Strain improvement of thermotolerant Saccharomyces cerevisiae VS strain for better utilization of lignocellulosic substrates

AIM: Pentose-utilizing yeast development by protoplast fusion and sequential mutations and ethanol fermentation using lignocellulosic substrate. METHODS AND RESULTS: Protoplasts of thermotolerant Saccharomyces cerevisiae and mesophilic, xylose-utilizing Candida shehatae were fused by electrofusion. The fusants were selected based on their growth at 42 degrees C and ability to utilize xylose. The selected best fusant was mutated sequentially and 3 mutant fusants obtained were tested for their stability. The mutant fusant CP11 was found to be stable and used for lignocellulosic fermentation. The Prosopis juliflora wood material was hydrolysed with 1% sulphuric acid initially for 18 h at room temperature and then for 20 min at 121 degrees C. The acid hydrolysate was separated and used for detoxification by ethyl acetate and overliming. The hard cellulosic fraction was hydrolysed with Aspergillus niger crude cellulase enzyme for 18 h at 50 degrees C. The substrate (15% w/v) yielded 84 g l(-1) sugars, representing 80% (w/w) hydrolysis of carbohydrate content present in the lignocellulosic material. The acid and enzyme hydrolysates were then equally mixed and used for fermentation with the developed fusant yeast (CP11). The fusant yeast gave an ethanol yield of 0.459 +/- 0.012 g g(-1), productivity of 0.67 +/- 0.015 g l(-1) h(-1) and fermentation efficiency of 90%. CONCLUSIONS: Protoplast fusion followed by sequential mutations method gave a stable and good performing fusant with maximum utilization of reducing sugars in the media. SIGNIFICANCE AND IMPACT OF THE STUDY: This new method could be applied to develop fusants for better biotechnological applications.     

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.     

玉米、小麦、水稻原生质体制备条件优化

玉米Zea mays L.、小麦Triticum aestivum L.、水稻Oryza sativaL.是三大重要粮食作物,对其原生质体制备条件的优化具有重要意义.以玉米(综3)、小麦(中国春)、水稻(日本晴)10日龄幼苗为材料,研究了叶肉细胞原生质体分离过程中的酶浓度、酶解时间和离心力大小等因素对产量和活力的影响.结果表明:酶浓度和酶解时间对原生质体产量影响显著,随着酶解液浓度和酶解时间的提高,原生质体产量增加,但细胞碎片同时增多.水稻经真空处理后,原生质体产量大幅度提高.通过正交实验设计得出如下结果:玉米叶肉细胞原生质体分离的最佳条件为:纤维素酶1.5％,离析酶0.5％,50 r/min酶解7h,100×g离心2 min收集,原生质体产量为7×106/g FW;小麦叶肉细胞原生质体分离的最佳条件为:纤维素酶1.5％,离析酶0.5％,50 r/min酶解5h,100×g离心2 min收集,原生质体产量为6×106/g FW;水稻叶肉细胞原生质体分离的最佳条件为:纤维素酶2.0％,离析酶0.7％,50 r/min酶解7h,1 000×g离心2 min收集,得到的原生质体产量为6×106/g FW.通过二乙酸荧光素染色发现原生质体活力均在90％以上.用PEG-Ca2+介导法将含有绿色荧光蛋白的质粒转化入原生质体,转化率可达50％ ～80％.     

Purification and biochemical properties of a galactooligosaccharide producing β-galactosidase from Bullera singularis

A beta-galactosidase, catalyzing lactose hydrolysis and galactooligosaccharide (GalOS) synthesis from lactose, was extracted from the yeast, Bullera singularis KCTC 7534. The crude enzyme had a high transgalactosylation activity resulting in the oligosaccharide conversion of over 34% using pure lactose and cheese whey permeate as substrates. The enzyme was purified by two chromatographic steps giving 96-fold purification with a yield of 16%. The molecular weight of the purified enzyme (specific activity of 56 U mg(-1)) was approx. 53 000 Da. The hydrolytic activity was the highest at pH 5 and 50 degrees C, and was stable to 45 degrees C for 2 h. Enzyme activity was inhibited by 10 mM Ag3+ and 10 mM SDS. The Km for lactose hydrolysis was 0.58 M and the maximum reaction velocity (V(max)) was 4 mM min(-1). GalOS, including tri- and tetra-saccharides were produced with a conversion yield of 50%, corresponding to 90 g GalOS l(-1) from 180 g lactose l(-1) by the purified enzyme.     

