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以海洋低温蛋白酶生产菌株YS-9412-130为研究对象，从传代、菌龄、溶菌酶浓度与酶解时间、甘氨酸与EDTA预处理以及稳定剂对该菌原生质体形成与再生的影响进行研究。结果表明，在相同条件下，第一代菌至第五代菌原生质体的形成率分别为86．9％、70．3％、66．8％、63．4％、60．2％，再生率分别为10．5％、18．6％、17．9％、18．2％、17．8％；取该菌对数生长前期、对数生长中后期、稳定期部分菌液制备原生质体，原生质体的形成率分别为72．1％、70．4％、60．5％，再生率为13．4％、18．9％、10．4％；溶菌酶浓度为2．5mg／mL、5mg／mL、10mg／mL、20mg／mL，酶解60min，原生质体的形成率分别为40．6％、70．8％、81．8％、95．6％，原生质体的再生率为19．0％、19．2％、19．0％、10．6％；使用10mg／mL溶菌酶酶解YS-9412-130菌30min、60min、90min、120min，原生质体的形成率分别为30．8％、81．3％、81．7％、82．9％，原生质体的再生率分别为19．2％、19．3％、16．9％、11．2％；在细菌培养液中添加终质量浓度为5mg／mL、10mg／mL、20mg／mL和40mg／mL的甘氨酸，培养该菌16h后制备原生质体，原生质体的形成率分别为81．0％、81．1％、90．3％、90．8％、90．6％，再生率为19．1％、19．0％、19．3％、12．0％、9．0％；EDTA对原生质体的形成稍有促进作用，但作用超过30min会影响原生质体的再生；分别以0．6mol／L Kcl、0．3mol／L KCl＋0．3mol／L蔗糖、0．6mol／L蔗糖作为稳定剂，原生质体的再生率分别为10．9％、19．6％、25．9％。结论认为，YS-9412-130原生质体制备的最佳条件为选用第2代YS-9412-130菌，在培养基中添加终质度浓度为10mg／mL的甘氨酸培养16h后，再将菌体置于含有0．05mol／LEDTA的高渗溶液中30℃预处理30min，用10mg／mL溶菌酶，30℃酶解60min，再用0．6mol／L蔗糖作为原生质体再生培养稳定剂，比较适合于YS-9412-130原生质体的制备与再生。这一试验结果将为通过原生质体技术对YS-9412-130菌进行遗传改良提供重要参数。 相似文献
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海洋细菌S-12-86的产溶菌酶条件 总被引：2，自引：0，他引：2
从中国东海海域底泥中筛选获得一株产溶菌酶的海洋细菌S-12-86,采用摇瓶发酵的方式,分别从培养基组分与发酵条件两个方面对海洋细菌S-12-86产酶的影响进行研究。结果表明,菌株S-12-86能够利用麦芽糖、淀粉、甘油和葡萄糖作为碳源,不能利用蔗糖与甘露醇;能够利用牛肉膏、酵母膏、蛋白胨和硫酸铵作为氮源,其中牛肉膏效果最佳,而硝酸钾、氯化铵和尿素几乎不能被利用;Zn2 、Mn2 、Cu2 对菌株S-12-86的生长和产酶均有抑制作用;Fe2 对菌体生长无明显影响,但明显抑制菌体产酶;Na 、K 对菌体的生长和产酶无明显影响;Ca2 对菌体生长有促进作用;Mg2 对菌体生长和产酶均有促进作用。菌株S-12-86最适发酵条件为:培养温度30℃、接种体积比4.0%、装液量体积比10.0%、产酶高峰在发酵开始后24 h。与白色链霉菌G(Streptomyces albusG)、灰色链霉菌P-51(S.griseusP-51)、枯草芽孢杆菌77(Bacillus subtilis77)等产溶菌酶微生物的产酶条件相比较,菌株S-12-86具有易培养、产酶量较高、生产成本较低等优点。因此,菌株S-12-86有较大的生产开发潜力。 相似文献
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产低温碱性蛋白酶黄海黄杆菌YS-9412-130高产菌株的选育 总被引：6，自引：0，他引：6
以黄海黄杆菌YS－9412－130菌株为出发株,经亚硝基胍、硫酸二乙酯、紫外线（UV）与微波（MI）复合诱变和自然选育,获得1株产低温碱性蛋白酶高产稳定的突变株YS－9412－130－SW1－104,其产低温碱性蛋白酶为出发株的16倍。在摇瓶发酵培养条件下,酶活力达2320U／ml(20℃测定）,该诱变株具有Ref^r、Str^r、Amp^r和Met^-、Lys^-标记。突变株与出发菌株在细胞形态上比较,结果表明二者没有显著差异。 相似文献
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大闸蟹肠道中产酶菌株及其胞外酶的研究 总被引：1，自引：1，他引：1
从健康成年大闸蟹肠道中分离出细菌36株。革兰氏染色结果表明，11株为革兰氏阳性菌，25株为革兰氏阴性菌。52．8％的菌株能分泌蛋白酶，44．4％的菌株能分泌脂肪酶，58．3％的菌株能分泌淀粉酶，41．7％的菌株能分泌纤维素酶。36株菌均能产酶，其中产4种酶的有1株，产3种酶的有9株，产2种酶的有14株。由于这些产酶菌株的作用，使得大闸蟹的肠道菌群能够维持平衡，提高饲料的消化利用率，促进大闸蟹生长。 相似文献
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尼罗罗非鱼肠道中产酶菌株的研究 总被引：4，自引：0，他引：4
从尼罗罗非鱼肠道中分离出41株好氧菌和6株厌氧菌。好氧菌中31株为革兰氏阴性菌，10株革兰氏阳性菌；厌氧菌中，革兰氏阳性菌5株，革兰氏阴性菌1株。41株好氧菌中有31．7％的菌株能分泌蛋白酶(13株菌)；有39．0％的菌株能分泌脂肪酶(16株菌)；有17．1％的菌株能产淀粉酶(7株菌)，有36．6％的菌株能产纤维素酶(15株菌)。其中，产3种酶的有7株菌，产2种酶的有9株菌，产1种酶的有12株菌，不产酶的有13株菌。好氧菌在尼罗罗非鱼消化食饵过程所起的作用大；6株厌氧菌中仅有1株菌能产脂肪酶和纤维素酶，对尼罗罗非鱼消化食饵所起作用相对不大。优化罗非鱼肠道中微生物菌群的结构很有必要。 相似文献
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从南极获得的260株低温细菌中筛选到107株具有蛋白酶活性菌株，其中5株菌所产蛋白酶的活性高于45U／mL。对其进行16S rRNA基因序列的同源性和系统发育分析，结果表明，菌株NJ276、NJ5—9、NJ16—70、NJ345属于假交替单胞菌属(Pseudoalteromonas)，NJ341属于科尔韦尔氏属(Colwellia)。对其中NJ276、NJ341、NJ16—70、NJ345这4株产蛋白酶南极嗜冷菌的生长及分泌蛋白酶的部分酶活特性进行研究，结果表明：(1)4株菌最适生长、产酶温度均为10℃左右；培养2～5d，嗜冷菌生长、产酶量一直处于较高的状态。(2)4株南极嗜冷菌分泌的蛋白酶的酶活反应最适pH值为9。(3)菌株NJ276、NJ5—9分泌的蛋白酶最适酶活温度为50℃；菌株NJ341、NJ345分泌的蛋白酶最适酶活温度为40℃；在0℃时蛋白酶活性是最高活性的30％左右，蛋白酶热稳定性较差，因此菌株NJ341、NJ345分泌的蛋白酶属于低温蛋白酶。 相似文献
