Alginate lyases are a group of enzymes that catalyze the depolymerization of alginates into oligosaccharides or monosaccharides. These enzymes have been widely used for a variety of purposes, such as producing bioactive oligosaccharides, controlling the rheological properties of polysaccharides, and performing structural analyses of polysaccharides. The algM4 gene of the marine bacterium Vibrio weizhoudaoensis M0101 encodes an alginate lyase that belongs to the polysaccharide lyase family 7 (PL7). In this study, the kinetic constants Vmax (maximum reaction rate) and Km (Michaelis constant) of AlgM4 activity were determined as 2.75 nmol/s and 2.72 mg/mL, respectively. The optimum temperature for AlgM4 activity was 30 °C, and at 70 °C, AlgM4 activity dropped to 11% of the maximum observed activity. The optimum pH for AlgM4 activity was 8.5, and AlgM4 was completely inactive at pH 11. The addition of 1 mol/L NaCl resulted in a more than sevenfold increase in the relative activity of AlgM4. The secondary structure of AlgM4 was altered in the presence of NaCl, which caused the α-helical content to decrease from 12.4 to 10.8% and the β-sheet content to decrease by 1.7%. In addition, NaCl enhanced the thermal stability of AlgM4 and increased the midpoint of thermal denaturation (Tm) by 4.9 °C. AlgM4 exhibited an ability to degrade sodium alginate, poly-mannuronic acid (polyM), and poly-guluronic acid (polyG), resulting in the production of oligosaccharides with a degree of polymerization (DP) of 2–9. AlgM4 possessed broader substrate, indicating that it is a bifunctional alginate lyase. Thus, AlgM4 is a novel salt-activated and bifunctional alginate lyase of the PL7 family with endolytic activity.

[Objective] A marine Vibrio X511 with strong ability to use alginate was isolated from the rotten Sargassum in the Weizhou Island of the North Sea.The metabolic pathway of using alginate by Vibrio X511 was studied by transcriptome sequencing.[Methods] Transcriptions sequencing was done by Illumina Hi Seq2500 sequencing platform and the transcriptome of the strains cultured in alginate and glucose were sequenced.Differential transcripts were compared and analyzed, and the sequencing results were confirmed by fluorescence quantitative PCR.The fluorescence quantitative PCR was applied to verify the sequencing results.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were used to annotate the function and pathway in differential transcripts.[Results] There were 2024 differentially expressed genes in the culture of alginate compared to those in glucose, with 1066 genes up-regulated and 958 genes down-regulated.Some genes in the ubiquitous metabolic pathway were also differentially expressed in different cultures.The transcriptions of all the genes involved in alginate utilization and the key genes of synthetic ethanol in Vibrio X511 were up-regulated.In addition, the strain had a unique way to utilize alginate and one of the metabolic processes has not been reported in Vibrio.[Conclusion] Our findings enrich the research of biological method for degradation of alginate, and provide valuable data for the research of large seaweed biomass-based energy.

The purpose of this work is to obtain a strain with an ability to degrade alginate. With sodium alginate as a sole carbon source, a strain of marine bacterium with the solid ability to degrade alginate was screened and purified from the cankered gulfweed,coded as X511. According to the morphological observation,physicochemical indexes and the techniques of molecular biology,the strain was identified as Vibrio,designated as Vibrio sp. X511. The exponential phase of strain X511 was about 5-16 h,and the suitable salinity for its growth was around 2%-6%(W/V). It was capable to grow in the culture medium with a sole carbon source like glucose,mannitol,or starch,et al. Also,4%-6%(W/V)of the salinity,the kelp powder,and the laminarin extended its stationary phase. The activity of intracellular crude alginate lyase reached 12.68 ± 0.13 U/mL when the X511 fermented in the screening medium for 24 h. The intracellular crude alginate lyase was extracted,and its ability to digest alginate sodium,poly mannurono acid,and poly guluronic acid was measured as such order :alginate sodium > poly mannurono acid > poly guluronic acid. The results of the Thin Layer Chromatography(TLC)showed that the enzymatic degradation product of alginate sodium was trisaccharide. The above results indicate that the X511 is a marine strain with strong salt tolerance, short growth cycle,and it simultaneously utilizes the sodium alginate,poly mannurono acid,and poly-guluronic acid as its growing carbon resource.