Production and ecological significance of yeast cell wall-degrading enzymes from oerskovia

Motile actinomycetes capable of degrading walls of viable yeast cells were isolated from soil and identified as Oerskovia xanthineolytica. A lytic assay based on susceptibility of enzyme-treated cells to osmotic shock was developed, and 10 of 15 strains of O. xanthineolytica, Oerskovia turbata, and nonmotile Oerskovia- like organisms from other collections were found to possess yeast lytic activities. All lytic strains produced laminaranase and alpha-mannanase, but the amounts, determined by reducing group assays, were not proportional to the observed lytic activities. The Oerskovia isolates demonstrated chemotactic, predatory activity against various yeast strains and killed yeasts in mixed cultures. Of 15 carbon sources tested for production of lytic enzyme, purified yeast cell walls elicited the highest activity. Glucose repressed enzyme production and caused cells to remain in the microfilamentous and motile rod stages of the Oerskovia cell cycle. Crude lytic activity was optimal at pH 5.6 to 7.0 and inactivated by heating for 6 min at 50 degrees C. Partial purification by isoelectric focusing showed that all lytic activity was associated with four beta-(1-->3)-glucanases. The absence of protein disulfide reductase, N-acetyl-beta-d-hexosaminidase, and phosphomannanase in crude preparations indicated that the principal enzyme responsible for yeast wall lysis was a beta-(1-->3)-glucanase that produced relatively little reducing sugar from yeast glucan.     

“红颜”草莓原生质体制备及瞬时转化体系的建立

为探索“红颜”草莓悬浮细胞系原生质体提取的最优条件,并建立“红颜”草莓原生质体瞬时转化体系,以“红颜”草莓悬浮细胞为材料,对酶液组成、酶解温度、酶解方式进行研究。用PEG介导的瞬时转化法将标记基因GFP转化到“红颜”草莓原生质体中。结果显示：以“红颜”草莓悬浮细胞系作为分离材料,酶液组合为CPW中含有0.5%PVP+0.1%MES+1%纤维素酶+0.5%离析酶+0.01%半纤维素酶+0.9 mol/L甘露醇,在低速（50 r/min）恒温（31 ℃）震摇下进行酶解反应,酶解10 h时,达到“红颜”草莓原生质体最佳分离效果,每克鲜重产量可得原生质体6×10⁸ 个,活力值可达93.0%。PEG介导法成功将含有绿色荧光蛋白（green fluorescent protein, GFP）的植物表达载体转化“红颜”草莓悬浮细胞原生质体,转化效率达44%。通过实验筛选得到“红颜”草莓悬浮细胞原生质体的最佳制备条件,建立“红颜”草莓悬浮细胞原生质体的瞬时转化体系,为进一步开展“红颜”草莓功能基因及合成生物学研究奠定基础。     

Protoplast isolation in the marine brown alga Dictyopteris prolifera (Dictyotales)

Protoplasts were isolated from thalli of Dictyopteris prolifera using a mixture of crude enzymes from vicera of live oysters (Crassostrea gigas) and the following commercial enzymes: an abalone enzyme, cellulase, polygalacturonase and hemicellulase. The enzyme mixtures produced up to 3.3 × 10⁷ cells per l g of tissue fresh weight. The conversion to protoplasts of the cells was about 100% using the oyster enzyme or the abalone enzyme alone. The optimum pH for protoplast isolation was 6.0 and 20 hours were required for conversion to protoplasts.     