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An improved enzyme preparation for rapid mass production of protoplasts as seed stock for aquaculture of macrophytic marine green algae 总被引：1，自引：0，他引：1
C.R.K. Reddy Shikh Dipakkore G. Rajakrishna Kumar Bhavanath Jha Donald P. Cheney Yuji Fujita 《Aquaculture (Amsterdam, Netherlands)》2006,260(1-4):290-297
Preparation of protoplasts and their subsequent applications for both basic and applied research of marine macroalgae remains largely under developed due to lack of development of reliable methods with consistent yields of viable protoplasts. An improved enzyme preparation with a single commercial enzyme, e.g. 2% Cellulase Onozuka R-10 in 1% NaCl solution, was developed to produce protoplasts rapidly from different green algal genera of Ulva, Enteromorpha and Monostroma. The simple dissolution of enzyme powder in 1% NaCl resulted in exclusion of 2% Macerozyme R-10 from the mixture consisting of 2% Cellulase Onozuka R-10 with 3% NaCl earlier reported as superior for the same algae. Optimal conditions for the isolation of maximum yields of viable protoplasts were found to be with 2% Cellulase Onozuka R-10 incubated at 20 °C for 2 h in 1% NaCl solution with 0.8 M mannitol adjusted to pH 6.0. The protoplast yield with optimized enzyme mixture was as high as 102.8 × 106 cells g− 1 f. wt for M. oxyspermum while it was in the range of 74.4–88.6 × 106 cells g− 1 f. wt thallus for seven species of Ulva, and 82.5–95.4 × 106 cells g− 1 f. wt for three species of Enteromorpha. The regeneration rate of protoplasts isolated using this method ranged from 89 to 92% with normal morphogenesis. The seeding of nylon threads with isolated protoplasts of M. oxyspermum was successful and after 3–4 weeks the entire frame with nylon threads became thick green in color with tiny germlings in laboratory culture. Thus, the method described in the present study allow for rapid mass production of viable protoplasts that could be potentially used as a source for seed material for mariculture and for other applied phycological research. 相似文献
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CHIAKI IMADA YUKO IKEMOTO TAKESHI KOBAYASHI NAOKO HAMADA ETSUO WATANABE 《Fisheries Science》2002,68(2):395-402
ABSTRACT: Protoplast fusion between different species of Streptomyces was performed using a liquid regeneration method developed for a rapid and simple preparation of the fusants. Consequently, new clones, which could not be obtained using the conventional agar regeneration method, were obtained. In the crosses between S. griseus and S. durhamensis , and between S. californicus and S. catenulae , eight and two recombinants, respectively, were obtained using the liquid regeneration method. Conversely, in the case of crosses between S. ornatus and S. catenulae , and between S. ornatus and S. vendargensis , seven recombinants each were obtained using only the agar method. The physiological characteristics, such as the assimilation of carbohydrate and antibiotic resistance, of these fusants differed considerably from those of their parental strains. Using the proposed liquid regeneration method, a simpler and quicker procedure for protoplast fusion is described. 相似文献
