Characterization on the Conformation of Glucoamylase from Rhizopus niveus and Rhizopus delemar

Using the pH-jump (neutral to above 11) for Tyr-OH ionization as a probe, two glucoamylases from Rhizopus niveus and Rhizopus delemar were found to be caused the change in conformation, which is in a kinetically single step. The pH titration at pHs below 12 was carried out on a multidimensional correlation spectrophotometer. Correlations among the spectrophotometric properties: fluorescence, absorption and circular dichroism are almost identical for the two enzymes. These results suggest that conformational characteristics of the niveus enzyme is almost the same as that of the delemar one.     

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。     

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。     

Multiplicity of Glucoamylase of Aspergillus oryzae Part 1. Separation and Purification of Three Forms of Glucoamylase

The glucoamylase system of Aspergillus oryzae cultured on wheat bran was separated into 3 active fractions by (NH₄)₂SO₄, rivanol and ethanol precipitation followed by ion exchange chromatography on DEAE-Sephadex A-25. One of these fractions, referred to as glucoamylase I was purified by further chromatography on hydroxyapatite gel and Sephadex G-200. The other two fractions referred to as glucoamylase II and III were purified by further gel filtration on Sephadex G-200. The purified glucoamylases were found to be homogeneous on 7.5% polyacrylamide gel electrophoresis and isoelectric focusing by carrier ampholites.     

Multiplicity of Glucoamylase of Aspergillus oryzae Part 2. Enzymatic and Physicochemical Properties of Three Forms of Glucoamylase

Three forms of glucoamylase of Aspergillus oryzae had optimum pH range between 4.5 to 5.0 at 40°C and optimum temperature range between 40 and 60°C. They were most stable at pH range between 4.0 and 7.0 and temperatures up to 40°C. The molecular weights were 76.000, 38.000 and 38.000, respectively. Hydrolysis activities of the three glucoamylases on different substrates were studied. They differed in their rate of attack on many substrates. Glucoamylase I had strong debranching activity and was highly active in raw corn starch digestion. Glucoamylase II and III had weak debranching activities and could hardly digest raw starch.     

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.     

Multiple Forms of Phenoloxidase

SUMMARY— The phenoloxidase system in the tissues of mushrooms, potatoes, and apples was investigated using a polyacrylamide electrophoretic technique. The enzyme system was shown to exhibit the phenomenon of multiple forms. The multiple form pattern obtained was highly characteristic for each individual species and variety studied, and substrate specificity was evident. The mushroom phenoloxidase system ( Agaricus campestris ) was shown to consist of at least nine distinct dl-dopa-reactive multiple forms, and at least three forms reacting with I-tyrosine. Potatoes (var. Rural Russet) showed at least 11 bands of dl-dopa activity, while apples (var. Golden Delicious) had at least three multiple forms of dl-dopa activity. By introducing the "multiphase" gel electrophoretic technique a better resolution of the multiple forms was obtained. A group of closely related dl-dopa multiple forms in mushrooms had the unique ability to withstand the temperature of 70°C for one hour. Sulfite, ethylenediamine-tetraacetic acid (EDTA) and other treatments affected the multiple forms differently. Each multiple form behaved as an individual entity upon repeated elutions and electrophoreses.     

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。     

Effect of Organic Nitrogen Sources on Raw Starch‐Digesting Glucoamylase Production of Rhizopus sp. MKU 40

In a liquid cultivation of Rhizopus sp. MKU 40, supplementation of the medium with 1.5% (w/v) organic nitrogen sources (neopeptone, casein from milk, and meat extract) had a slightly positive effect on glucoamylase (GA) (EC 3.2.1.3) activity compared with the medium lacking organic nitrogen sources. The addition of organic nitrogen sources induced production of protease. Supplementation of the medium with 1.5% (w/v) organic nitrogen sources resulted in an acid and neutral protease activity of 11 — 25 U/mL and 12 — 20 U/mL, respectively. The co‐existence of GA‐I [a highly raw starch‐digesting glucoamylase (RSDG)] and protease in the same medium leads to the production of Ga‐II (an extremely weak RSDG) from GA‐I. As a result the RSDG activity in the medium decreases. Raw starch adsorption rates of a medium without organic nitrogen sources were 100%, because the medium contained only GA‐I. In contrast, the media supplemented with organic nitrogen sources had low starch adsorption rates because the media contained both GA‐I and GA‐II. The results presented in this paper indicate that supplementation of the culture medium of Rhizopus strains with organic nitrogen sources negatively affects GA‐I production.     