Enzymatic Removal of Diacetyl from Beer : III. Enzyme Protection and Regeneration of Cofactor

Use of diacetyl reductase, a reduced nicotinamide adenine dinucleotide (NADH)-requiring enzyme, to eliminate diacetyl off-flavor in beer was studied. The crude enzyme was extracted from Aerobacter aerogenes and partially purified by ammonium sulfate precipitation or Sephadex chromatography. In the semipure state, the enzyme was inactivated by lyophilization; in a crude state, the lyophilized extract remained stable for at least 4 months at - 20 C. A 50% reduction in specific activity within 5 min was observed when crude diacetyl reductase was suspended (5 mg of protein/ml) in phosphate buffer at pH 5.5 or below; a similar inactivation rate was observed when the crude enzyme was dissolved in a 5% aqueous ethyl alcohol solution. Effective crude enzyme activity in beer at a natural pH of 4.1 required protection of the enzyme in 10% gelatin. Incorporation of yeast cells with the gel-protected enzyme provided regeneration of NADH. Combinations of yeast, enzyme, and gelatin were tested to obtain data analyzed by regression analysis to determine the optimal concentration of each component of the system required to reduce the level of diacetyl in spiked (0.5 ppm) beer to less than 0.12 ppm within 48 hr at 5 C. The protected enzyme system was also effective in removing diacetyl from orange juice (pH 3.8) and some distilled liquors.     

沙冬青子叶原生质体瞬时表达体系的建立及其AmDREB1蛋白的亚细胞定位

以沙冬青(Ammopiptanthus mongolicus(Maxim.ex Kom.)Cheng f.)幼苗的子叶为材料,对其原生质体的分离、纯化和瞬时表达体系进行了研究。结果表明,子叶原生质体分离的最佳酶解液组成为CPW溶液+3.0%纤维素酶R-10+0.5%离析酶R-10+0.3%半纤维素酶+9.0%甘露醇(p H5.8);最佳酶解条件为室温、避光、40 r/min轻摇14 h。采用W5溶液作为漂洗液将酶解物稀释后进行过滤,将过滤液在4℃、700 r/min条件下离心5 min,所得纯化原生质体的产量约为2.50×106cells/g,活力达到90%;以纯化的原生质体作为受体,利用聚乙二醇(PEG)介导法成功将植物瞬时表达载体p BI-GFP导入其中,转化效率达到50.8%。利用本研究建立的原生质体瞬时表达体系,检测到沙冬青脱水应答转录因子Am DREB1定位于细胞核内。     

Isolation,Culture and Plant Regeneration of Protoplasts from an Embryogenic Callus Tissue of Gossypium hirsutum

Protoplasts were isolated and cultured from hypocotyl embryogenic callus tissue of Gossypium hirsutum L. cv. "Lumian 6". The highest yields of viable protoplasts were obtained from a vigorous embryogenic callus 7 to 9 d old subcultured on MS medium supplemented with 2 mg/L IAA and 1 mg/L KT using a solution of 1% cellulase Onozuka R-10, 1% pectinase, 0.7 mmol/L KH2PO4, 2.5 mmol/L Ca2+ , and 0.5 mol/L osmoticum (mannitol), at pH 5.8 and at a temperature of 30 ℃. After separation and purification (in 21% sucrose floatation medium), the protoplasts were laid up in a quiet liquid protoplast culture medium containing K3 salts, NT vitamins with 0.1 mg/L 2,4-D, 0.2 mg/L KT and 0.45 mol/L glucose for 10 to 15 min. The protoplasts were fractioned into an upper and a lower layer in the centrifugal tube. Most of the protoplasts in the lower layer were smaller, round and rich in cytoplasts in which contain many granular substances. When this kind of protoplasts were cultured in the thin liquid protoplast culture medium with a density of 1 x l0s to 5 x los protoplasts/mL, the division and the callus formation of the regenerated cells were easily observed. The first divisions occurred in 3 days and small cell clusters could be seen after 2 to 3 weeks in the culture. At this moment, the addition of the protoplast culture medium with decreased osmoticum once or twice is needed for the continuous protoplasts division to form calli. Regenerated calli, 3 to 5 mm in diameter, were transferred in succession on MS medium with 2 mg/L IAA and 1 mg/L KT for the initiation of embryogenesis. The embryoids germinated on the hormonefree MS medium and a number of plantlets were obtained. It seems that using vigorous embryogenic callus and decreasing osmoticum are the two critical factors for plant regeneration of cotton protoplasts.     

