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.     

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%。     

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.     

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。     

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.     

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.     

糖化酶稳定性的研究

用大分子亲水性多糖黄原胶,可明显地提高糖化酶的耐热性及储存稳定性。在含有黄原胶、ＮａＣｌ、甘油的缓冲体系中,糖化酶液于室温放置６个月,其酶活只损失１５．２％,而对照的却损失了３９．８％。     

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.     

从土壤中快速筛选出产乳酸根霉的研究

本文报道了适用于根霉乳酸生产菌的筛选米饭富集培养法、平板和摇瓶培养初筛法。用上述富集培养法可使土样中产酸菌的筛出率提高2倍，而利用溴甲酚绿平板和摇瓶培养法与高效液相结合的方法则使快速准确地检出乳酸产生菌，经筛选的根霉菌的产酸率达到49．26g／L．     

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.     

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.     

从日本根霉中提取壳聚糖的初步研究

报道了一种从日本根酶中提取壳聚糖的简单方法。正交试验研究显示,以2%淀粉为碳源,1%蛋白胨为氮源,在起始pH5.0,26℃下培养60h时产量最高,菌丝体干重为8.43g/L。经NaOH间歇处理,HCl抽提,得到天然壳聚糖为895mg/L,壳聚糖产量占菌丝体干重的10.58%。产品壳聚糖纯度为90.5%。     

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)