米根霉葡萄糖淀粉酶的分离纯化及特性研究

葡萄糖淀粉酶是米根霉在淀粉质培养基中诱导分泌的一类胞外酶，其表达对米根霉转化淀粉生成L-乳酸的效率，以及L-乳酸分批发酵周期的长短有很大影响。本文对米根霉葡萄糖淀粉酶进行分离纯化，并研究其酶学性质，结论如下：经硫酸铵盐析、透析脱盐、Sephadex G-100柱层析等纯化步骤，葡萄糖淀粉酶比活力提高23倍。SDS-PAGE显示纯化后的酶为一条带，相对分子量约75．5kD。该酶以可溶性淀粉为底物时最适催化反应pH值为4．0～6．0，最适催化反应温度为40-50℃，在50℃下保持稳定。钙离了和锰离子对该酶活力有一定的增强作用，而铅离子和亚铁离子对其活力有明显的抑制作用。     

Subsite Structure of Rhizopus niveus Glucoamylase,Estimated with the Binding Parameters for Maltooligosaccharides

Based on K_d and K_i for maltosaccarides G_n (n = 1 ± 7) and on a specified mode of their binding, the intrinsic affinities A₁, A₂, –, A_i at subsites i (i = 1 ± 5), were tried to estimate for glucoamylase from Rhizopus niveus. Previously, we have evaluated the apparent values A_i using K_m and k₀ on the enzyme-catalyzed hydrolysis for G_n, where A₁ is nearly 0 kcal/mol. Thus A₁, which was found here 3.3 kcal/mol, may be cancelled out with the heat, which is consumed for the hydrolytic cleavage of a substrate glucosyl bond.     

根霉脂肪酶降解壳聚糖条件研究

以德氏根霉(Rhizopus delemar)为菌种,采用固态发酵法生产脂肪酶,并用此酶对壳聚糖进行非专一性水解.研究了脂肪酶降解壳聚糖的多种影响因素,包括温度、pH、底物浓度、酶浓度、反应时间和常见金属离子等.结果表明,在45℃、pH5.0务件下,该脂肪酶能显著地降解壳聚糖,反应6h后,壳聚糖溶液的粘度即下降为原溶液的8%.适当增加酶浓度可使降解速度加快,但此酶促反应不遵循Michealis-Menten动力学方程.     

Multiple Forms of Glucoamylase of Rhizopus Species

The glucoamylase system of Rhizopus sp. was fractionated into two kinds of glucoamylase by using the corn starch adsorption technique supplemented by CM-cellulose chromatography. One of these fractions, referred to as glucoamylase I, has a strong debranching activity and is highly active in raw starch digestion. The raw waxy corn starch digestion by glucoamylase I was accelerated by adding a-amylase from Rhizopus sp. or from Aspergillus oryzae. The raw non-waxy corn starch digestion by glucoamylase I was more difficult than the raw waxy corn starch digestion, which was also accelerated by adding α-amylase from Aspergillus oryzae. On the other hand, another fraction, referred to as glucoamylase II, has a weak debranching activity and can not be adsorbed on raw starch. The raw waxy corn starch digestion by glucoamylase II was very weak, but was accelerated by adding α-amylase from Rhizopus sp.     

玉米芯诱导米根霉生成葡萄糖淀粉酶的研究

本研究发现,玉米芯能够使菌株CL-6生成葡萄糖淀粉酶的时间缩短为原来的1/3。于是,对菌株CL-6进行鉴定,并分析了玉米芯中的糖类成分以探究其中诱导因子的化学成分。菌株的鉴定采用形态学和ITS序列分子鉴定方法,葡萄糖淀粉酶酶活测定方法采用葡萄糖氧化酶法,发酵体系采用液体摇瓶发酵法,玉米芯中糖类成分的测定采用薄层层析和高效液相示差折光检测法。结果表明,菌株CL-6为米根霉属。玉米芯中水溶性成分中的糖类成分为蔗糖、葡萄糖、果糖和麦芽糖,其含量为520.33、242.67、228.67、30.33 mg/2.5 g湿基。通过分析在米根霉CL-6液体发酵体系中加入玉米芯、玉米芯水溶性成分、玉米芯水溶性成分中的糖类成分以及玉米芯水溶性成分中分子量高于8~14.4 KD成分对其葡萄糖淀粉酶酶活的影响,本研究推测,玉米芯中的诱导因子为分子量高于8~14.4 KD的水溶性物质而非糖类成分。     