Plant Regeneration from Protoplasts of Ligusticurn wallichii Franch

The hypocotyls of the embryoid derived plantlets of Ligusticum wallichii Franch were used for protoplast preparation. Protoplasts were obtained with the enzyme mixture containing 1.5% Onozuka R-10, 0.3% Macerozyme R-10, 0.5% Snailase, 5 mmol/l CaCl2, 1 mmol/l KH2PO4, 0.6 mol/l manitol, at pH 5.6–5.8 and 27℃. Protoplasts were cultured in a modified MS liquid medium containing 1 mg/l 2,4-D + 0.5 mg/l 6- BA. The first divisions were found after twelve days, and the dividing cells formed cell colonies of 0.5–1 mm after about fourty days. When they were transferred to MS agar medium (with half quantity of macronutrients) supplemented with 2,4-D (0.5mg/l) and 6-BA(0.5mg/l), they grew into calli. At last, on the medium without any phytohormones, the growing calli differentiated embryoids which developed into plantlets with many green leaves and roots.     

Some characteristics of human adrenal microsomal 21-hydroxylase activity

The following general characteristics of 21-hydroxylase activity were determined using pooled microsomes obtained from three glands. Enzyme activity exhibited a broad pH dependence, being optimal between pH 7.4-pH 7.8, and was maximal with NADPH in the range 2 to 4.75 X 10(-4)mol/l. No microsomal 21-hydroxylase activity was detected in the absence of NADPH or substrate and when heat denatured microsomes were employed. Enzyme activity was depressed by greater than 75% in the presence of 100% oxygen or nitrogen. In a second set of experiments, microsomal fractions were prepared individually from 7 glands. In the presence of 17 alpha-hydroxy progesterone (2.0 X 10(-7) and 2.0 X 10(-6)mol/l) product formation was linear with time for up to 90 s when the microsomal protein concentration was 5, 10 and 20 micrograms/ml. Between 5 and 30% of the substrate was converted during the first 60 s. In 5/7 of the glands the addition of the autologous cytosol (20 micrograms protein/ml) was without effect, and enzyme activity (using a 60 s reaction and either 2.0 X 10(-7) or 2 X 10(-6)mol/l 17 alpha-hydroxy progesterone was directly proportional to the microsomal protein concentration (range 0-20 micrograms/ml). With the other 2 adrenals 21-hydroxylation was not proportional to the same range of microsomal protein concentrations, although it became so upon the addition of cytosol, which significantly augmented activity. There was considerable variation in enzyme activity between glands from different individuals (Vmax ranging from 2.6 to 16.6 X 10(-9) mol/min/mg protein) and in the apparent Km's (from 0.22 to 1.1 X 10(-6)mol/l). In the two preparations sensitive to cytosol, the Vmax increased 2-fold, and the Km was 3 times lower. Cytosol was without effect upon the kinetic characteristics of the other 5 microsomal preparations. Ascorbic acid (1 X 10(-3) mol/l) depressed enzyme activity by 25-43% whereas oxidised and reduced glutathione (1 X 10(-3) mol/l) showed a slight and variable effect upon 21-hydroxylation.     

