白腐真菌原生质体制备和再生条件的研究

对影响白腐真菌(5．776)原生质体制备和再生的条件：包括菌龄、水解酶液的种类及浓度、酶解温度、酶解时间、再生培养基的稳渗剂的选择进行了研究。通过单因素比较分析和正交实验得到最适合的白腐真菌原生质体制备和再生条件。结果表明：当菌龄为58h,采用1%纤维素酶和1%蜗牛酶(2：1)混合液,酶解温度30℃,酶解时间180min,用0．7mol/L氯化钠作渗透压稳压剂,白腐真菌(5．776)原生质体的形成数和再生率均比优化前大为提高,原生质体形成量为8．36×10~5个/mL,原生质体再生率为9．12%。     

酶制剂研制的国内外进展和发展策略

简述了酶工程的研究范围,国际上酶制剂工业发展的研究,特点。概述了近年来我国酶制剂工业发展中存在的问题,包括产品结构,技术含量,研发能力。提出我国酶制剂工业今后发展的一些建议：充分发挥资源优势,加强现代生物技术的应用,调整酶制剂产品的结构。     

松茸菌丝原生质体形成与再生条件的研究

采用多因素实验正交优选法比较不同酶、渗透压稳定剂、菌丝生理状况、菌丝培养基、预处理液和酶解时间等因素对松茸原生质体形成的影响和再生培养基、明胶、酪蛋白对松茸原生质体再生的影响.以0.6MMgSO4作为渗透压稳定剂,2-硫基苏糖醇为预处理液,用2%蜗牛酶在30℃酶解6h,每克菌丝可以得到1×106个原生质体.再生最优条件0.6MMgSO4,PDY再生培养基,2.5%明胶,松茸菌丝再生率最高4.97%.     

兽用博落回散对饲用酶制剂酶活性的影响

博落回散能抑菌消炎、改善畜禽肠道内环境,兼具营养和药用双重作用,已经成为一类新型饲料添加剂。酶制剂是目前已在养殖业广泛使用的饲料添加剂,它能提高动物对饲料的消化利用率、提高饲料转化率、降低饲喂成本。博落回散能否和酶制剂同时使用目前尚未有相关的研究报道。本文研究了不同浓度博落回散溶液对8种常用饲用酶制剂活性的影响,实验结果表明,在正常使用浓度(30～50 mg/L)范围内,博落回散不会明显影响淀粉酶、纤维素酶、果胶酶、甘露聚糖酶和木聚糖酶这5种糖基水解酶的活性;对植酸酶、酸性蛋白酶和脂肪酶这3种非糖基水解酶的活性影响不大,这说明博落回散能和酶制剂同时添加到饲料中。本研究为兽用博落回散饲料添加剂在养殖行业的广泛应用提供了实验支持。     

Studies on enzymes in the developing sea urchin egg

The relative activity of a number of different enzymes has been determined in different developmental stages of normal and Li treated sea urchin eggs.Some enzymes show a constant activity throughout development, whereas the activity of other enzymes is constant up to mesenchymeblastula stage and then increases. A short review of enzymatic changes in other embryonic material is presented. This demonstrated a striking agreement with the sea urchin egg in essential points.It has been suggested that the enzymes which rise in activity at mesenchymeblastula stage are localized in mitochondria, whereas enzymes with constant activity are non-mitochondrial.Li treatment during cleavage stages, which causes vegetalization, inhibits the rise in activity of apyrase, cathepsin II, glutaminase, and according to preliminary experiments also of succinodehydrogenase.The authors conclude that Li treatment during cleavage stages interferes with the later development of mitochondria, possibly by inhibiting the development of their precursors.According to these results, the ectodermal development is characterized by a more important mitochondrial activity, i.e. a more elaborate mechanism of synthesis, than the mesentodermal development. This is at least the case during earlier stages of development.     

