酵母菌絮凝的分型及其生理生化特性的研究

通过对410余株酵母菌进行絮凝测定,从中筛选到5株强絮凝菌。依据不同糖对其絮凝水平的抑制,将5株强絮凝菌分为Flo 1型和NewFlo型。对这两种絮凝型菌株的相关生理生化特性进行了研究。结果表明,Flo 1型菌絮凝只受甘露糖抑制,它对高温(70℃)、蛋白酶E、胰蛋白酶敏感,而对蛋白酶K、糜蛋白酶、Ca\+\{2+\}\,pH有一定耐受性。NewFlo型菌絮凝受甘露糖等多种糖抑制,它对高温(70℃)、各种蛋白酶、Ca\+\{2+\}、pH均较敏感。这两种类型菌株絮凝的最适Ca\+\{2+\}浓度为10mmol/L～1mol/L,最适pH为3.0～45。     

嗜热芽孢杆菌XJT9503高温中性蛋白酶的研究──Ⅰ．XJT9503菌的特性

从新疆吐鲁番盆地土壤里分离到一株嗜热芽孢杆菌,代号ＸＪＴ９５０３．能产生胞外高温中性蛋白酶,该菌在旋转式摇床上１１０ｒ／ｍｉｎ,４５℃下培养６０小时,发酵液产酶为５６００单位／ｍｌ,但在４５℃静止培养产酶可达７４００单位／ｍｌ,Ｃａ２＋、Ｍｇ２＋对产酶有明显促进作用,Ｃｕ２＋等抑制产酶。     

高及低转移潜能小鼠肝癌细胞株糖蛋白凝集素结合特征的研究

为探讨肿瘤转移与细胞表面的糖结构的关系,对小鼠肝癌细胞的高、低淋巴道转移株Ｈｃａ－Ｆ和Ｈｃａ－Ｐ进行了蛋白质电泳及经蛋白质印迹术后的５种凝集素（ＣｏｎＡ、ＷＧＡ、ＵＥＡ、ＳＢＡ、ＰＮＡ）结合糖蛋白谱的对比分析．结果表明：高、低转移两株细胞的ＳＤＳ－ＰＡＧＥ谱基本相同;ＣｏｎＡ特异结合糖蛋白共有５种（～７２,８０～９０,～１０４,～１５０,～２００ｋＤ）;其中较明显的差异为～７２ｋＤＣｏｎＡ特异结合糖蛋白,它在Ｈｃａ－Ｐ细胞的表达明显高于Ｈｃａ－Ｆ细胞．ＷＧＡ特异结合糖蛋白１种（～１５０ｋＤ）,在Ｈｃａ－Ｐ细胞的表达略高于Ｈｃａ－Ｆ细胞．此外,实验发现两种性质未明的蛋白质（～７９,～１３０ｋＤ）,后者在Ｈｃａ－Ｐ细胞的含量明显高于Ｈｃａ－Ｐ细胞．结果提示Ｈｃａ－Ｆ和Ｈｃａ－Ｐ细胞不同的转移表型可能与其糖蛋白的表达有一定的关联．     

钙调素对花粉萌发和花粉管生长的效应

牛脑和玉米胚ＣａＭ能显著促进花粉萌发和花粉管生长（图１）,而ＣａＭ抑制剂ＴＦＰ、ＣＰＺ及另外两个专一性更强的抑制剂Ｃｏｍｐｏｕｎｄ４８／８０和Ｗ７均严重抑制甚至阻止花粉的萌发（图２,３）。用对ＣａＭ亲和性较低的Ｗ７同系物Ｗ５,在与Ｗ７同样浓度下,对花粉萌发和花粉管生长无明显影响。此外,Ｗ７对花粉萌发和花粉管生长的抑制效应可被外源ＣａＭ所消除（图４）。在花粉萌发过程中,其内源ＣａＭ含量显著上升,在花粉萌发率接近最大值时,花粉ＣａＭ含量达最高水平（图５）。上述结果表明ＣａＭ对花粉萌发和花粉管生长的调控起重要作用。     