雪白根霉脂肪酶基因在毕赤酵母中的高效表达及其酶学性质研究

采用同源克隆法获得了雪白根霉的脂肪酶基因(rnl),将其连接到pPICZαA载体上,得到重组表达载体pPICZαA-rnl。将表达载体pPICZαA-rnl用限制性内切酶SacI线性化后,电击转入毕赤酵母X-33中。雪白根霉脂肪酶基因在毕赤酵母X-33中成功表达,重组菌株摇瓶培养144 h后,酶活可达48 U/mL。重组酶最适pH值为8.5,最适反应温度为35℃。1 mmol/L的Mn2+,Ca2+和K+以及1%的表面活性剂吐温20,吐温80,曲通100对重组脂肪酶具有激活作用。重组酶的最适底物为三月桂酸甘油酯(C12)。     

Purification and Characterization of a Pullulan-Hydrolyzing Glucoamylase from Sclerotium rolfsii

The pullulan-hydrolyzing enzyme from the culture filtrates of Sclerotium rolfsii grown on soluble starch as a carbon source has been purified by ultrafiltration (Amicon, PM-10), ion-exchange chromatography (DEAE-Cellulose DE-52) and gel filtration chromatography (Bio-Gel P-150). The enzyme moved as a single band in non-denaturing polyacrylamide gel electrophoresis carried out at pH 2.9 and 7.5. The relative molecular mass of the enzyme was estimated to be 64.000 D by SDS-PAGE and 66.070 D by gel filtration on Bio-Gel P150. The enzyme hydrolyzed pullulan optimally at 50°C between pH 4.0–4.5, whereas, soluble starch was optimally hydrolyzed at a pH of between 4.0–4.5 and at 65°C. The Michaelis constant (Km) for pullulan was 5.13mg·ml⁻¹ (V_max 1.0U · mg⁻¹) and for soluble starch, it was 0.6mg · ml⁻¹ (V_max 8.33 U · mg⁻¹). The enzyme was observed to be a glycoprotein (12–13% carbohydrate by weight) and had a strong affinity for Concanavalin A. The enzyme hydrolyzed α-D-glucans in an exo-manner, which resulted in the release of glucose as the sole product of hydrolysis. Acarbose, a maltotetraose analog, was found to be a potent inhibitor of both pullulan and starch hydrolysis (100% inhibition at 0.06 μM). The enzyme has been characterized as a glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) showing a significant action on pullulan.     

Construction of a Rhizopus delemar genomic library and screening for direct lipase gene expression

Recombinant DNA methods were used to begin a molecular biological study of the biotechnically important lipase produced by the fungus Rhlzopus delemar. Pure, high molecular weight DNA was isolated, mildly digested with Mbo I, ligated into pBR322, and introduced into E. coli by transformation. Transformants were recovered by ampicillin selection. The frequency of insertional inactivation of tetracycline resistance in the transformants was 95%. The cloned DNA inserts ranged in size from approximately 0 to 14 kilobases (average: 4.7). Colony hybridization using fungal genomic DNA as a probe verified the presence of fungal sequences in the transformed cells. A rapid, sensitive assay for lipase production was developed and applied to a sufficient number of transformants to represent several genomic equivalents of fungal DNA. No lipase‐producing clones were detected.     

利用Native制备电泳分离来源于Rhizopus microsporus var. chinensis的葡萄糖淀粉酶同工酶

将Native制备电泳应用于Rhizopus microsporus var. chinensis中2个葡萄糖淀粉酶同工酶的分离,考察了电泳缓冲系统、凝胶浓度和凝胶长度对分离效果的影响。结果表明:先使用Ornstein-Davis系统分离同工酶,再使用0.02mol/L,pH6.2的NaAc.CH3COOH缓冲液洗脱,在长度为6cm的7%的Native电泳胶上,2个性质相近仅电泳迁移率略有差别的同工酶能有效分离。分离回收的蛋白质可保持其活力,便于后续研究。     

优化密码子及诱导温度提高雪白根霉脂肪酶在毕赤酵母中的表达

根据毕赤酵母密码子的偏好性,通过在线软件对雪白根霉脂肪酶基因(rnl)进行密码子优化。优化后的脂肪酶基因(rnl-opt)GC含量由原来的46%提高到49%,碱基A,T,C,G均匀分布,减少了AT和GC富集区,适应指数由0.82提升到0.85。将rnl-opt连接到表达载体p PICZαA并转入毕赤酵母X33中。将含有rnl和rnlopt的重组工程菌在摇瓶和50 L发酵罐中进行诱导表达。摇瓶培养条件下,含有rnl和rnl-opt重组工程菌的最大酶活分别为458、956 U/m L。50 L发酵罐培养条件下,含有rnl和rnl-opt重组工程菌的最大酶活和总蛋白浓度分别为14 856、30 500 U/m L和3.61、7.8 g/L。为了进一步提高含rnl-opt重组工程菌的表达酶活,对其在50L发酵罐培养的诱导温度进行优化,当诱导温度为22℃时,含rnl-opt重组工程菌的酶活和细胞湿重均达到最大值分别为39 520 U/m L和461 g/L。相对于30℃,酶活和细胞湿重分别提高了30%和16%。     

