克鲁维酵母高渗培养与原生质体制备与再生

研究了高渗预培养条件对克鲁维酵母Ｙ０３４原生质体形成与再生的影响。结果表明,同等条件下,经高渗预培养的原生质体再生率为常规培养的１．７倍,为改造菌株遗传特性而制备高活性原生质体探索了新的途径。     

毕氏酵母营养缺陷株P315株原生质体形成和再生的研究

通过单因子试验和正交试验摸索了一株毕氏酵母(Pichia sp.)原生质体形成和再生的较优条件。培养对数生长早期的细胞(2—7×10~7个/ml),在2％蜗牛酶和0.1％β-巯基乙醇的作用下,60—80分钟原生质体形成率达97—100％,再生率为3—4％。并试验了不同渗透压稳定剂对再生的影响。     

酿酒酵母与球形红假单胞菌原生质体跨界融合研究

测定了球形红假单胞菌原核细胞与酿酒酵母真核细胞的原生质体形成、再生及融合最佳组合条件。以溶菌酶ＥＤＴＡ反应系统处理球形红假单胞菌Ｐ９４７９,最适脱壁条件为：溶菌酶浓度０.５ｍｇ／ｍｌ,ＥＤＴＡ浓度为０.２％,蔗糖浓度２０％,酶作用时间为４０ｍｉｎ;此条件下原生质体形率为７８.９％,再生率为１１.２％。用蜗牛酶巯基乙醇反应系统处理酿酒酵母Ｙ９４０７,最适脱壁条件为：蜗牛酶浓度１.０％,巯基乙醇浓度０.１％,蔗糖浓度２０％,酶作用时间为３０ｍｉｎ;此条件下酵母原生质体形成率为９９.８％,再生率为９.７％。用聚乙二醇诱导Ｐ９４７９与Ｙ９４０７的原生质体发生融合,当聚乙二醇的浓度为３０％,Ｃａ２＋浓度为５０ｍｍｏｌ／Ｌ,ｐＨ为６.５,时间为１０ｍｉｎ时,有最高的融合率为７.６×１０－６。     

香菇单核菌丝9101（12）原生质体再生条件的研究

本文对香菇（Ｌｅｎｔｉｎｕｓｅｄｏｄｅｓ）单核菌丝９１０１（１２）原生质体制备后的再生条件,进行了探索。结果表明,原生质体经过合０．６Ｍ．甘露醇的麦芽糖酵母粉培养基（ＭＹＧ）液体培养５天,用双层培养法,在含０．６ＭＭｇＳＯ４的高渗ＭＹＧ平板上经再生,再生率达５×１０－５。本文将Ｋｎｏｗｌｔｏｎ等于１９８４年创造的一种分离高频再生衍生株的方法首次运用至食用菌中,所得９１０１（１２）再生株Ｒ３,再生率提高２～３倍,为香菇原生质体融合操作打下基础。     

假丝酵母原生质体形成与再生的研究

本文研究了菌龄、酶系统、脱壁促进剂、渗透压稳定剂对假丝酵母Y002 原生质体形成与再生的影响。综合考虑形成率与再生率后,选用对数生长中期的菌体于1%蜗牛酶、 35℃下作用60分钟制备原生质体,并于以17%蔗糖作渗透压稳定剂的再生培养基中再生效果为好。     

原生质体融合构建耐温酵母菌株(英文)

耐温的克鲁维酵母（Ｙ０３４）与产酒率高的酿酒酵母（Ａ００１）进行属间原生质体融合构建耐温酵母菌株,经ＤＴＴ分段预处理获得大量具再生活性原生质体对融合株ＡＹ０２３等进行了乙醇脱氢酶同工酶,麦芽糖同化,遗传稳定性及高温发酵分析,融合株ＡＹ０２３表达了双亲遗传特性。在４５℃高温发酵条件,乙醇产率高达７．４％,是目前已见文献报道的产酒率最高的耐温（４５℃）酵母菌株     