丙型肝炎病毒C33_c单克隆抗体的研制和鉴定

用丙型肝炎病毒重组蛋白Ｃ３３_ｃ抗原免疫ＢＡＬＢ／ｃ小鼠,运用杂交瘤技术成功地建立了７株能稳定分泌抗Ｃ３３_ｃ单克隆抗体的杂交瘤细胞１Ｈ６Ｄ２、２Ｇ１Ａ６、３Ａ４Ａ８、３Ｅ３Ｅ７、４Ｇ１２Ｃ１０、４Ａ１０Ｃ２、５Ｆ４Ｂ６．试验结果表明,７株ＭｃＡｂｓ具有良好的ＨＣＶ特异性,间接ＥＬＩＳＡ法测得小鼠腹水ＭｃＡｂ效价为１：１０￣４－１：４×１０￣４;竞争抑制实验和相加指数测定证实７株ＭｃＡｂｓ识别相关的抗原表位;７株ＭｃＡｂｓ中１株为ＩｇＭ（５Ｆ４Ｂ６）,其它６株为ＩｇＧ（２ａ）。     

地衣芽孢杆菌碱性蛋白酶的研究：I.碱性蛋白酶高产菌的筛选及产酶条件初探

从成都佳丰食品厂等处采集的样品中平板分离初筛到１２４株碱性蛋白酶产生菌,进一步复筛出一株高产,且稳定的碱性蛋白酶产生菌株Ｂ．Ｌ．ＪＦ－ｌｄ初步鉴定为地衣芽孢杆菌（Ｂａｃｉｌｌｕｓｌｉｃｈｅｎｉｆｏｒｍｉｓ）。该菌的最适产酶条件为：培养基（％）为麦芽糖７．５,酵母膏３,ＮａＣｌ０．５,Ｋ２ＨＰＯ４·３Ｈ２Ｏ０．５３,ＮａＨ２ＰＯ４·２３Ｈ２Ｏ０．０３,Ｎａ２ＣＯ３０．０５６,ＭｎＳＯ４ｌ×１０＾－４     

粘质赛氏菌胞外蛋白酶的氨基酸组成、N端序列及其部分性质

终浓度为５ｍｍｏｌ／Ｌ的ＥＤＴＡ可彻底抑制粘质赛氏菌４１００３（２）胞外蛋白酶活力,而Ｃａ２＋可以回复部分酶活力,表明Ｃａ２＋为酶活力所必需;Ｚｎ２＋、Ｍｎ２＋、Ｆｅ２＋等金属离子对酶具有不同程度的抑制作用;５－磷酸吡哆醛及对甲氧基苯甲醛对酶具有明显的激活作用;以酪蛋白为底物,采用双倒数法求得酶的Ｋｍ值为６．６７ｍｇｍｌ－１;Ｎ,Ｎ－二甲基酪蛋白也是酶的理想底物;经酸水解,测定出酶的氨基酸组成;利用蛋白质自动分析仪测定了酶的Ｎ端９个氨基酸序列．比较来源不同的粘质赛氏菌胞外蛋白酶的Ｎ端序列,发现它们之间存在一定程度的同源性,并且存在特定的通读结构Ａｌａ…Ａｓｐ…Ｇｌｙ．     

FD耐热逆转录酶的部分酶学性质研究

ＦＤ耐热逆转录酶（ＦＤ－ＴＲＴ）来自一株栖热杆菌菌株．通过ＲＴ－ＰＣＲ方法,论文对ＦＤ－ＴＲＴ的部分酶学性质进行了研究．ＦＤ耐热逆转录酶能耐受９５℃的高温,最适反应温度在６５～７５℃左右,这时ＲＮＡ的高级结构能解开,可以提高逆转录反应的效率;同时引物和ＲＮＡ模板识别的专一性强,可大大提高逆转录的特异性．实验表明ＦＤ－ＴＲＴ逆转录反应的最适条件为：２５ｍｍｏｌ／ＬＴｒｉｓ－ＨＣｌ（ｐＨ８．８,１５ｍｍｏｌ／Ｌ（ＮＨ４）２ＳＯ４,１００μｇ／ｍｌ明胶,５００μｍｏｌ／ＬｄＮＴＰｓ,２５ｐｍｏｌ逆转录引物,１ｍｍｏｌ／ＬＭｎＣｌ２,２ＵＦＤ耐热逆转录酶,反应在６５～７０℃下进行．在上述条件下,用ＲＴ－ＰＣＲ可检出少于５ｐｇ外周血总ＲＮＡ中特异的α珠蛋白ｍＲＮＡ．     

猪生殖—呼吸道综合征病毒（PRRSV）CH—1a株糖蛋白基因的遗传变异分析

新近发现的猪生殖－呼吸道综合征病毒（ＰＲＲＳＶ）是单股ＲＮＡ病毒,属于不久前成立的动脉炎病毒科,为了比较国内分离的ＣＨ－１ａ株与国外毒株的分子遗传学关系,扩增并克隆了ＰＲＲＳＶ ＣＨ－１ａ株糖蛋白基因ＯＲＦ２￣５,测定了其核苷酸序列。用序列分析软件分析了其编码产物的分子量、等电点、疏水性、抗原性和有关位点,并与国外毒株进行了序列比较和系统发育进化分析。结果表明：ＣＨ－１ａ株与ＶＲ－２３３２株尽管密     

