Thermostable ethanol tolerant xylanase from a cold-adapted marine species Acinetobacter johnsonii

A xylanase-producing bacterium, isolated from deep sea sediments, was identified as the cold-adapted marine species Acinetobacter Johnsonii. A cold-adapted marine species Acinetobacter Johnsonii could grow at 4 ℃. The optimum temperature and pH of xylanase from a cold-adapted marine species Acinetobacter Johnsonii were 55 ℃ and pH 6.0. Xylanase from a cold-adapted marine species Acinetobacter Johnsonii remained at 80% activity after incubation for 1 h at 65 ℃. The xylanase activity was 1.2-fold higher in 4% ethanol solution than in ethanol free solution. Gibbs free energy of denaturation, ΔG, was higher in 4% ethanol solution than in ethanol free solution. Thermostable ethanol tolerant xylanase was valuable for bioethanol production by simultaneous saccharification and fermentation process with xylan as a carbon source.

Extracellular enzymatic activities of cold-adapted bacteria from polar oceans and effect of temperature and salinity on cell growth

The potential of 324 bacteria isolated from different habitats in polar oceans to produce a variety of extracellular enzymatic activities at low temperature was investigated. By plate assay, lipase, protease, amylase, gelatinase, agarase, chitinase or cellulase were detected. Lipases were generally present by bacteria living in polar oceans. Protease-producing bacteria held the second highest proportion in culturable isolates. Strains producing amylase kept a relative stable proportion of around 30% in different polar marine habitats. All 50 Arctic sea-ice bacteria producing proteases were cold-adapted strains, however, only 20% were psychrophilic. 98% of them could grow at 3% NaCl, and 56% could grow without NaCl. On the other hand, 98% of these sea-ice bacteria produced extracellular proteases with optimum temperature at or higher than 35℃, well above the upper temperature limit of cell growth. Extracellular enzymes including amylase, agarase, cellulase and lipase released by bacteria from seawater or sediment in polar oceans, most expressed maximum activities between 25 and 35℃. Among extracellular enzymes released by bacterial strain BSw20308, protease expressed maximum activity at 40℃, higher than 35℃ of polysaccharide hydrolases and 25℃ of lipase.

Fermentation Performance and Characterization of Cold-Adapted Lipase Produced with Pseudomonas Lip35

Strain of Pseudomonas Lip35 producing lipase was isolated in a refrigerator. Lipase production and characterization of this strain were investigated under different conditions. The Pseudomonas was cultivated in shaking flasks in a fermentation medium in various nutritional and physical environments. Lipase production has been influenced by the presence of yeast-extract, soybean powder, NaCI, and Tween-80. Maximum lipase productivity was obtained when the physical environment of the fermentation medium was optimal for 67 h. The production of lipase reached 58.9 U·mL^-1. The lipase of Pseudomonas Lip35 can be considered to be inducible, but the inducer had little influence on the production of lipase. The lipase was characterized and showed high lipolytic activity from pH 7.5-8.0. The optimum temperature was observed at 20℃ and the thermal inactivation of lipase was obvious at 60℃. The lipase activity was inhibited by K＋, stimulated by Ca^2＋, and thermostability decreased in the presence of Ca^2＋, therefore the lipase was Ca^2＋ -dependent cold-adapted enzyme.

Optimization of four types of antimicrobial agents to increase the inhibitory ability of marine Arthrobacter oxydans KQ 11 dextranase mouthwash

We adopted the response surface methodology using single factor and orthogonal experiments to optimize four types of antimicrobial agents that could inhibit biofilm formation by Streptococcus mutans, which is commonly found in the human oral cavity and causes tooth decay. The objective was to improve the function of marine Arthrobacter oxydans KQll dextranase mouthwash （designed and developed by our laboratory）. The experiment was conducted in a three-level, four-variable central composite design to determine the best combination of ZnSO4, lysozyme, citric acid and chitosan. The optimized antibacterial agents were 2.16 g/L ZnSO4, 14 g/L lysozyme, 4.5 g/L citric acid and 5 g/L chitosan. The biofilm formation inhibition reached 84.49%. In addition, microscopic observation of the biofilm was performed using scanning electron microscopy and confocal laser scanning microscopy. The optimized formula was tested in marine dextranase Arthrobacter oxydans KQ11 mouthwash and enhanced the inhibition of S. mutans. This work may be promoted for the design and development of future marine dextranase oral care products.