原生质体融合构建耐温酵母菌株

耐温的克鲁维酵母与产酒率高的酿酒酵母进行属间原生质体融合构建耐温酵母蓖株,经ＤＴＴ分段预处理获得大量具再生活性原生质体对融合株ＡＹ０２３等进行了乙醇脱氢酶同工酶,麦芽糖同化,遗传稳定性及高温发酵分析,融合株ＡＹ０２３表达了双亲遗传特性,在４５℃高温发酵条件,乙醇产率高达７．４％,是目前已见文献报道的产酒率最高的耐温酵母菌株。     

克鲁维酵母种间原生质体融合的研究

乳酸克鲁维酵母(Kluyueromyces lactis Y12—1)和脆壁克鲁维酵母(K.fragilis8554)是乳糖酶生产菌株。应用原生质体融合技术进行了两菌株种问融合的研究。通过试验．原生质体形成及再生的最佳条件为：对数期的细胞,2％的蜗牛酶．30℃酶解30分钟．原生质体形成率90％以上,再生率20%左右。原生质体融合由聚乙二醇(PEG)诱导。K.lactisY12-l不能旋酵菊糖;K.fragilis 8554不能同化D－松三糖和麦芽糖;利用二菌株自身的营养缺陷性质获得融合子。融合子既能发酵菊糖又能同化D－松三糖和麦芽糖;融合子的DNA含量约为二亲株之和;融合子的菌落形态与亲株相比有一定差别．在以乳糖为碳源的培养基中,融合子的乳糖酶产量提高14一l6％;连续15次传代,融合子稳定。     

利用原生质体诱变育种选育富硒能力强的酵母菌株

利用原生质体诱变育种技术选育富硒能力强的酵母菌株,从13株啤酒酵母中筛选出一株富硒量高的诱变出发菌株,采用溶壁酶进行破壁,确定了原生质体制备的最适条件为酶浓度1g／100mL,酶解处理时间为120min,原生质体形成率为95．2％,再生率为21．8％,诱变后筛选出富硒量为821mg／kg,酵母干菌体收获量为0．88g／100mL的酵母菌Al。     

欧文氏菌和棒杆菌的属间隔合研究

研究了用原生质体融合技术获得欧文氏菌和棒杆菌的融合细胞,串联发酵Ｄ－葡萄糖产生２－酮基－Ｌ－古龙酸的第一步发酵菌株欧文氏菌ＳＣＢ２４７经０．８ｍｇ／ｍＬ溶菌酶酶解０．５ｈ后,原生质体的形成率和再生率分别为９９．８％和２７．８％。第二步发酵菌株棒杆菌ＳＣＢ３０５８经预处理后由１．３ｍｇ／ｍＬ溶菌酶酶解２ｈ,原生质体的形成率和再生率分别为９９．５％和５６．３％,用携带氨苄青霉素抗性标记的ＳＣＢ２４７和     

Isolation and Characterization of Protoplasts from Saccharomyces rouxii

Cells of the osmotolerant yeast Saccharomyces rouxii were transformed to protoplasts in good yield (85%) by digesting cell walls with snail-gut enzyme in the presence of 10 mM dithioerythritol, 0.1 M sodium phosphate buffer (pH 6.8), and 2.0 M KCl. The requirement for 2.0 M KCl compares with that for S. bisporus var. mellis (another osmotolerant species) and contrasts with the 0.3 to 0.8 M KCl concentrations used in the preparation of most yeast protoplasts. Short digestions (60 min or less) produced mostly spheroplasts; longer incubations (90 min or more) yielded mostly protoplasts as judged by electron micrographs. These protoplasts could be transferred to 1.0 M KCl or 2.0 M sorbitol without lysing, but lysis was pronounced in 0.5 M KCl or 1.0 M mannitol and complete in 0.02 M KCl. Protoplasts were separated from isolated cell wall remnants and debris by centrifugation on a linear gradient of Ficoll 400 (35 to 17.5%, wt/vol) containing 2.0 M KCl. Both crude and fractionated protoplast preparations contained vesicles which were identified with the periplasmic bodies of whole cells. Some of the periplasmic bodies were connected to protoplasts by fine pedicels; others appeared free. Independent degeneracy of periplasmic bodies was occasionally observed. beta-Fructofuranosidase (EC 3.2.1.26) activity is cryptic (physically) in cells of S. rouxii in contrast to the expressed enzyme (periplasmic space) of other Saccharomyces species. This enzyme remains cryptic in protoplast preparations of S. rouxii but is expressed upon lysis. The same specific activities were found per unit cell or protoplast. The possible association of the cryptic enzyme with periplasmic bodies is discussed.     