新生儿颊粘膜定植菌株的拮抗性研究

本文分离在新生儿颊粘膜上皮细胞表面形成微菌落、且初代培养时菌落较纯的１５株ａ溶血细菌,进行对Ｂ链球菌、金黄色葡萄球菌、绿脓杆菌、大肠杆菌４种共７株菌的体外拮抗实验。１５株菌中包括８株ａＳｔｒｅｐ．,其中Ｓｔｒｅｐ．ｍｉｔｉｓ４株,Ｓｔｒｅｐ．ｏｒａｌｉｓ２株、Ｓｔｒｅｐ．Ｓａｌｉｖ．Ｓａｌｉｖａｒｉｕｓ１株、Ｓｔｒｅｐ．ｉｎｔｅｒｍｅｄｉｕｓ１株;ＬＣ．ｌａｃｔｉｓ．ｃｒｅｍｏｒｉｓ５株、Ｇｅｍｅｌａｍｏｒｂｉｌｏｒｕｍ１株、Ｇｅｍｅｌａｈａｅｍｏｌｙｓａｎｓ１株。结果约６０％（９／１５）的颊粘膜定植株对两株及两株以上的病原菌株或阴性杆菌株有拮抗作用,只对１株菌有损坏抗作用的ａ溶血菌４株,完全无拮抗作用的菌株２株;ａＳｔｒｅｐ．、ＬＣ．ｌａｃｔｉｓ．ｃｒｅｍｏｒｉｓ的拮抗作用较强;拮抗作用的有无、强弱有很强的菌株特异性     

Characteristics of Flo11-dependent flocculation in Saccharomyces cerevisiae

The FLO11-encoded flocculin is required for a variety of important phenotypes in Saccharomyces cerevisiae, including flocculation, adhesion to agar and plastic, invasive growth, pseudohyphae formation and biofilm development. We present evidence that Flo11p belongs to the Flo1-type class of flocculins rather than to the NewFlo class. Both Flo1-type and NewFlo yeast flocculation are inhibited by mannose. NewFlo flocculation, however, is also inhibited by several other carbohydrates including glucose, maltose and sucrose. These differences have in at least one case been shown to reflect differences in the structure of the carbohydrate-binding site of the flocculins. We report that Flo11p-dependent flocculation is inhibited by mannose, but not by glucose, maltose or sucrose. Furthermore, Flo11p contains a peptide sequence highly similar to one that has been shown to characterise Flo1-type flocculins. Further characterisation of the properties of Flo11p-dependent flocculation revealed that it is dependent on calcium, occurs only at cell densities greater than 1 x 10(8) ml(-1), and only occurs at acidic pH.     

Deletion of tandem repeats causes flocculation phenotype conversion from Flo1 to NewFlo in Saccharomyces cerevisiae

A gene, FLONS, conferring NewFlo-type flocculation ability in yeast was cloned. The 3,396-bp ORF encoded a peptide of 1,132 amino acids with high identity to Flo1 protein. Aligned with the FLO1 gene, two repeated regions (675 and 540 bp) were lost in the middle of FLONS, revealing that this gene was a derived form of the FLO1 gene. The missing repeated sequence contained three highly homologous repeat units. Although the flocculation phenotype of the transformant YTS-S with the FLONS gene was inhibited by both mannose and glucose, it exhibited some distinguished physiological characteristics from the reported typical NewFlo-type flocculation during detailed investigation. The deletion of repeats was suspected to cause conversion of the flocculation phenotype from Flo1 to NewFlo, suggesting that intragenic tandem repeats generated functional variability in Flo1 protein.     

Modeling of orthokinetic flocculation of Saccharomyces cerevisiae.