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.     

根霉曲一，二级菌种接种方法

传统的根霉酒曲生产是以纯根霉菌、纯酵母菌 ,经过试管（一级）、三角瓶（二级） ,用麦麸皮、麦芽汁、琼脂制成培养基 ,逐级转接扩大培养 ,再投入大批生产。这就是典型的微生物接种培养 ,它将一种微生物移到新的灭过菌的培养基上 ,使它生长繁殖。质量好的根霉酒曲首先要选好菌种 ,其次做好试管、三角瓶菌种的接种与培养工作。现将根霉酒曲试管、三角瓶菌种的接种与培养工艺操作简述如下 ,供同行参考。１菌种Ｑ３０３麸皮试管菌种 :贵州省轻工业科研所。３．８６６麸皮试管菌种 :中国科学院菌种组。ＹＧ５ -５麸皮试管菌种 :重庆市酒类科研所…     

糖化酶及糖化酶基因在酿酒酵母中表达的研究进展

介绍了糖化酶的性质和结构,同时介绍了糖化酶基因引入酿酒酵母中构建酿酒酵母工程菌和糖化酶高效分泌到酿酒酵母胞外的研究进展;展望了糖化酶今后的研究方向以及应用前景.     

Properties of Gel-Entrapped Glucoamylase

Glucoamylase from Aspergillus niger was immobilized by entrapping in glutaraldehyde crosslinked gelatin. Preparations were prepared in the form of beads and thin layers on activated glass, polyester and aluminium foils. Immobilization did not cause changes of pH and temperature optimum. However, it increased thermal stability additionally stabilized by the presence of a substrate. The most stable preparations acted without change in activity for 34 days at a temperature of 50°C. Obtained preparations hydrolyzed α-amylase liquefied starch to a DE value of about 97%. The preparations of glucoamylase immobilized in a thin layer of gelatin on the polyester and aluminium foil were used in a thin layer flow reactor for hydrolysis of 10% starch solution.     

Glucoamylase of Amylomyces rouxii

The production of glucoamylase by Amylomyces rouxii, a mold used in rice and cassava fermentations in the Orient, was demonstrated under solid substrate culture conditions. The enzyme purified by ammonium sulfate fractionation, gel filtration, and Sephadex ion exchange column chromatography appears homogeneous. A. rouxii glucoamylase is a glycoprotein and has an optimum pH around 4.5, optimum temperature 60°C, a molecular weight of 55,600 daltons, and K_m values of 15.8, 27.6, 16.8 mg/mL for soluble starch, glycogen, and amylopectin, respectively. Unlike the other fungal glucoamylases, which were found (from culture filtrates) to exist in multiple forms, A. rouxii glucoamylase, isolated from solid substrate fermentation, displayed only one form.     

上海型根霉与川黔型根霉的特点

方心芳等老前辈将根霉分为“上海型”和“川黔型”。这两类根霉都具有很强的糖化力．都具有糖化和酒精发酵的功能,但它们又各自具有特点。上海型根霉在发酵过程中产酸能力强．适于酿造薯类原料,而不同的上海型根霉菌种又各具特色;川黔型根霉产酸很少．适于高粱原料的发酵。(陶然)     

Immobilization of Glucoamylase on Cellulose

The effectiveness of immobilization of glucoamylase on cotton linters and beech wood pulp activated in a number of manners was examined as well as the kinetic properties of immobilized enzyme.     

Adsorption of Glucoamylase on DEAE-Cellulose

The adsorption of glucoamylase on DEAE-cellulose and the properties of the immobilized enzyme in respect to its use in hydrolysis of starch are described.     

Glucoamylase Research: An Overview

This paper reviews the main features of glucoamylase research describing essentially more recent developments on microorganisms, production and properties of glucoamylases. It is an important industrial enzyme and is widely used in starch saccharification, brewing and distilling industry. Glucoamylase can be derived from a wide variety of sources including plants, animals and microorganisms. Most of the production techniques essentially, however, are based on microbial sources. It is an extracellular enzyme and has got second place (next to proteases) in world's distribution and sales among industrial enzymes. Most of the production of glucoamylase is carried out using liquid fermentation process. Technique of solid state fermentation, however, have also been employed for its production and recently this technique is gaining renewed interest from researchers for its production. The paper also describes aspects related with the purification and characterization of glucoamylase enzyme.