The alginate lyase gene in marine Vibrio sp.X511 was expressed by Escherichia coli to realize the large-scale preparation of alginate lyase and research enzymatic properties.By analyzing the sequence,the first 20 amino acids of the enzyme were predicted as the signal peptide,the protein molecular mass without the signal peptide was 30 254.28 Da,the isoelectric point was 8.75,the molecular formula was C1368H2100N364O401S6,and the corresponding gene sequence was named as alg1987.The construction program of bacteria recombination was as follows:the specific nucleic acid primers were designed and the alg1987 was obtained by PCR;the recombinant plasmid pET-30(a)-alg1987 was introduced into E.coli Trans5t for sequencing;the positive plasmid pET-30(a)-alg1987 was extracted and introduced into E.coli BL21,then expressed by the induce of IPTG.The supematant of recombinant bacteria cytosol was induced and detected by SDS-PAGE,it was found that the molecular mass of the alginate lyase by heterologous expression was similar to the predicted value.The expression conditions of alginate lyase in recombinant bacteria were optimized.The results showed that optimum parameters were as follows:induction temperature 16 ℃,time 16 h,induction agent IPTG concentration 0.8 mmol/L.

采用响应面法优化海洋弧菌X511的产酶发酵培养基,提高其胞内褐藻胶裂解酶产量.通过单因素试验研究了不同碳源、不同氮源、海藻酸钠、氯化钠、蛋白胨、硫酸亚铁、硫酸镁和磷酸氢二钾对菌株产胞内酶活力的影响,在此基础上,利用Plackett-burman试验确定海藻酸钠、氯化钠、蛋白胨和硫酸镁对胞内酶产量的影响.通过响应面试验构建回归方程,结果表明,最佳发酵培养基成分为海藻酸钠9.0 g/L,NaC1 31.6 g/L,蛋白胨15.0g/L,MgSO4·7H2O 1.5 g/L.在此条件下,该菌株的胞内褐藻胶裂解酶的活力为(20.65±0.14) U/mL,较优化前的酶活提高了64.4％.

Objective]Alginate lyase in Shewanella haliotis BP-1 strains was studied illustrate its biological activity of degrading alginate.[Methods]The gene cloning technology and the Escherichia coli heterologous expression technology were applied to overexpress the alginate lyase;And the enzyme activity was analyzed after the crude enzyme was separated and purified by DEAE Sepharose FF chromatogra-phy.[Results]The alginate lyase gene Alg 1 7S , with a size of 2 1 5 7 bp,was cloned from S. haliotis BP-1 strain genomic DNA and encoded an alginate lyase Alg17S,which belonged to pol-ysaccharide lyase(PL)1 7 family and had a size of 79 726 Da protein(including an N-terminal signal peptide of 26 amino acid signal peptide).Alg17S showed high sequence identity of 5 2% with PL-17 protein sequence Alg17C from Saccharophagus degradans 2-40.Both the purified recombi-nase Alg17S and the △snAlg17S(without the N-terminal signal peptide of 26 amino acids)can degrade alginate,but the enzymatic activity of △snAlg17S revealed a specific activity of 9 635 U/mg,which was more efficient than Alg17S.[Conclusion]The recombinant alginate lyase △s-nAlg17S that has both high-level expression and high enzymatic activity could be a potential en-zyme for further researching on the alginate saccharification and the biofuels production.

Abtract:[Objective]The aim of this study is to analyze the interaction between the different groups ofβ-carotene in the heating process.[Methods]Two-dimensional correlation spectrosco-py was applied to study the dynamic spectral changes ofβ-carotene from 30℃ to 100℃.[Re-sults]The changes of absorption characteristic peaks ofβ-carotene were inconspicuous in the conventional FTIR spectra and second derivative FTIR spectra during 30℃ to 100℃,which in-dicated that they had no oxidation reation.Two-dimensional correlation analysis showed that the changes of absorption peaks at 968 cm-1 ,1 442 cm-1 ,2 9 6 6 cm-1 and 3 0 1 2 cm-1 were more sen-sitive to temperature.Meanwhile,the order of different groups changes induced by temperature were as follows:the spectral changes of methyl-ene were faster than methyl,the spectral changes of methyl C-H symmetric stretching vibration in low wavenumber were faster than methyl anti-symmetric stretching vibration in high wavenumber,and olefin hydrocarbon symmetric stretching vibration were prior to olefin hydrocarbon anti-symmetric stretching vibration.[Con-clusion]This provides experimental basis for the mechanism of the conformational change ofβ-carotene in heating process.