This study examined the flocculation behavior of two Saccharomyces cerevisiae strains expressing either Flo1 (LCC1209) genotype or NewFlo (LCC125) phenotype in a laminar flow field by measurement of the fundamental flocculation parameter, the orthokinetic capture coefficient. This orthokinetic capture coefficient was measured as a function of shear rate (5.95-223 s(-1)) and temperature (5-45 degrees C). The capture coefficients of these suspensions were directly proportional to the inverse of shear rate, and exhibited an increase as the temperature was increased to 45 degrees C. The capture coefficient of pronase-treated cells was also measured over similar shear rate and temperature range. A theory, which predicts capture coefficient values due to zymolectin interactions, was simplified from that developed by Long et al. [Biophys. J. 76: (1999) 1112]. This new modified theory uses estimates of: (1) cell wall densities of zymolectins and carbohydrate ligands; (2) cell wall collision contact area; and (3) the forward rate coefficient of binding to predict theoretical capture coefficients. A second model that involves both zymolectin interactions and DLVO forces was used to describe the phenomenon of yeast flocculation at intermediate shear ranges, to explain yeast flocculation in laminar flow.     

Flocculation of industrial and laboratory strains ofSaccharomyces cerevisiae

Summary A comparative study has been made of different laboratory and industrial wild-type strains ofSaccharomyces cerevisiae in relation to their flocculation behavior. All strains were inhibited by mannose and only one by maltose. In regard to the stability of these characters in the presence of proteases and high salt concentrations, a relevant degree of variation was found among the strains. This was to such an extent that it did not allow their inclusion in the Flol or NewFlo phenotypes. Genetic characterization of one wild-type strain revealed that the flocculation-governing gene was allelic toFLO1 found in genetic strains.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Deletion of Intragenic Tandem Repeats in Unit C of FLO1 of Saccharomyces cerevisiae Increases the Conformational Stability of Flocculin under Acidic and Alkaline Conditions

Flocculation is an attractive property for Saccaromyces cerevisiae, which plays important roles in fermentation industry and environmental remediation. The process of flocculation is mediated by a family of cell surface flocculins. As one member of flocculins, Flo1 is characterized by four families of repeats (designated as repeat units A, B, C and D) in the central domain. It is generally accepted that variation of repeat unit A in length in Flo1 influences the degree of flocculation or specificity for sugar recognization. However, no reports were observed for other repeat units. Here, we compared the flocculation ability and its sensitivity to environmental factors between yeast strain YSF1 carrying the intact FLO1 gene and yeast strains carrying the derived forms of FLO1 with partial or complete deletion of repeats in unit C. No obvious differences in flocculation ability and specificity of carbohydrate recognition were observed among these yeast strains, which indicates the truncated flocculins can stride across the cell wall and cluster the N-terminal domain on the surface of yeast cells as the intact Flo1 thereby improving intercellular binding. However, yeast strains with the truncated flocculins required more mannose to inhibit completely the flocculation, displayed broad tolerance of flocculation to pH fluctuation, and the fewer the repeats in unit C, the stronger adaptability of flocculation to pH change, which was not relevant to the position of deletion. This suggests that more stable active conformation is obtained for flocculin by deletion the repeat unit C in the central domain of Flo1, which was validated further by the higher hydrophobicity on the surface of cells of YSF1c with complete deletion of unit C under neutral and alkaline conditions and the stabilization of GFP conformation by fusion with flocculin with complete deletion of unit C in the central domain.     

Identification and functional study of a new FLO10-derivative gene from the industrial flocculating yeast SPSC01

Yeast flocculation is an important property for the brewing industry as well as for ethanol fermentation to facilitate biomass recovery by sedimentation from the fermentation broth, which is cost-effective. In this study, a new flocculating gene FLO10 (spsc) of 4,221 bp homologous to FLO10 was identified in the industrial flocculating yeast SPSC01. Sequence analysis indicated that the N- and C-terminus of the deduced protein of this new FLO gene are 99 % identical to that of FLO10, but more intragenic repeats are included. The study on the function of FLO10 (spsc) by its integrative expression in the non-flocculating industrial yeast indicated severe inhibition in the flocculation of the transformant by mannose and maltose, moderate inhibition by sucrose and glucose and no inhibition by xylose and galactose, and thus the NewFlo type was established. Meanwhile, the flocculation of the transformant was stable when the temperature was below 50 °C and the pH was in the range of 4.0-6.0. Furthermore, the medium containing 250 g/l glucose was completely fermented within 48 h by the transformant, with about 110 g/l ethanol and 5.5 g(DCW)/l biomass produced, and no significant difference in ethanol fermentation performance was observed compared to its wide-type strain. Therefore, the FLO gene and corresponding transformation strategy provide a platform for engineering yeast strains with the flocculation phenotype to facilitate biomass recovery.     