Gene cloning and sequence analysis of the cold-adapted chaperones DnaK and DnaJ from deep-sea psychrotrophic bacterium Pseudoalteromonas sp. SM9913

Pseudoalteromonas sp. SM9913 is a phychrotmphic bacterium isolated from the deep-sea sediment. The genes encoding chaperones DnaJ and DnaK of P. sp. SM9913 were cloned by normal PCR and TAIL - PCR （GenBank accession Nos DQ640312, DQ504163 ）. The chaperones DnaJ and DnaK from the strain SM9913 contain such conserved domains as those of many other bacteria, and show some cold-adapted characteristics in their structures when compared with those from psychro-, meso-and themophilic bacteria. It is indicated that chaperones DnaJ and DnaK of P. sp. SM9913 may be adapted to low temperature in deep-sea and function well in assisting folding, assembling and translocation of proteins at low temperature. This research lays a foundation for the further study on the cold-adapted mechanism of chaperones DnaJ and DnaK of cold-adapted microorganisms.

Decomposition and removal of dental model plaque by using toothpaste containing dextranase

Objective:The purpose of this study is to demonstrate the decomposition and removal effects of dextranase-containing toothpaste on dental plaque. Method:In the decomposition test,the supernatant of three times diluted toothpaste was applied to a dextran solution (as a dental model plaque),and samples were evaluated by colorimetric reaction with Fehling’s test solution. In the removal test,the supernatant of three times diluted toothpaste was applied to a dental model plaque prepared with Streptococcus mutans and the optical density at 550nm (hereinafter referred to as OD_(550)) was measured as the remaining plaque. Results:In the test solution of toothpaste containing dextranase,a red-brown precipitate was observed. On the other hand,a precipitate was not observed in the test solution of the placebo toothpaste which did not contain dextranase. The plaque removal effect of the test toothpaste was 2. 7 times higher than that of the placebo toothpaste. Conclusion:Our findings suggest that the test toothpaste containing dextranase has a higher plaque removal effect by cuttingα-1,6-linkages inside the plaque. Therefore,the test toothpaste might be helpful to prevent dental caries.

Cold-adaptive alkaline protease from the psychrophilic Planomicrobium sp.547: enzyme characterization and gene cloning

A psychrophilic bacterium strain 547 producing cold-adaptive alkaline protease was isolated from the deep sea sediment of Prydz Bay, Antarctica. The organism was identified as a Planomicrobium species by 16S rRNA analysis. The optimal and highest growth temperatures for strain 547 were 15~C and 30~C, respectively. The extracellular protease was purified by ammonium sulfate precipitation and DEAE cellulose-52 chromatography. The optimal temperature and pH for the activity of the purified enzyme were 35~C and pH 9.0, respectively. The enzyme retained approximately 40% of its activity after 2 h of incubation at 50℃. The enzymatic activity was inhibited by 1 mmol/L phenylmethyl sulfonylfluoride （PMSF） and hydrochloride 4-（2-aminoethyl）-benzenesulfonyl fluoride （AEBSF）, indicating that it was a serine protease. The presence of Cae＋ and Mnz＋ increased the activity of the enzyme. The protease gene with a size of 1 269 bp was cloned from Planomicrobium sp. 547 using primers designed based on the conserved sequences of proteases in GenBank. The Planomicrobium sp. 547 protease contained a domain belonging to the peptidase S8 family, which has a length of 309 amino acid （AA） residues. The alignment and phylogenetic analysis of the AA sequence indicated that the protease belonged to the subtilisin family.