Analysis of viability and cell types of macroalgal protoplasts using flow cytometry

The ability to rapidly distinguish viable sub-populations of cells within populations of macroalgal protoplast isolations was demonstrated using flow cytometry. Viable protoplasts from Ulva sp. and Porphyra perforata J. Ag. were distinguished from non-viable protoplasts based on differential fluorescein accumulation. The identities of cortical and epidermal protoplasts from Macrocystis pyrifera (L.) C. Ag. were inferred based on light-scattering and chlorophyll a autofluorescence. Three cell types could be distinguished among protoplasts released from thalli of P. perforata based on chlorophyll a and phycoerythrin autofluorescence. Mixed protoplast populations of Ulva sp. and P. perforata were also discernable based on relative chlorophyll a and phycoerythrin autofluorescence. The ability to screen heterogenous protoplast populations rapidly, combined with the cell sorting capabilities of many flow cytometers, should prove valuable for seaweed biotechnology.     

酶制剂工业概况及其应用进展

概述了国内外酶制剂工业的生产和市场的情况,并介绍了酶制剂应用的进展,对于我国酶制剂工业的发展也作了展望。     

Degradation of agarophytic red algal cell wall components by new crude enzyme preparations

Several new crude enzyme preparations were isolated from a marine association of the agarolytic bacterium Cytophaga diffluens and the infusorium Uronema marinum, an axenic culture of Cytophaga diffluens, some species of land micro- and macromycetes adapted to assimilate red algal biomass and from the marine mollusc Littorina littorea. Fungal and mollusc enzyme preparations were shown to have cellulase, xylanase, protease and agarase activities. Fungal agarase activity was revealed only after 3–4 passages of the culture on the medium containing algal biomass. Enzyme preparations from the association and the pure bacterial culture growing on the medium with bactoagar as the sole carbon source contained only agarase activity. The maximum specific agarase activity was found in a preparation from the marine association. The preparations obtained can be used for isolating protoplasts and single cells from red seaweed thalli. This revised version was published online in June 2006 with corrections to the Cover Date.     

重组逆转录病毒的制备、浓缩及保存研究

选择含有新霉素磷酸转移酶Ⅱ(Neo^R)的重组逆转录病毒载体,用包装细胞进行包装,得到了含有重组逆转录病毒粒子的病毒上清,该病毒上清经差速离心法浓缩后,－80℃无任何添加物的情况下保存3个月,体外感染靶细胞NIH3T3以测定其滴度,冻存前后的浓缩病毒上清的感染滴度无明显降低,经过冻存的浓缩病毒上清的感染滴度仍可达到10^6cfu/ml,具有理想的感染靶细胞的能力。     

“大豆分离蛋白”工艺用复合酶制剂的研究

大豆分离蛋白的碱浸提酸沉淀工艺,目前普遍存在蛋白质回收率低,分离蛋白成品得率低,纯度低的问题,利用复合酶制剂的作用可改变这一状况。针对工艺需要,从十余种工业酶制剂中筛选出两种蛋白酶活低、多糖降解酶活高的酶制剂;Ａｎ－７６半纤维酶和果胶酶ｕｌｔｒａｚｙｒｍ＾ＴＭ。对两种酶制剂的酶活组分、主体酶的部分酶学性质,以及酶的用量与蛋白质浸出率的关系等方面进行了大量基础实验。经优化实验确定了复合酶制剂的复配比     

Improvement of DNA preparation and of PCR cycling in RAPD analysis of marine macroalgæ

In order to avoid the effects of bacterial contamination and the excess of RNA and polysaccharides coming from the cell walls, algal DNA for PCR cycling in RAPD analysis was extracted from protoplasts and purified in a CsCl gradient. Results indicate that RAPD can be efficiently applied to marine algæ and useful to distinguish between different strains or between different species.     