【Objective】The apoptosis of yeast cells was induced by ultraviolet and its process was studied in order to understand the changes of biological macromolecules and the molecular mechanism.【Methods】Laser tweezers Raman spectroscopy(LTRS)was used to monitor the dynamics of the intracellular biological macromolecules in real-time during the apoptosis process of yeast cells stressed with UV at both cellular population level and single-cell level,respectively.【Results】Lethal doses of UV irradiation could cause cell apoptosis.The intensities ofRaman peaks,which were assigned to nucleic acids(1085cm-1,1300cm-1),proteins(850cm-1,1440cm-1,1604cm-1,1655cm-1)and lipids(1085cm-1,1300cm-1,1440cm-1),decreased significantly as a function of the duration of cell apoptosis at cellular population level,suggesting that the content of nucleic acids,proteins and lipids reduced gradually during the time that the yeast cells were undergoing apoptosis induced by UV.The peak of 1604cm-1,which was called the Raman spectroscopic signature of life in yeasts,was a marker of Raman band for cell metabolic activity.Its intensity had the sharpest decline by 60%at the late stage of apoptosis,implying that the energy metabolism and breathing capacity were decreased during the process of cell apoptosis,which related to the changes of ergosterol structure and function theoretically.However,the changes of the intensities of Raman peaks at 850cm-1,1085cm-1,1300cm-1,1440cm-1 and 1665cm-1 between the group cells and the single cells were different significantly in the period of 90~120min and 125~167min,demonstrating that the heterogeneities of the single cells were masked by the average spectroscopy of the population cells.【Conclusion】LTRS can be used to directly and truthfully detect the kinetics of apoptotic process of yeast cells under UV irradiation at the single cell level and probe cellular heterogeneity.

A modified procedure of Percoll density gradient centrifugation was developed to isolate and fractionate synchronous cells from stationary phase (sp) cultures of different yeast strains, as well as Raman spectra discrimination of single yeast cells was reported. About 1.75 mL Percoll solution in 2 mL polypropylene centrifugal tube was centrifuged at 19,320 g, 20 °C with an angle rotor for 15 min to form continuous densities gradient (1.00~1.31 g · mL(-1)), approximately 100 μL sample was overlaid onto the preformed continuous density gradient carefully, subsequently, centrifuged at 400 g for 60 min in a tabletop centrifuge equipped with a angle rotor at 25 °C. Yeast samples could be observed that the suspensions were separated into two cell fractions obviously. Both fractions of different yeast strains were respectively determined by differential interference contrast (DIC), phase contrast microscope and synchronous culture to distinguish their morphological and growth trait. The results showed that the lower fraction cells were unbudded, mostly unicellular, highly refractive, homogeneous and uniform in size, and represented growth characteristic synchronously; Their protoplasm had relatively high density, and contained significant concentrations of glycogen; all of which were accordant with description of quiescent yeast cells and G0 cells in previously published paper. It was shown that lower fraction was quiescent cells, synchronous G0 cells as well. A Raman tweezers setup was used to investigate the differences between two fractions, G0 cells and non G0 cells, at a single cell level. The result showed that both G0 cells and the non G0 cells had the same characteristic peaks corresponding biological macromolecules including proteins, carbohydrates and nucleic acids, but all characteristic peak intensities of G0 cells were higher than that of non G0 cells, implied that the macromolecular substance content of G0 cells was more higher. Principal component analysis (PCA) was performed between G0 cells and non G0 cells, the results showed that the chemical composition content among the synchronization G0 cells has less difference, and G0 cells were homogeneous but non G0 cells were heterogeneous, indicating single cell optical tweezers Raman spectroscopy could identify the synchronous and asynchronous cells. The modified method is feasible, economical and efficient highly. G0 synchronous cells of most yeast strains could be isolated by a modification of Percoll density gradient centrifugation.