黑曲霉突变菌株产糖化酶的酶学特性表征

以电子束诱变黑曲霉突变菌株为对象,通过最适pH试验、最适作用温度试验、热稳定性试验、酸碱稳定性试验和金属离子对糖化酶活力影响的试验,探明黑曲霉电子束突变菌株产糖化酶的酶学特性。结果表明:突变菌株产糖化酶酶最适作用温度为63℃,且最高酶活较原始菌株提高26%,在80℃原始菌株所产糖化酶失活时,仍有8.4 kU/mL酶活剩余,突变菌株所产的糖化酶的热稳定性明显提高。突变菌株所产糖化酶最适pH为4.6,且最高酶活较原始菌株提高24%,在原始菌株所产糖化酶失活时仍有6.9 kU/mL酶活剩余,突变菌株糖化酶的pH稳定性有着明显提高。K⁺、Mg²⁺、Ca²⁺可在一定程度上增强其活力;Ag⁺、Fe²⁺、Cu²⁺则在不同程度上抑制糖化酶活力;Zn²⁺、EDTA对其酶活力影响较小或无明显现象。     

利用玉米芯水解液产苹果酸的戴尔根霉突变菌株选育及代谢分析

诱变筛选发酵玉米芯水解液高产苹果酸的根霉属菌株,并对其进行代谢分析,研究其高产苹果酸的机理。对分离得到的菌株进行ITS序列鉴定,进一步利用软X射线辐射对菌种进行诱变筛选、对出发菌株和突变菌株的相关酶活力进行测定及代谢通量分析。利用软X射线辐射结合丙烯醇平板筛选乙醇脱氢酶缺陷型菌株,得到突变株-1,发现其乙醇脱氢酶基因的3 处密码子突变为“TAA”,阻断了突变菌株的乙醇代谢途径。进而利用软X射线辐射结合氟乙酸平板筛选乙醛酸循环缺陷型菌株,得到复合突变株-2,降低了副产物富马酸及琥珀酸的产量。复合突变株-2的葡萄糖-6-磷酸脱氢酶中几处NADP（H）结合位点发生突变,增加了糖酵解和磷酸戊糖两种途径的相互作用,促进了其戊糖代谢。经过两步分离筛选,得到的复合突变株-2能发酵玉米芯水解液高产苹果酸,且减少了副产物乙醇、富马酸、琥珀酸的生成。复合突变株-2的苹果酸产量占代谢物总产量的比例由出发菌株的71%增加到91%,苹果酸产量增大1 倍,研究成果对工业化利用突变菌株生产苹果酸具有重要意义。     

Rhizopus oryzae产酸性蛋白酶条件及其酶学性质研究

本文以少孢根霉(Rhizopus oligosporus)为对照研究了豆豉中的一个分离菌株米根霉(Rhizopus oryzae)产生酸性蛋白酶的条件及所产蛋白酶的性质,结果表明这个菌株的产酶条件和蛋白酶的性质与少孢根霉相比有相似性但也存在一些差异:米根霉在水分含量57%~59%、pH2.5~3.0的酸性介质中、28~31℃下培养36h时产酸性蛋白酶能力最强,所分泌的蛋白酶系在pH4.0和pH6.0附近有最强的催化活性,在pH3.0~6.0的范围内有较好的稳定性,催化反应的最适作用温度为50℃,它的温度稳定性很差,在50℃保温30min已完全失活;少孢根霉在水分含量52%~55%、pH2.5~3.0的酸性介质中、31℃下培养48h时产酸性蛋白酶能力最强,在35℃条件下培养36h也能产生较高的酶活力,少孢根霉分泌的蛋白酶系在pH3.0和pH6.0附近有最强的催化活性,在pH4.0～6.0范围内很稳定,催化反应的最适作用温度可达55～60℃,但它的温度稳定性较差,在50℃保温30min,酶活力损失达到90%,保温120min酶几乎完全失活。     

Kinetic Properties of the Rhizopus Glucoamylase and Bacillus α-Amylase,which are Immobilized on Cellulofine

Glucoamylase from Rhizopus niveus was immobilized on Cellulofine, a kind of cellulose gel, to construct an enzyme-Cellulofine structure, of which the molar activities k₀ for maltose and for soluble starch were found almost equal to 4 and 1/9 times, respectively, of those found with the intact enzyme. Liquefying Bacillus α-amylase was fixed to make another enzyme-Cellulofine structure, of which ratio of the molar activities, k₀ (modified)/k₀ (intact) for an oligomer substrate maltohexaose is much larger than those for high-polymer substrates, amylose and soluble starch. These findings suggest that the substrate specificity of the amylase-Cellulofine structure is improved to be useful for the enzyme-catalyzed hydrolysis of saccharides having small degree of polymerization.     