基于右旋糖酐酶制备小麦多孔淀粉及其特性

生物酶法制备多孔淀粉条件温和、工艺简单和绿色环保。本研究选用特异性水解α-1,6糖苷键的右旋糖酐酶制备小麦多孔淀粉。通过X-晶体射线衍射、傅里叶红外光谱、扫描电镜和粒度分析仪对淀粉的结构进行表征,同时测定了淀粉膨胀率、吸水和吸油率、药品负载率和体外溶出等指标。结果表明:在温度为50℃条件下处理16 h、小麦淀粉的表面形成孔洞,淀粉粒径减小,多孔淀粉的晶型结构无显著变化。多孔淀粉的比容积为2.11±0.02 cm^(3)/g、溶解度为9.02%±0.21%、膨胀度达到了9.91%±0.22%、吸水率和吸油率分别提高到80.12%和78.23%;多孔淀粉与姜黄素、甲磺酸达比加群酯、制霉菌素和氧氟沙星四种药品在160:1的比例下负载率平均达到了75.07%。体外试验显示,小麦多孔淀粉负载姜黄素的释放率显著降低。在胃液中2.5 h的释放率约为30%。在肠液中5 h的释放率约为40%。研究结果为小麦多孔淀粉的制备和应用提高的依据。

绳状青霉菌发酵产右旋糖酐酶的条件研究

研究绳状青霉菌(Penicillium funiculosum)发酵培养产右旋糖酐酶的条件。研究碳源、氮源对绳状青霉菌产右旋糖酐酶的影响,结果表明最适合发酵产酶的碳源、氮源分别是右旋糖酐和蛋白胨。采用正交试验对绳状青霉菌产右旋糖酐酶的发酵条件进行优化,结果表明最适条件为:发酵瓶装液量为80mL/250mL,发酵培养基的初始pH值为5.5,培养温度为28℃,在该条件下,右旋糖酐酶的酶活力为18.963U/mL。

淡紫拟青霉右旋糖酐酶的形成条件

比较了各种碳水化合物对淡紫拟青霉(Paecilomyces lilacinus)右旋糖酐酶形成的影响,右旋糖酐是最好的碳源,也是最佳诱导物。不同分子量(17.2—1000kD)的右旋糖酐对酶形成的诱导作用不同,酶的产生随右旋糖酐分子量的增大而增加。用分子量为1000kD 的右旋糖酐作碳源时比用17.2kD 的右旋糖酐作碳源时的产酶量高40%以上。用右旋糖酐和其它糖的混合物作碳源时,酶的形成受到不同程度的抑制。右旋糖酐酶形成的其它适宜条件:氮源为牛肉蛋白胨,培养基初始 pH6.0—7.0,种龄为48小时,在250ml 三角瓶中装50ml 培养基,于28℃在200r/min 摇床上培养6天。

α-葡聚糖酶在毕赤酵母中的组成型表达

为避免α-葡聚糖造成的制糖过程中的糖分损失、制炼难度增加以及糖产品质量下降,利用α-葡聚糖酶分解而直接去除α-葡聚糖是目前最佳的选择.文中扩增了α-葡聚糖酶基因dex,构建组成型表达载体pGAPZαA-dex;以毕赤酵母KM71H为受体菌,将表达载体整合进入受体菌基因组,构建了组成型表达α-葡聚糖酶的毕赤酵母工程菌.摇瓶发酵120h,α-葡聚糖酶酶活达到60.4U/mL,从而实现了α-葡聚糖酶在毕赤酵母中的组成型表达,使发酵过程无需使用甲醇进行诱导,提高了生产和使用安全性.

葡聚糖酶高产菌株的分离鉴定及其培养基优化的研究

进行高产分解葡聚糖酶的优良菌株的分离、鉴定及其培养基的优化.经过对土壤样品的分离、诱变筛选和一系列的形态、生理生化试验后,获得一株葡聚糖酶活力为97μ/ml的菌株并鉴定为枯草芽孢杆菌属.其在摇瓶发酵的优化培养基为:葡萄糖4%,磷酸氢二铵0.03%,磷酸二氢钾0.15%,硫酸镁0.1%,其它适量无机离子及生长因子.