柑桔种间体配融合及培养研究

“平户”文旦（柚）（Ｃｉｔｒｕｓｇｒａｎｄｉｓ）Ｏｓｂｅｃｋ的四分体经酶解,分离出原生质体。ＰＥＧ（聚乙二醇）诱导这类原生质体与二倍体“伏令夏”甜橙（Ｃ．ｓｉｎｅｎｓｉｓ）胚性悬浮细胞系的原生质体融合。融合后的原生质体培养于ＢＨ３／ＥＭＥ培养基中。２天后,观察到花粉管生长现象。不同处理的结果显示,这一现象来源于异核体细胞。这种具花粉管生长的细胞可进一步分裂,形成多细胞团及球形和心形胚状体。对再生的胚状体进行染色体数检查,证明１３．１％的胚状体为三倍体,２ｎ＝３ｘ＝２７。而起始悬浮细胞系为二倍体,检查的３９２个细胞,未发现有染色体倍性变异。     

碱性普鲁兰酶产生菌选育和发酵条件的研究

从儿童食品中分离筛选到1株产碱性普鲁兰酶活性较高的菌株,编号为SX－12,初步鉴定为芽孢杆菌Bacillus sp.,研究了SX－12原生质体制备与再生最佳条件,其原生质体经紫外线诱变处理,选育出产碱性普鲁兰酶的高产菌株SX－12C67,酶活由出发菌株的2．42U/mL提高到6．87U／mL,提高了约1．8倍,在此基础上对产酶条件进行了优化,优化后的最佳发酵培养基为：可溶性淀粉3％,蛋白胨1．0％,酵母膏0．5％,K2HPO42％,MgSO4.7H2O0.05%MnCl20.0001%,最适p;H9.5,最适温度40℃,初步研究了酶的部分性质,酶反应的最适pH,温度分别为10．0－10．5和55℃,在55摄氏度反应条件下,酶在pH6.0-11.0的范围内都具有一定的活性,Ca^2 ,Mn^2 ,Mg^2 等离子是酶的激活性,Zn^2 ,Hg^2 等离子是抑制剂。     

Studies on the effect of protoplast density and genotype mixing on cell regeneration

Genotypes of Solanum tuberosum, assessed for their differential response to a protoplast regeneration protocol, were identified. This enabled comparisons to be made in relation to their abilities to regenerate into dividing cells and colonies. Protoplasts from genotypes with contrasting regeneration responses were plated as single or mixed genotype cultures at various plating densities in replicated, randomised experiments, and the effect on the regeneration of particular cell types observed. Protoplasts from single genotype cultures showed intergenotypic variation in the extent of cell regeneration and there were significant effects of genotype mixing and density × genotype mixing interactions. Overall, the effect of mixing was beneficial to a regenerating culture but significant density genotype mixing interactions showed only a positive benefit at the lowest plating density. The protoplast mixing phenomenon was not correlated with the behaviour of the same genotypes at the plantlet level in experiments with in vitro meristem cultures.     

Studies on the preparation and properties of ribonucleoprotein particles from rat liver nuclei

Ribonucleoprotein particles of 38 S were extracted from rat liver nuclei with isotonic salt buffer under concomitant sonication. The fate of the endogeneous nuclear RNAases assayed with poly(A), high molecular weight yeast RNA and rapidly labeled hnRNA was followed during the preparation of 38-S nuclear ribonucleoprotein (nRNP) particles. Essentially all the RNAase activity could be removed from the particle preparation. The effect of synthetic RNAase inhibitors on the nRNP particles was studied. Upon extraction of nuclei with 0.14 M NaCl, approximately 38% of the total nuclear radioactivity was found in the 38-S nRNP particles. By two successive extractions of the remaining chromatin with either isotonic or 0.22 and 0.3 M NaCl, an additional 25 and 9% of rapidly labeled hnRNA of 38 S particle were dissociated from chromatin, respectively. The chromatin components, DNA, nonhistone proteins, histones and RNA were determined after successive salt extractions. Particularly alterations in the nonhistone proteins and RNA were found. The protein patterns upon SDS-acrylamide gel electrophoresis of the salt-extracted chromatin preparations were compared with those of the 38-S nRNP particles. Particularly proteins in the molecular weight range of 32 000-43 000 were dissociated from chromatin after treatment with 0.22 or 0.3 M NaCl.     