米根霉AS 3.819基因启动子片段的克隆及功能鉴定

从L-乳酸生产菌米根霉（Rhizopus oryzae）菌株AS 3.819基因组DNA中分别扩增得到了乳酸脱氢酶基因（ldhA）、丙酮酸脱氢酶基因（pdcA）、淀粉糖化酶基因（amyA）以及磷酸甘油酸酯激酶基因（pgk1）的启动子片段,并构建启动子探针载体pUKMR,以β-内酰胺酶基因（bla）为报告基因在大肠杆菌JM109中对这些启动子片段进行筛选及启动活性检测。结果表明：4 种启动子片段成功启动报告基因表达;在非底物诱导情况下,ldhA和pgk1启动子启动活性较强;在有合适碳源底物诱导情况下pdcA和amyA启动子拥有更高的启动活性;ldhA基因的启动子启动活性随着启动子片段长度的增加有一定提高,而在长度为500 bp以上时,其启动活性变化不明显。本研究为Rhizopus oryzae提供了一种快速简便的启动子捕获分离及启动活性检测方法。     

少根根霉发酵液纤溶酶的分离纯化及部分性质鉴定

以产纤溶酶的根霉菌8B进行发酵,发酵液通过蒸发浓缩、(NH_4)_2SO_4分级沉淀、透析除盐、DEAE阴离子交换纤维素层析、Sephadex G—75凝胶过滤层析对该酶进行纯化,纯化浓缩后酶液活力达到3 978.5U/ mL。结果表明,该纤溶酶在-18～37℃,pH 5～7时性质稳定。该酶即能直接分解纤维蛋白,又能激活血纤维蛋白溶酶原,间接分解纤维蛋白。对家兔血栓的溶解实验表明,8B纤溶酶6h对血栓的溶解率达到52.4%,并且对兔血细胞没有明显损伤,在体外是安全有效的。     

Production,Purification, and Characterization of Thermoanaerobacterium thermosaccharolyticum Glucoamylase

Glucoamylase from Thermoanaerobacterium thermosaccharolyticum ATCC 7956 (DSM 571) was produced in extracellular form. It was purified to homogeneity by two separate methods, one with two chromatographic steps and the other with three. This glucoamylase is closely related to glucoamylases from Clostridium sp. G0005 and T. thermosaccharolyticum DSM 572. Activities and K_M values with maltose substrate are less than one‐tenth and about fourfold, respectively, those of Aspergillus niger glucoamylase. T. thermosaccharolyticum glucoamylase is about twenty times as thermostable as A. niger glucoamylase and its optimal pH is somewhat higher at 4.9; however, it is produced in much lower activities. Sorbitol strongly stabilizes A. niger glucoamylase.     

Characterization of extra- and intracellular phytases from Rhizopus oligosporus used in tempeh production

Rhizopus oligosporus strain CT11K2, commonly used in tempeh (fermented soybean) production produced both extra- and intracellular phytases. The enzymes were isolated from growth media and the cultured mould and partially purified by acetone fractionation, gel filtration on Sephadex G-100 and DEAE-cellulose chromatography. Intracellular phytase activity was higher than that of the extracellular enzyme. Both enzymes showed maximum activity at pH 4.5 and 55 degrees C, suggesting relatively high thermostability. The enzymes were partially inhibited by high concentrations of substrate. The Km and Vmax values of the extracellular phytase were 0.15 mM and 0.076 mumol Pi per min per ml DEAE-cellulose purified enzyme, respectively, and for the intracellular phytase were 0.17 mM and 0.34 mumol Pi per min per ml enzyme, respectively. Extracellular phytases showed inactivation and activation energies for the hydrolysis of phytic acid of approximately 28,300 cal per mol and 6100 cal per mol, respectively, while inactivation and activation energies for the intracellular phytase were approximately 33,200 per mol and 9500 cal per mol, respectively.     

Fractionation of the Glucoamylase System from Black-koji Mold and the Effects of Adding Isoamylase and Alpha-amylase on Amylolysis by the Glucoamylase Fractions

The glucoamylase system of black koji mold was fractionated into four kinds of glucoamylase by using the corn starch adsorption technique supplement by DEAE-cellulose column chromatography. One of these fractions, referred to as glucoamylase I, had a strong debranching activity and is highly active in raw starch digestion. The raw waxy corn starch digestion by glucoamylase I was accelerated by adding α-amylase or isoamylase. The raw non-waxy corn starch digestion by glucoamylase I was accelerated by α-amylase but not so much by isoamylase as by α-amylase. On the other hand, another fraction, referred to as glucoamylase II, had very weak debranching activity and was most feeble in the digestion of raw starch. The raw waxy corn starch digestion by glucoamylase II was very weak, but was accelerated extremely by adding isoamylase, but not by α-amylase.