Purification and some properties of thiosulphate-cleaving enzyme from Thiobacillus novellus

A thiosulphate-cleaving enzyme was purified from Thiobacillus novellus and some of its properties studied. The enzyme showed an absorption peak at 279 nm and no peaks between 300 and 650 nm. Its Mr was 38,000. Although the crude enzyme cleaved thiosulphate to form sulphite without addition of cyanide, the purified enzyme required cyanide to cleave thiosulphate. The Km values for thiosulphate and cyanide of the purified enzyme were 1.0 mM and 0.3 mM, respectively. One mol of the enzyme formed 10 mol of thiocyanate per s from thiosulphate and cyanide. The thiosulphate-cleaving activity of the enzyme was strongly inhibited by cysteine, while beta-mercaptoethanol was less inhibitory. The factor which accepted sulphur from thiosulphate in the crude preparation of thiosulphate-cleaving enzyme seemed to be a relatively labile compound with an Mr of 10,000 x 20,000.     

辣椒子叶原生质体分离条件的研究

以不同基因型的辣椒子叶为供体组织进行辣椒原生质体分离条件的研究,结果表明：幼龄子叶的原生质体产量与活力均高于老龄子叶;酶解过程中酶液渗透压、酶液浓度、酶解时间均对原生质体分离效果产生重要影响。对于辣椒子叶原生质体,最佳分离条件为酶液甘露醇浓度０．５ｍｏｌ／Ｌ,纤维素酶Ｃｅｌｌｕｌａｓｅ Ｏｎｚｕｋａ Ｒ－１０ １．５,果胶酶Ｍａｃｅｒｏｚｙｍｅ Ｒ－１０ ０．６％,酶解时间８－１０ｈ。不同基因型辣     

Biomass production from glutamate fermentation wastewater by the co-culture of Candida halophila and Rhodotorula glutinis

In this study, the biomass production and pollutant removal from high-strength glutamate fermentation wastewater (GFW) using yeast isolates was investigated. Following enrichment culture, two species of yeasts, Candida halophila and Rhodotorula glutinis, were isolated from raw GFW with chemical oxygen demand (COD) and ammonia-nitrogen levels of 40 and 16 g l(-1), respectively. The binary mixed yeast culture was cultivated batchwise in 2.5-fold diluted GFW from which 85% of COD and 96% of reducing sugar were removed. The resulting yeast biomass contained 56% crude protein, 36.0% carbohydrate and 0.4% crude lipid. The amino acid composition of mixed yeast cells was balanced and was comparable with that of C. utilis and soybean.     

斑玉蕈酸性磷酸酯酶的酶学性质研究

以斑玉蕈为材料分别从菌盖和菌柄中提取一种酸性磷酸酯酶（ACPase,EC.3.1.3.2）,进一步用硫酸铵沉淀分离,Sephadex G-200柱纯化,从菌盖中分离到3个酶组分,从菌柄中分离到4个酶组分,分别对菌盖和菌柄的酶Ⅰ和酶Ⅰ′进行聚丙烯酰胺凝胶（PAGE）电泳纯度鉴定,均呈现单一酶蛋白带。SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定酶Ⅰ和酶Ⅰ′的相对分子量均为65kDa,SDS-聚丙烯酰胺凝胶电泳及Sephadex G-75凝胶过滤测定分析,酶Ⅰ和酶Ⅰ′均为单亚基蛋白。紫外吸收光谱（UV）测     

红曲霉原生质体的制备、再生及其遗传转化系统

原生质体是研究和建立真菌遗传转化系统的重要工具。为了建立原生质体介导的红曲霉遗传转化系统,考察了各种细胞壁裂解酶和渗透压稳定剂等对红曲霉原生质体形成和再生的影响。将红曲霉分生孢子在铺有玻璃纸的平板上30℃培养30~40 h收获的菌丝体最有利于原生质体的形成和释放。红曲霉菌丝体形成和释放原生质体最适裂解酶和酶解时间分别为：0.3 % lysing enzyme、0.1 % cellulase和1 % snailase的酶组合,30℃作用2.5 h;最适渗透压稳定剂是：1mol /L MgSO4。最适合原生质体再生的培养基为含0.6 mol/L蔗糖的CM培养基。原生质体液涂布单层再生培养基的方法,再生率最高,菌株M34和N18分别为8.5 %和36.4 %。在PEG和CaCl2存在下,以潮霉素B为抗生素选择标记,用质粒pBC-Hygro和pNL1共转化菌株M34原生质体,每微克DNA克获得100个稳定转化子。     

N-terminal glycation of cholecystokinin-8 abolishes its insulinotropic action on clonal pancreatic B-cells.