Distribution of immobilized enzymes on porous membranes

In this article, the results from a theoretical and experimental investigation of enzyme immobilization in porous membranes are reported. A theoretical model of the immobilization process, which accounts for restricted diffusion of enzyme in the pores of the membrane, has been developed. The model predicts the effect of immobilization kinetics and time of immobilization on the enzyme distribution in the pores of the membrane. The immobilization of glucose oxidase and glucose oxidase-biotin conjugate on porous alumina membranes was experimentally investigated. Enzyme uptake data was correlated to the theory to determine the rate constant of imobilization and the distribution of the enzyme in the pore. Immobilization studies were carried out for enzyme adsorption and for enzyme attachment by covalent coupling. The distribution of enzyme was experimentally studied by assembling five membranes in the diffusion cell. Following immobilization, the membranes were separated and each was assayed for activity. The amount of active enzyme present in each membrane yielded a discrete distribution that compared well with that predicted by theory. (c) 1992 John Wiley & Sons, Inc.     

原人参二醇型皂苷水解酶及制备人参皂苷Compound K的研究进展

人参皂苷Compound K (CK)是一种具有抗癌抗炎等药理活性的化合物。目前在天然人参中暂未鉴定出,工业上主要通过原人参二醇型皂苷的去糖基化进行制备。相对于传统的物理、化学的去糖基化法,利用原人参二醇型皂苷水解酶制备CK具有特异性强、绿色环保和高效稳定的优点。本文根据水解酶作用的糖基连接碳原子的差异将原人参二醇型皂苷水解酶分成了3类,发现大多数能制备CK的水解酶为Ⅲ型原人参二醇型皂苷水解酶。此外,对水解酶在制备CK中的应用进行了总结评估,旨在为人参皂苷CK的大规模制备及其在食品和药品行业中的开发提供参考。     

Catalytic power of enzymes decreases with temperature: New insights for understanding soil C cycling and microbial ecology under warming

Most current models of soil C dynamics predict that climate warming will accelerate soil C mineralization, resulting in a long‐term CO₂ release and positive feedback to global warming. However, ecosystem warming experiments show that CO₂ loss from warmed soils declines to control levels within a few years. Here, we explore the temperature dependence of enzymatic conversion of polymerized soil organic C (SOC) into assimilable compounds, which is presumed the rate‐limiting step of SOC mineralization. Combining literature review, modelling and enzyme assays, we studied the effect of temperature on activity of enzymes considering their thermal inactivation and catalytic activity. We defined the catalytic power of enzymes (E_power) as the cumulative amount of degraded substrate by one unit of enzyme until its complete inactivation. We show a universal pattern of enzyme's thermodynamic properties: activation energy of catalytic activity (EA_cat) < activation energy of thermal inactivation (EA_inact). By investing in stable enzymes (high EA_inact) having high catalytic activity (low EA_cat), microorganisms may maximize the E_power of their enzymes. The counterpart of such EAs’ hierarchical pattern is the higher relative temperature sensitivity of enzyme inactivation than catalysis, resulting in a reduction in E_power under warming. Our findings could explain the decrease with temperature in soil enzyme pools, microbial biomass (MB) and carbon use efficiency (CUE) reported in some warming experiments and studies monitoring the seasonal variation in soil enzymes. They also suggest that a decrease in soil enzyme pools due to their faster inactivation under warming contributes to the observed attenuation of warming effect on soil C mineralization. This testable theory predicts that the ultimate response of SOC degradation to warming can be positive or negative depending on the relative temperature response of E_power and microbial production of enzymes.