Natural transformation of Streptococcus crista

Abstract Most brewing strains of Saccharomyces cerevisiae flocculate following growth in beer wort. However, many do not flocculate in laboratory culture media, because their initial pH and buffering capacity do not correspond to the pH range within which these yeasts flocculate. Many, though not all, NewFlo phenotype brewing yeasts flocculate within a narrow pH range only; this is indicative of the existence of more than one NewFlo flocculation phenotype. Such strains may be flocculated by small alterations of pH to within the flocculation range. Induction of flocculation by pH change may be used to separate cells from media at any stage during fermentation.     

Characterization of certain proteinase isoenzymes produced by benign and virulent strains of Bacteroides nodosus

Three proteinase isoenzymes from one benign strain of Bacteroides nodosus and five proteinase isoenzymes from each of two virulent strains of B. nodosus were purified by horizontal slab polyacrylamide gel electrophoresis. The purified isoenzymes hydrolysed casein, collagen I, collagen III, elastin, alpha-elastin, fibrinogen, gelatin, haemoglobin and alpha-keratin. The pH optima of all the isoenzymes lay between 7.25 and 9.5, the range of 8.75-9.25 being common to all. The isoenzymes were inhibited by phenylmethylsulphonyl fluoride, diphenylcarbamyl chloride, L-(1-tosylamide-2-phenyl)ethyl chloromethyl ketone, EGTA and EDTA, indicating that they were chymotrypsin-like serine proteinases that require a metal ion for stability or activity. EDTA inhibition was not reversed by addition of Ca2+ or Mg2+. Some isoenzymes were activated by Mg2+, Ca2+, Cr3+ and Se4+ and all were inhibited by Fe2+, Co2+, Cu2+, Zn2+, Cd2+ and Hg2+. Isoenzymes from benign strains had a lower temperature stability, losing all activity at 55 degrees C, whereas those from virulent strains lost all activity at 60 degrees C.     

Region of Flo1 Proteins Responsible for Sugar Recognition

Yeast flocculation is a phenomenon which is believed to result from an interaction between a lectin-like protein and a mannose chain located on the yeast cell surface. The FLO1 gene, which encodes a cell wall protein, is considered to play an important role in yeast flocculation, which is inhibited by mannose but not by glucose (mannose-specific flocculation). A new homologue of FLO1, named Lg-FLO1, was isolated from a flocculent bottom-fermenting yeast strain in which flocculation is inhibited by both mannose and glucose (mannose/glucose-specific flocculation). In order to confirm that both FLO1 and Lg-FLO1 are involved in the yeast flocculation phenomenon, the FLO1 gene in the mannose-specific flocculation strain was replaced by the Lg-FLO1 gene. The transformant in which the Lg-FLO1 gene was incorporated showed the same flocculation phenotype as the mannose/glucose-specific flocculation strain, suggesting that the FLO1 and Lg-FLO1 genes encode mannose-specific and mannose/glucose-specific lectin-like proteins, respectively. Moreover, the sugar recognition sites for these sugars were identified by expressing chimeric FLO1 and Lg-FLO1 genes. It was found that the region from amino acid 196 to amino acid 240 of both gene products is important for flocculation phenotypes. Further mutational analysis of this region suggested that Thr-202 in the Lg-Flo1 protein and Trp-228 in the Flo1 protein are involved in sugar recognition.     

Flocculation in ale brewing strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: re-evaluation of the role of cell surface charge and hydrophobicity

Flocculation is an eco-friendly process of cell separation, which has been traditionally exploited by the brewing industry. Cell surface charge (CSC), cell surface hydrophobicity (CSH) and the presence of active flocculins, during the growth of two (NCYC 1195 and NCYC 1214) ale brewing flocculent strains, belonging to the NewFlo phenotype, were examined. Ale strains, in exponential phase of growth, were not flocculent and did not present active flocculent lectins on the cell surface; in contrast, the same strains, in stationary phase of growth, were highly flocculent (>98%) and presented a hydrophobicity of approximately three to seven times higher than in exponential phase. No relationship between growth phase, flocculation and CSC was observed. For comparative purposes, a constitutively flocculent strain (S646-1B) and its isogenic non-flocculent strain (S646-8D) were also used. The treatment of ale brewing and S646-1B strains with pronase E originated a loss of flocculation and a strong reduction of CSH; S646-1B pronase E-treated cells displayed a similar CSH as the non-treated S646-8D cells. The treatment of the S646-8D strain with protease did not reduce CSH. In conclusion, the increase of CSH observed at the onset of flocculation of ale strains is a consequence of the presence of flocculins on the yeast cell surface and not the cause of yeast flocculation. CSH and CSC play a minor role in the auto-aggregation of the ale strains since the degree of flocculation is defined, primarily, by the presence of active flocculins on the yeast cell wall.