Monoglycated cholecystokinin octapeptide (Asp(1)-glucitol CCK-8) was prepared under hyperglycaemic reducing conditions and purified by reverse phase-high performance liquid chromatography. Electrospray ionisation mass spectrometry and automated Edman degradation demonstrated that CCK-8 was glycated specifically at the amino-terminal Asp(1) residue. Effects of Asp(1)-glucitol CCK-8 and CCK-8 on insulin secretion were examined using glucose-responsive clonal BRIN-BD11 cells. In acute (20 min) incubations, 10(-10) mol/l CCK-8 enhanced insulin release by 1.2-1.5-fold at 5.6-11.1 mmol/l glucose. The stimulatory effect induced by 10(-10) mol/l CCK-8 was abolished following glycation. At 5.6 mmol/l glucose, CCK-8 at concentrations ranging from 10(-11) to 10(-7) mol/l induced a significant 1.6-1.9-fold increase in insulin secretion. Insulin output in the presence of Asp(1)-glucitol CCK-8 over the concentration range 10(-11)-10(-7) mol/l was decreased by 21-35% compared with CCK-8, and its insulinotropic action was effectively abolished. Asp(1)-glucitol CCK-8 at 10(-8) mol/l also completely blocked the stimulatory effects of 10(-11)-10(-8) mol/l CCK-8. These data indicate that structural modification by glycation at the amino-terminal Asp(1) residue effectively abolishes and/or antagonises the insulinotropic activity of CCK-8.     

麦角菌和雀稗麦角菌原生质体的形成与再生

本文报道了快速制备麦角菌(Claviceps purpurea)和雀稗麦角菌(Claviceps paspali)原生质体的方法及影响原生质体形成的若干因素。用适当方法培养菌丝体,利用商品溶壁酶制剂(10mg/ml),以0.7mol/l KCl为原生质体高渗液,28℃为酶解温度,处理时间仅需1—2h,菌丝体细胞壁即被彻底消化而释放出大量原生质体。在适当的固体再生培养基上,两种麦角菌原生质体的再生频率分别为7.7%和13.2%左右。培养基中添加25mg/l脱氧胆酸钠有利于形成易于计数和分离的小菌落。本文还就CaCl_2,MgCl_2及低温保藏(3—4℃,30天)对于原生质体稳定性与再生的影响作了初步探讨,并证实溶壁酶对原生质体有明显的破坏作用。     

Lead toxicity in Saccharomyces cerevisiae

The effect of Pb on Saccharomyces cerevisiae cell structure and function was examined. Membrane integrity was assessed by the release of UV-absorbing compounds and by the intracellular K⁺ efflux. No leakage of UV₂₆₀-absorbing compounds or loss of K⁺ were observed in Pb (until 1,000 μmol/l) treated cells up to 30 min; these results suggest that plasma membrane seems not to be the immediate and primary target of Pb toxicity. The effect of Pb on yeast metabolism was examined using the fluorescent probe FUN-1 and compared with the ability to reproduce, evaluated by colony-forming units counting. The exposition of yeast cells, during 60 min to 1,000 μmol/l Pb, induces a decrease in the ability to process FUN-1 although the cells retain its proliferation capacity. A more prolonged contact time (120 min) of yeast cells with Pb induces a marked (> 50%) loss of yeast cells metabolic activity and replication competence through a mechanism which most likely requires protein synthesis.