交替假单胞菌LP621菌株产右旋糖苷酶的培养条件优化

从江苏连云港海域分离和筛选到1株产右旋糖苷酶的海洋细菌交替假单胞菌Pseudoalteromonas tetraodonis LP621,通过单因素试验和正交试验对该菌株产右旋糖苷酶培养条件进行优化。单因素试验结果表明,最佳培养时间为24 h,最适产酶温度为25℃;产酶pH范围为5.0~11.0,最适产酶pH为6.0;产酶NaCl浓度范围为1%~10%,NaCl浓度为4%时产酶较高;装液量在25%。麦芽糖、胰蛋白胨和酵母膏促进产酶。利用响应面方法对LP621产右旋糖苷酶的发酵条件进行优化。选择培养基pH、时间、麦芽糖浓度和装液量4因素进行优化,结果为pH 7.07,发酵时间21.94 h,麦芽糖浓度0.42%,装液量为21.88%,酶活为270.1U/mL。

酶法合成右旋糖酐相对分子质量的控制

用海藻酸盐固定的肠膜状明串珠菌所产右旋糖酐蔗糖酶合成右旋糖酐，在合成过程中加入右旋糖酐酶，使超高相对分子质量的右旋糖酐裂解，以调节产物的相对分子质量；用凝胶渗透色谱（GPC）作为表征手段，对右旋糖酐相对分子质量与右旋糖酐酶的添加量及反应时间的关系等进行了研究。结果表明，右旋糖酐酶在最初的2h就能使右旋糖酐的重均相对分子质量从8．52×10^5降至2．00×10^5，随后作用减缓；右旋糖酐的重均相对分子质量随右旋糖酐酶用量的增加而减小，并且酶用量对产物相对分子质量的影响是非线性的；右旋糖酐蔗糖酶与右旋糖酐同时进入反应体系所得右旋糖酐的收率最高可达86．8％。在100mL蔗糖质量浓度为100g／L的底物溶液中，加入15u右旋糖酐蔗糖酶液，同时加入0．19～0．75U右旋糖酐酶，反应24h，可以得到相对分子质量为1．0×10^5～2．0×10^4的右旋糖酐。

发酵耦合酶解高效制备右旋糖酐工艺研究

【目的】改进肠膜明串珠菌（Leuconostoc mesenteriodes 31208）发酵蔗糖制备右旋糖酐的工艺，实现灵活控制分子量及高效制备右旋糖酐的目标。【方法】采用单因素实验法研究蔗糖浓度对蔗糖转化的影响，确定蔗糖流加工艺的各个参数，以及右旋糖酐酶添加耦合蔗糖流加的工艺条件。【结果】通过调控右旋糖酐酶添加工艺，可灵活控制右旋糖酐的分子量，无需再经酸水解及乙醇分级沉淀就可以得到不同分子量的右旋糖酐，节省无水乙醇的用量；且确定的最佳蔗糖流加工艺参数：初始蔗糖浓度为130 g／L，流加速度为12 g·L－1·h－1，起始流加时间为16 h。与蔗糖单批发酵结果相比，该工艺可使蔗糖转化效率提高约75％，特定分子量（5000～7500 Da）右旋糖酐浓度可达108 g／L，相应提高2．3倍，收率稳定在32％左右。【结论】该方法具有发酵易调控、高效和成本低的优点，对大规模生产右旋糖酐具有重要的参考价值和应用价值。

一株产低温右旋糖苷酶海洋细菌的筛选和鉴定

从连云港海域筛选得到一株产低温右旋糖苷酶的菌株LP621,经形态特征、生理生化特征以及16S rDNA序列分析和鉴定,该菌株为Pseudoalteromonas tetraodonis。该菌产生低温右旋糖苷酶的最适作用温度为30℃,在80℃保温2.5 h后该酶仍具有40%以上的活性。目前尚无Pseudoalteromonas tetraodonis产生低温右旋糖苷酶的报道。菌株LP621产生的右旋糖苷酶作用温度低,耐热性好,有较大的潜在工业应用价值。