酵母菌Y17吸附Cu2+的影响因素及吸附机理研究

以高效吸附Cu2+的酵母菌Y17为材料, 对其吸附Cu2+过程中的主要影响因素, 包括溶液pH、Cu2+初始浓度、菌体添加量、吸附时间和温度以及吸附机理进行了探讨。结果表明, 对吸附过程影响较大的因素依次为吸附液pH值、Cu2+初始浓度、菌体添加量和吸附时间。正交试验得到最佳吸附条件为溶液pH5.0, 吸附时间40 min, 加菌量5.0 g湿菌/L时, 对初始浓度为8 mmol/L的Cu2+达到最佳吸附率为82.7%。通过对Y17菌体不同处理及解吸实验, 初步确定Y17吸附Cu2+的位点在细胞壁, 细胞壁表面的-NH2, -COOH基团在其吸附过程中起着重要作用。     

酵母菌Y17吸附Cu2+的影响因素及吸附机理研究

以高效吸附Cu2 的酵母菌Y17为材料,对其吸附Cu2 过程中的主要影响因素,包括溶液Ph、Cu2 初始浓度、菌体添加量、吸附时间和温度以及吸附机理进行了探讨.结果表明,对吸附过程影响较大的因素依次为吸附液Ph值、Cu2 初始浓度、菌体添加量和吸附时间.正交试验得到最佳吸附条件为溶液Ph5.0,吸附时间40min,加菌量5.Og湿菌/L时,对初始浓度为8mmol/L的Cu2 达到最佳吸附率为82.7%.通过对Y17菌体不同处理及解吸实验,初步确定Y17吸附Cu2 的位点在细胞壁,细胞壁表面的-NH2,-COOH基团在其吸附过程中起着重要作用.     

Response to chilling of tomato mesophyll protoplasts

Freshly isolated protoplasts from tomato leaves show two completely different responses to a chilling treatment of 12 h at 7° C prior to culture at 29° C, depending on the presence or absence of glucose in the medium. In the culture medium with glucose as osmoticum, where the rate of cell divisions under optimal culture conditions is relatively high (about 20% plating efficiency), protoplasts were drastically injured by the chilling procedure and died. In the medium with mannitol as the osmoticum instead of glucose, where the plating efficiency even under optimal conditions is rather low (about 8%), protoplasts withstand the chilling procedure. More-over, after the chilling treatment when the protoplasts were transferred to the optimal culture temperature of 29° C, the plating efficiency was raised to about 20%, which is the same level as in the glucose-containing medium without chilling. This effect was not observed when the medium in which the protoplasts were suspended during the chilling period was replaced with fresh medium. This suggests that under these conditions tomato protoplasts produce and excrete a factor in the cold that improves the vitality of the cells or stimulates cell division. The possible relationship between chilling sensitivity of tomato protoplasts and their ability to divide will be discussed.     

Wall mannoproteins of the yeast and mycelial cells of Candida albicans: nature of the glycosidic bonds and polydispersity of their mannan moieties

Zymolyase released between 20 and 25% of the total protein from purified walls of yeast (Y) and mycelial (M) cells of Candida albicans. The material released contained 92% carbohydrate (86% mannose and 6% glucose) and 7% protein. Over 85% of the carbohydrate was N-glycosidically linked to the protein and the rest (less than 15%) was linked O-glycosidically. Highly polydisperse, high molecular mass mannoproteins, resolved by electrophoresis as four defined bands in Y cells and two bands in M cells, had both types of sugar chains. A 34 kDa species found in both types of cells had a single 2.5 kDa N-glycosidically linked sugar chain and a 31.5 kDa protein moiety. Polydispersity in the high molecular mass mannoproteins was due to the N-linked sugar chains (mannan) with a molecular mass between 500 kDa and 20 kDa (average 100 kDa) in Y cells and between 400 kDa and 20 kDa (average 50 kDa) in M cells. Three mannoproteins of 34, 30 and 29 kDa secreted by protoplasts were associated with the high molecular mass mannoproteins, suggesting that this type of interaction might be related to the regeneration of the cell wall.     