Laser tweezers Raman spectroscopy (LTRS) as a non-invasive tool for mitochondria analysis, combined with oxygen electrode and ultraviolet spectrophotometric method, was used to investigate the Raman spectra of mitochondria of yeast cells in vitro and in vivo after induced with acetic acid. The results showed that when the mitochondria of yeast cells in vivo was induced by acetic acid, the spectral peaks of the nucleic acid (1081 and 1301 cm−1), proteins (872, 1604, 1445 and 1657 cm−1), lipids (1125, 1301, 1445 and 1657 cm−1), cytochrome c (750 and 1125 cm−1) and mitochondria respiration (1604 cm−1) were significantly decreased as a function of the duration of the acetic acid stress, and the obtained physiological and biochemical indexes of respiration rate, phosphorous/oxygen (P/O) ration and cytochrome c content were similar to those by conventional method. Furthermore, when the mitochondria of yeast cells in vitro was induced by acetic acid, the spectral peaks of the nucleic acid (1081 and 1301 cm−1), proteins (872, 1604, 1445 and 1657 cm−1), lipids (1125, 1301, 1445 and 1657 cm−1), cytochrome c (750 and 1125 cm−1) and mitochondria respiration (1604 cm−1) were significantly decreased as the function of the duration of the acetic acid stress, and the obtained physiological and biochemical indexes of respiration rate, P/O ration and cytochrome c content were similar to those by conventional method. The results indicated that acetic acid could penetrate into the cell interior and directly impacted the mitochondria possibly, resulting in the release of inclusions from the mitochondria, subsequently, causing the apoptosis of yeast cells via mitochondrial pathway-induced apoptosis.

运用激光镊子拉曼光谱技术（LTRS）研究铝胁迫下土生隐球酵母细胞和线粒体拉曼光谱变化。细胞和线粒体中与核酸、蛋白质和脂类相关的特征峰强度随着铝处理浓度和处理时间的增加而逐渐降低,表明细胞凋亡过程中细胞和线粒体内的核酸、蛋白质和脂类生物大分子逐渐减少。线粒体内与细胞色素c（Cyt c）相关的特征峰强度随铝处理浓度增大和处理时间的延长而显著降低,说明线粒体中的细胞色素c释放到线粒体外。线粒体的呼吸峰随着铝浓度的增加和处理时间的延长而降低,说明线粒体的活性不断减弱,能量代谢受阻。因此,拉曼光谱实时监测了铝胁迫细胞凋亡的动态过程及其线粒体的生理生化变化过程,并且揭示了铝诱导土生隐球酵母凋亡过程中线粒体内细胞色素c的释放行为,这些结果有助于了解酸性土壤中铝对生物的毒害机理。

应用激光镊子拉曼光谱系统（LTRS）,实时分析不同温度下蚕豆14-3-3b可溶性蛋白和包涵体蛋白在重组大肠杆菌细胞中的动态表达水平。结果显示,14-3-3b可溶性蛋白与包涵体蛋白有明显不同的拉曼光谱特征峰,反映出两者在主链和侧链构象上的差异性;在28℃时,包涵体蛋白特征峰900,1033,1328,1415和1446cm-1强度随着诱导时间延长而增加的幅度,明显大于可溶性蛋白的特征峰763,1002,1363,1451和1665cm-1的增加幅度,16℃时诱导表达结果正好相反,可溶性蛋白的特征峰增强幅度显著大于包涵体蛋白,说明28℃诱导培养条件下,重组菌蛋白质过量表达以形成包涵体为主,16℃较低温度条件下以形成可溶性蛋白为主,正确折叠蛋白增加的信息可以通过光谱变化反映出来,在该温度下,蛋白质的正确折叠有利于细胞形成稳定的可溶性蛋白;另外,重组菌在相对低温条件下更多地表达含胱氨酸的非重组蛋白,可能与蛋白质折叠相关。LTRS技术可以在单个细胞水平上对大肠杆菌细胞过量表达可溶性蛋白和包涵体蛋白过程进行无损害、无标记的实时分析。

Laminaria japonica containing abundant carbohydrates but less lignin and cellulose have enormous potential for bioethanol production. Polysaccharides and monosaccharides extracted from L. japonica as well as the monosaccharides that constitute the polysaccharides of L. japonica were used as substrates to produce ethanol with Pichia angophorae. And the objective of this study is to evaluate the ethanol bioconversion of the great mass of polysaccharides and monosaccharides in L. hyperborean by P angophorae directly. The results showed that fucoidan and laminarin, which are polysaccharides in L. japonica extracts, could be used as a only carbon source to produce ethanol by P angophorae directly, and the ethanol conversion rate reached a maximum of 0.222 g and 0.309 g ethanol (g substrate)(-1) respectively. Likewise, this strain could readily produce ethanol from mannitol, mannose and glucose with a high ethanol conversion rate between the range of 0.42 to 0.45 g ethanol (g substrate)(-1), both from fucose and xylose with a lower rate of 0.331 g and 0.185 g ethanol (g substrate)(-1). Moreover, guluronic acid and mannuronic acid derived from sodium alginate could be converted to ethanol with conversion rates of 0.324 g and 0.361 g ethanol (g substrate)(-1). Furthermore, kelp extracts were fermented by P angophorae for preliminary research on ethanol production from L. japonica, the highest ethanol conversion rate was 0.415 g ethanol (g reducing sugar)(-1). These results provided a reference for further optimization of fermenting L. japonica extracts to ethanol by P angophorae, and illustrated the great potential of P angophorae as the fermentative strain for ethanol production with kelps as a feedstock.

