanjing University of Chinese MedicineNanjing210023China

alginate,an acidic polysaccharide,is formed byβ-D-mannuronate(M)andα-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 ***,the application of alginate lyase has been limited due to its low catalytic efficiency and poor temperature *** 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 *** this review,we summarized the structural features and catalytic mechanisms of alginate *** modification strategies,such as rational design,directed evolution,conserved domain recombination,and non-catalytic domain truncation,are also described in ***,the application of alginate lyase is *** comprehensive summary can inform future applications of alginate lyases.

关键词： alginate lyase Structure Catalytic mechanism Molecular modification Application

Optimization of Culturing Condition and Medium Composition for the Production of alginate lyase by a Marine Vibrio sp. YKW-34

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Journal of Ocean University of China 2008年第1期7卷 97-102页

作者： FU Xiaoting LIN Hong KIM Sang Moo College of Food Science andEngineering Ocean University of China Qingdao 266003 P. R. China Faculty of Marine Bioscience and Technology Kangnung National University Gangneung 210-702 Korea

Carbohydrases secreted by marine Vibrio sp. YKW-34 with strong Laminaria cell wall degrading ability were screened, and among them alginate lyase was found to be dominant. The effects of medium composition and culturing condition on the produc- tion of alginate lyase by marine Vibrio sp. YKW-34 in flask were investigated in this study. In the culture medium of marine broth, no alginate lyase was produced. The activity of the alginate lyase, after being induced, reached 5 UmL–1. The best inoculum volume and inoculum age were 10% and 12 h, respectively. The optimal temperature for alginate lyase production was 25℃. The fermentation medium was composed of 0.5% of Laminaria powder and 0.2% of KNO3 with an initial acidity of pH 8.0. alginate could induce alginate lyase production but not as efficiently as Laminaria powder did. The addition of fucoidan, cellulose and glucose had nega- tive effect on the alginate lyase production. Other kinds of nitrogen sources, such as yeast extract, beef extract and peptone, had posi- tive effect on the growth of the microorganism and negative effect on alginate lyase production. In addition, the time course of algi- nate lyase production under the optimized condition was described. The optimal harvest time was 48 h.

关键词： alginate lyase Vibrio sp. YKW-34 medium optimization

Purification and Characterization of alginate lyase from Marine Vibrio ***

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Acta Biochimica et Biophysica Sinica 2006年第9期38卷 633-638页

作者： Yuan-Hong WANG* Guang-Li YU Xin-Min WANG Zhi-Hua LV Xia ZHAO Zhi-Hong WU Wei-Shang JI Marine Drug and Food Institute,Key Laboratory of Marine Drugs of Shandong Province,Key Laboratory of Marine Drugs,Ministry of Education,Ocean University of China,Qingdao 266003,China Marine Drug and Food Institute Key Laboratory of Marine Drugs of Shandong Province Key Laboratory of Marine Drugs Ministry of Education Ocean University of China Qingdao 266003 China. yhwang@***

Extracellular alginate lyase secreted by marine Vibrio ***,isolated from decayedLaminaria japonica,was purified by a combination of ammonium sulfate precipitation and diethylaminoethyl-Sephacel column *** results show that the molecular mass of alginate lyase wasapproximately 62.5 kDa,with an optimal pH and temperature at pH 7.0 and 25℃,*** wasapproximately 72.73 g/*** activity of the enzyme was enhanced by EDTA and Zn2+,but inhibited by Ba2+.The substrates specificity analysis shows that it was specific for hydrolyzing poly-β-D-1,4-mannuronate inalginate.

关键词： marine Vibrio alginate alginate lyase purification characterization

Isolation of Protoplasts from Undaria pinnatifida by alginate lyase Digestion

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Journal of Ocean University of Qingdao 2003年第1期2卷 58-61页

作者： HU Xiaoke JIANG Xiaolu GUAN Huashi Fishery College Ocean University of ChinaQingdao 266003P..R.China

The aim of this study is to isolate protoplasts from Undaria pinnatifida. Protoplasts of the alga were isolated enzymatically by using alginate lyase, which was prepared by fermenting culture of a strain Vibrio sp. 510. Monofacterial method was applied for optimizing digestion condition. The optimum condition for protoplast preparation is enzymatic digestion at 28 ℃ for 2 h using alginate lyase at the concentration of 213.36 U (8 mL) every 0.5 g fresh thalline with NaCl 50 and at the shaking speed of 150 r min -1 during digestion. The protoplast yield can reach 2.62±0.09 million per 0.5 g fresh leave under the optimum condition. The enzyme activity is inhibited by Ca 2+ and slightly enhanced by Fe 2+ and Mn 2+ at concentrations of 0.05, 0.08 and 0.10 mol L -1.

关键词： alginate lyase Undaria pinnatifida protoplast

Biochemical Characteristics and alginate-Degrading Properties of a Novel alginate lyase AlgL-5 from Flammeovirga ***04

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Biochemical Characteristics and Alginate-Degrading Propertie...

P_(luxI)mutants with different promoting period and their application for quorum sensing regulated protein expression

2015中国酶工程与糖生物工程学术研讨会

作者： Wenjun Han Jingyan Gu Yuanyuan Cheng Huihui Liu Yuezhong Li Fuchuan Li National Glycoengineering Research Center ＆ State Key Laboratory of Microbial Technology Shandong University

＜正＞alginate is the most important polysaccharide component that contributes to the cell wall of brown seaweeds or the extracellular matrix of some bacteria *** lyases can degrade alginate via theβ-elimination mech... 详细信息

关键词： alginate lyase Flammeovirga Gene truncation Genome Molecular modification Oligosaccharide

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Food Science and Human Wellness 2023年第5期12卷 1841-1849页

作者： Zhuoning Cao Zhen Liu Guilin Zhang Xiangzhao Mao College of Food Science and Engineering Ocean University of ChinaQingdao 266003China Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology Qingdao 266237China

Quorum sensing(QS)system can dynamically control the expression of proteins along with the cell *** promoting period of QS system has been little focused on until *** this study,a self-induced dynamic regulated expression(SIDRE)system was constructed in Escherichia *** enable the system suitable for the expression of enzymes,promoter engineering was used to obtain P_(luxI)*** test the SIDRE system,alginate lyase AL493 and esterase Est7 were used as target protein for *** enzyme activity of alginate lyase and esterase reached 96.38%and 106.71%of the control strains containing the T7 *** high-density fermentation,the activity of alginate lyase expressed by the SIDRE system with P_(luxI)(T-38C)as promoter was 4.34-fold of that expressed by the T7 ***,the P_(luxI)mutants with different promoting periods and/or different strengths show great potential in both laboratory and industrial scale for protein expression.

关键词： Quorum sensing Promoter engineering Enzymes alginate lyase Esterase High-density fermentation