浆水直投式增香酵母冻干菌剂制备工艺的优化

以一株分离自甘肃天水传统酿造浆水中的产香酵母——异常汉逊酵母（Hansenula anomala）Y16为供试菌株,经高密度发酵后,采用Plackets-Burman试验、最陡爬坡实验及响应面法（response surface methodology,RSM）,对其直投式增香酵母冻干菌剂的制备工艺进行优化。结果表明,经高密度增殖培养后,酵母Y16细胞浓度可达9.2×1010 CFU/mL;收集Y16发酵液的最佳离心条件：Y16发酵液经1 600 g离心10 min,离心沉降活细胞率为95.5%;冻干保护剂最佳组合配方：11%脱脂奶粉,9%蔗糖,12%海藻糖,在该条件下,冻干菌剂中酵母存活率为91.54%。经上述工艺制备的增香发酵剂保存3个月仍具有较好的贮藏稳定性。此外,浆水模拟发酵试验的结果表明,浆水风味优良。研究结果为浆水工业化生产提供了良好的增香发酵剂来源。     

Optimization of single-cell-protein production from cassava starch using Schwanniomyces castellii

Schwanniomyces castellii B5285 grew faster and produced greater biomass and higher protein yield than either S. alluvius ATCC 26074 or S. alluvius 81Y when these amylolytic yeasts were grown with 2% (w/v) cassava starch as sole C source. With 0.5% (w/v) glutamate as N source, S. castellii reached 7.12 g cell dry mass/l, with a protein yield of 6.4 g/100 g starch. The optimal agitation speed, aeration rate and pH for growth of this yeast in a fermenter were 400 rev/min, 1.67 vol./vol.min. and 5.0, respectively. Tween 80 at 0.1% increased cell dry mass to 8.90 g/l, cell yield to 44 g/100 g starch and protein yield to 7.4 g/100 g starch.The authors are with the Department of industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai 90110, Thailand     

Lysis of Saccharomyces cerevisiae with 2-deoxy-2-fluoro-D-glucose, an inhibitor of the cell wall glucan synthesis

The effect of a synthetic glucose analogue, 2-deoxy-2-fluoro-d-glucose (FG) on growth and glucose metabolism of Saccharomyces cerevisiae was studied. The addition of FG (0.005-0.05%) to a 2% glucose medium resulted in reduction of the initial growth rate and, after several hours, in a complete cessation of the culture growth. These two events were due to extensive lysis of the population which continued long after the period when no more growth was recorded. Electron microscope examination of lysed cells showed that the lysis was a consequence of a dissolution of the cell walls. FG inhibited to a similar extent the initial growth rate and the incorporation of radioactivity from labeled glucose into growing population. The inhibition of radioactivity incorporation from glucose by growing protoplasts was much less. The yeast was found to be extremely FG sensitive whenever the synthesis of new cell wall material was involved. All observations imply that FG interferes mainly with the cell wall formation of S. cerevisiae. A comparison of the FG effects on metabolic activity of protoplasts, simultaneous secretion of mannan-proteins into the growth medium, and the formation of glucan fibrils on the surface of protoplasts demonstrated that the cell wall glucan synthesis is the most FG-sensitive process and evidently the growth-limiting factor in intact cells. FG-resistant cells were selected during growth experiments. They exhibited an altered mode of cell division when grown in the presence of FG.     

Reversion of protoplasts and spheroplasts in the yeast Nadsonia elongata

Summary The time rate of regeneration of the cell wall and reversion of protoplasts of the yeast Nadsonia elongata to cells of normal shape and size has been compared with the capability for regeneration of spheroplasts of this yeast. Nearly all protoplasts in a given culture were able to regenerate new walls and had usually reverted to cells of normal appearance by the 30th h of cultivation. Spheroplasts required only half this time to do this. These results can be interpreted as evidence that regeneration of a wall by protoplasts does not depend upon the presence of a cell wall primer, because the proportion of reverting protoplasts (which lack wall remnants) was the same as that of reverting spheroplasts (which possess them). The presence of wall remnants in spheroplasts appears to have merely an accelerating effect on the formation of a new wall and on subsequent reversion of the spheroplasts to complete cells of normal shape and size.