At group cells level and a single yeast level,laser tweezers Raman spectroscopy was used to monitor the dynamic of the intracellular biological macromolecular in real-time during the apoptosis process of yeast cells stressed with high concentrations of ethanol.The results show that the intensities of Raman peaks of nucleic acids(721,1 083,1 301cm-1),proteins(721,858,1 001,1 301,1 445,1 608,1 657cm-1)and lipids(1 083,1 301,1 445cm-1)decreased significantly along with the extension of treatment time,indicates that the contents of nucleic acids,proteins and lipids are reduced gradually while the yeast cells are undergoing apoptosis induced by high concentrations of ethanol.However,the results of single factor analysis and repeated measures analysis show that the changes of the intensities of Raman peaks between the group cells and the single cells are difference significantly,implies that the heterogeneities of the single cells are covered by the average spectroscopy of the population cells.The results display that laser tweezer Raman spectroscopy can be used to directly and truthfully detect the kinetic apoptosis process under high concentrations of ethanol stress at the single cell level and probe cellular heterogeneity.

To research the lethal mechanism of spores stressed by alkali, laser tweezers Raman spectroscopy (LTRS) combined with principal components analysis (PCA) was used to study the physiological process of single spore with alkali stress. The results showed that both spores and germinated spores had tolerance with alkali in a certain range, but the ability of spores was obviously lower than that of spores due to the release of their Ca2+ -DPA which plays a key role in spores resistance as well as spores resistance to many stresses; A small amount of Ca2+ -DPA of spores was observed to release after alkali stress, however, the behavior of release was different with the normal Ca2+ -DPA release behavior induced by L-alanine; The data before and after alkali stress of the spores and g. spores with PCA reflected that alkali mainly injured the membrane of spores, and alkali could be easily enter into the inner structure of spores to damage the structure of protein backbone and injure the nucleic acid of spores. We show that the alkali could result in the small amount of Ca2+ -DPA released by destroying the member channel of spores.

To explore a simple and rapid method without cell destruction to detect the real. time expression levels of the soluble and inclusionbody proteins in Escherichia. coli, Raman Laser Tweezers spectroscopy (LTRS) was used to investigate the structural differences and expression levels of the soluble and inclusionbody proteins of broad bean (Vicia faba L.) 14-3-3b in E. coli cells induced under different temperatures. The results showed that 14-3-3b soluble and inclusionbody protein had different Raman lines, indicating that LTRS could be used to identify the soluble and inclusionbody protein of 14-3-3b. Raman lines in 1002, 1451 and 1665 cm(-1) of the soluble protein was enhanced evidently in the recombinant E. coli induced at 16 degrees C and were reduced significantly in the recombinant E. coli induced at 28 degrees C. In contrast, Raman lines in 900 and 1446 cm(-1) of the inclusionbody protein were decreased evidently in the recombinant E. coli induced at 16 degrees C and were increased significantly in the recombinant E. coli induced at 28 degrees C. Changes in the soluble and inclusionbody protein of 14-3-3b expression levels reflected by these Raman lines was consistent with those obtained by the analysis of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This evidence confirms that LTRS is an effectively and rapid method for detection of the expression levels of soluble and inclusionbody proteins in the living single E. coli cell.

The detection of the expression of formate dehydrogenase (FDH) recombination proteins in E. coli by molecular methods is time-consuming and hard-working and needs to destruct E. coli cells. To explore a simple and rapid method without cell destruction to detect the real-time expression of FDH recombination proteins in E. coli, Laser Tweezers Raman spectroscopy (LTRS) was used to investigate the recombinant protein expression of formate dehydrogenase (FDH) in the single living E. coli cell at different culture times following the induction with isopropyl thiogalactoside (IPTG). The result showed that the characteristic peaks corresponding to the recombinant FDH protein were gradually enhanced with an in increase in IPTG induction time, indicating the expression and the accumulation of FDH protein in recombinant E. coli cells. This result is consistent with that obtained by the analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This evidence confirms that LTRS is an effectively method for detection of the real-time expression of FDH recombination proteins in the living single E. coli cell without cell destruction.