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为减少加工废弃物蛋白所造成的环境污染,提高水产品的利用率,探索了回收利用鲅鱼(Spanish mackerel)加工后富含蛋白质的废弃物,以寻找制备鲅鱼抗氧化肽的最佳生产条件。借助Design-Expert数据处理软件对苏云金芽孢杆菌Hy-4发酵产抗氧化肽的条件进行优化;在单因素试验的基础上,利用PlackettBurman试验设计法对影响发酵制备鲅鱼抗氧化肽的培养条件进行筛选。确定了影响发酵液总抗氧化活性的3个主要影响因素(P0.5)为发酵温度、培养基初始p H和料液比;在此基础上,利用最陡爬坡实验逼近3个关键因素的最大响应区域,再利用Box-Behnken试验设计及响应面分析法进行回归分析,通过求解回归方程得到产抗氧化肽的最优条件为:发酵时间48 h,发酵温度30℃,培养基初始p H 6.8,接种量2%,料液比1.45 g/50 m L,菌龄24 h。经过验证,发酵液总抗氧化活性达到537.73 U,较优化前提高了57.2%,与回归方程的预测值570.11 U相比,相对误差为5.7%,说明模型能够较好地预测菌株Hy-4发酵产抗氧化肽发酵液的总抗氧化性。 相似文献
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为了提高水产加工副产物的利用率和附加值,以鲣加工副产物为原料,采用胰蛋白酶结合超声处理制备蛋白胨,并分析了鲣加工副产物超声辅助酶解过程中蛋白酶活性、水解度、氮回收率、TCA-可溶性氮回收率、酶解产物分子量分布等指标的变化,探究了超声辅助酶解的作用时间、温度以及超声功率等因素对鲣加工副产物酶解产物的影响,通过单因素实验优化确定了蛋白胨制备的最佳酶解工艺条件。结果显示,超声处理造成胰蛋白酶酶活发生显著变化,在功率为120 W的超声场下,胰蛋白酶酶活在30 min内呈上升趋势,但在超声场处理30 min后,胰蛋白酶的酶活呈明显下降趋势,至4 h后酶活变化趋势趋于平缓。超声功率和酶解温度都会影响胰蛋白酶对鲣加工副产物的酶解效率,在超声功率240 W和酶解温度50°C条件下,氮回收率比无超声处理组提高了18.66%,水解度提高了30.43%,TCA-可溶性氮回收率提高了33.20%,且超声处理有效提高了酶解液中小分子肽的含量。超声辅助酶解鲣加工副产物的最佳工艺条件为温度55°C,超声功率240 W,酶解时间4 h,此时氮回收率为81.40%,TCA-可溶性氮回收率为40.75%。 相似文献
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Eight light‐intensity treatments (natural light, continuous darkness, and 15, 30, 60, 125, 250 and 500 lx under LD 12:12 cycle) were used to investigate the effects of light intensity on the daily activity of 30.27±3.08 g sea cucumber Apostichopus japonicus (Selenka). Cyclic nocturnal activity patterns of behaviour were observed at different light intensities in the range of 15–500 lx under LD 12:12 cycle. And an ongoing nocturnal cycle persisted in DD cycle for up to 8 days, but longer feeding time and less marked rhythm occurred at continuous darkness. Under poor light conditions (I<5.18 lx), the daily activity rhythm of A. japonicus was governed by an innate biological clock and the effect of light intensity was not significant among different treatments. And more individuals tended to retreat to shelters (from 56.04% to 91.83%) with the increase of light intensity within the weak light condition (from 5.18 to 278 lx). However, the daily behaviours of A. japonicus were influenced under strong light conditions (>278 lx). Less than 8.17% individuals kept actively feeding and the proportion was not decreased with the increase of light intensity. 相似文献
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Enshi Zhang Chiaki Imada Masazumi Kamata Takeshi Kobayashi Naoko Hamada-Sato 《Fisheries Science》2008,74(6):1290-1296