葡聚糖对制糖工业的影响及对策(下)

3 葡聚糖的测定方法 3．1 常用的测定方法 3．1．1 酒精Haze法及改进后的酒精Haze法 1959年Nicholson和Horsley根据葡聚糖溶液中加入酒精后产生沉淀，然后用分光光度计测量所产生沉淀浊度的原理提出Haze法测定葡聚糖含量。国际糖品统一分析方法委员会（ICUMSA）在第十七届会议上把此法列为试行方法。

朱黄青霉α-葡聚糖酶在毕赤酵母中的高效表达

【目的】提高朱黄青霉(Penicillium minioluteum)α-葡聚糖酶(Dextranase)基因在毕赤酵母(Pichia pastoris)中的表达水平。【方法】根据毕赤酵母的密码子偏爱对酶基因进行优化与合成。优化后的基因片段与载体pGAPZαA连接,转化毕赤酵母KM71H。【结果】获得组成型分泌表达α-葡聚糖酶的工程菌KM71H/pGAPZαA-dex。发酵工艺试验中,摇瓶培养144h,酶活为153U/mL。6.8L发酵罐补料分批培养92h,酶活达到1218U/mL。【结论】该工程菌以甘油作为碳源,发酵调控简单,产酶水平较高,具有适用于大规模生产的潜力。

焦曲霉右旋糖酐酶的纯化和性质

用制备凝胶电泳从焦曲霉(Aspergilluts ussus)菌株的培养液中分离纯化了右旋糖酐酶(EC 3.2.1.11)。纯酶经聚丙烯酰胺凝胶电泳分析为均一的,比活力为820 u/mg,提高20.5倍。酶作用最适 pH 为5.0—5.5,最适温度为50℃,在 pH 4.5—7.5和40℃以下稳定,在50℃保温30分钟酶活力损失55%。用 SDS-凝胶电泳测定酶的分子量为68000,用聚丙烯酰胺凝胶薄层等电聚焦电泳测得 pI 值为5.5。紫外吸收光谱在274nm 有最大吸收值,低谷在252 nm。纯酶只作用于α-1,6-葡萄糖苷键,水解右旋糖酐的产物为异麦芽糖、异麦芽三糖、葡萄糖和少量的较高聚合度的异麦芽寡糖,该酶为内切型的。作用于交联葡聚糖凝胶(Sephadex)时,随其交联度的增加酶作用能力减弱。纯酶作用于不同分子量的右旋糖酐(MW:2×10~4、1×10^(?)、4×10~4)的K_m值分别为0.30%、0.61%和0.70%。Hg^(2+)、Ag^+对酶完全抑制,Cu^(2+)、Mn^(2+)、Pb^(2+)、Be^(2+)、Fe^(3+)和 F^-对酶有一定的抑制作用,金属螯合剂 EDTA 对酶活性无影响,N-溴代琥珀酰亚胺(NBS)对酶强烈抑制。

产右旋糖酐酶埃氏交替单胞菌的筛选、鉴定及酶活分析

从连云港港口海泥样本筛选到一株产右旋糖酐酶的埃氏交替单胞菌A.espejiana YSN412,对其所产的右旋糖酐酶AesDEX酶学性质进行了分析,对其产酶条件进行了摸索。AesDEX最适反应温度为30℃,最适pH值为8。在30℃下保温2.5h,残余相对酶活在95%以上,而在50℃下保温2.5h后只有14%的残余酶活力。该菌株产酶培养的最佳碳源和氮源,分别是纤维二糖和酵母粉,最适产酶培养温度为30℃,NaCl浓度为2%,初始pH为8时培养液酶活力最高。该菌株培养12h时培养液酶活力达到最高值9U/mL。本实验筛选到的A.espejiana YSN412菌株产酶较为迅速,所产的右旋糖酐酶AesDEX酶活最适温度较低,在工业生产上具有潜在降低成本、提高生产效率的优势。