An attempt was made to improve the stability toward centrifugation of protoplast fusion between Shewanella sp. and Escherichia coli. Stability of the cell membrane is an important factor in protoplast fusion. In order to change the fatty acid composition
of the cell membrane phospholipids, eight fatty acids [caprylic acid, capric acid, palmitic acid, oleic acid, linoleic acid,
linolenic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid] were added to each nutrient medium of Shewanella sp. and E. coli. The protoplasts were treated with lysozyme, and fusion occurred in the presence of a polyethylene glycol solution. The stability
of the protoplast of Shewanella sp. decreased after EPA was added to the culture medium, and the stability of the protoplast of E. coli increased after the addition of linoleic acid or linolenic acid. Some fusant colonies that developed on the regenerated medium
selected for E. coli with antibiotic tolerance. The efficiency of this fusion was higher than that of initial condition using protoplasts from
Shewanella sp. and E. coli incubated without fatty acids. Protoplasts improved the fatty acid composition of the phospholipids. Cell membrane stability
can change in order for the weak cells to be taken in by strong cells. These results suggested that the fatty acid composition
of cell membrane phospholipids affected the fusant yield of the fusion of these bacteria. 相似文献
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盐度对黄鲷胚胎发育及早期仔鱼生长的影响 总被引：16，自引：0，他引：16
The effect of salinity on embryo and early larva development of Dentex tumiforns was discussed. Floating forms of egg in different salinities, optimal salinity for embryonic development and early larva growth was studied. The results showed: 1. In immobile condition, all eggs sank at salinity of 32.0, most of egg ssuspended in the middle of water at 34.0, and all eggs floated on the surface when salinity above 36.0. 2. Eggs did not hatch out at salinity of below 10.0 or above 60.0 and the death time of egg gradually moved up with increase or decline of salinity. Eggs hatched out at salinity range of 15.0 and 50.0, including abnormal larva. There was indistinct difference in speed of embryonic development (about 36-40h) within the salinity of 15.0 and 50.0. However the salinity had a great effect on larval survival after hatching and abnormal rate. The relation between hatching rate and salinity variation showed parabola but abnormal rate showed inverted parabola. The hatching rate was 81%-86% and abnormal rate of yolk sac larva was 27%-30% in suitable salinities of 27.0-39.0. The hatching rate was 89%-91% and abnormal rate was 13%-16% in optimal salinities of 33.0-36.0. The oil ball of abnormal larva was located in the central or front position. With increased extent of salinity, spondyle of larva bended and number of larva with arrhythmia increased. 3. The optimal salinities were 30.0-35.0 based on SAI. The test with SAI showed that SAI of early larvae was 41.25-47.53 at salinities of 30.0-35.0, the yolk and oil ball of 7-8 days larva was completely absorbed with a survival rate of 88%. 相似文献