黑曲霉β-葡萄糖苷酶的酶学特性研究

研究黑曲霉β-葡萄糖苷酶的酶学特性,采用酶学研究方法,通过硫酸铵沉淀、Sephadex G-25脱盐和Sephadex G-100纯化了β-葡萄糖苷酶,并进行了黑曲霉β-葡萄糖苷酶的最适反应温度、最适pH、热稳定性、pH稳定性及米氏常数等特性研究,采用SDS-PAGE凝胶电泳测定了分子量。研究表明,β-葡萄糖苷酶的最适反应温度为70℃、最适反应pH为4.5;在40、50和60℃下较稳定,80℃以上稳定性差;β-葡萄糖苷酶在pH为3、7、8、9的缓冲液中的稳定性很差,在pH为4、5、6的缓冲液中稳定性较好,其中在pH为5时,稳定性最好;酶的Km=41.67 mmol/L,Vmax=23.81 U/L;其分子量为65.2 ku。β-葡萄糖苷酶在饲料工业具有良好的应用前景。     

固定化β淀粉酶的制备及其性质

对β-硫酸酯乙飘基苯胺(SESA)与环氧氯丙烷交联琼脂糖反应,制得对氨基苯砜乙基(ABSE)交联琼脂糖,经重氯化后与β-淀粉酶偶联制成固定化酶。研究了载体的苯胺基含量、PH)、巯基乙醇等因素对酶偶联反应的影响。尤其是巯基乙醇的存在,可使固定化酶活力明显提高。固定化酶活力可达120u／ml,活力回收为38％,相对活力为45％。固定化β-淀粉酶的最适pH和最遗温度与自然酶相似,以可溶性淀粉为底物时,固定化酶的米氏常数是自然酶(Km=0．0057(％))的8倍。将固定化酶装柱,连续水解可溶性淀粉,在45℃下连续操作;50天后,酶活力未见下降,在50℃下28天后,还保留活力50%左右。     

米曲霉F-81产中性蛋白酶的固定化条件及其特性

以海藻酸钠为载体,戊二醛为交联剂固定化米曲霉F-81产中性蛋白酶,研究了固定化条件及固定化酶的性质。结果表明,固定化的最佳条件为:固定化时间1 h、海澡酸钠浓度4%、戊二醛浓度9%、CaCl2浓度0.7 mol/L。在此条件下固定化的中性蛋白酶活力为游离酶活力的68%。固定化酶的最适作用温度为65℃,最适作用pH值为7.0。60℃下酶稳定性较好,80℃下处理60 min,粗酶中几乎检测不到酶活力;中性蛋白酶pH稳定范围为6.5-9.5。Km值为24.83 mg/mL,最大反应速率Vmax为0.043 12 mg/min。     

一种固定化酶的新载体——对氨基苯砜乙基(ABSE-)交联琼脂

1.8%琼脂经环氧氯丙烷交联后,在碱性条件下与对β-硫酸酯乙砜基苯胺(SESA)反应制得了对氨基苯磺酰乙基-(ABSE)-交联琼脂。醚化反应最适pH是10。控制SESA加入量制得含有107～1216微克分子苯胺基/克干重琼脂载体。2.ABSE-交联琼脂经重氮化后可在pH6.4～8.0偶联核酸酶P_1,每克琼脂可结合105～120毫克核酸酶P_1,固定化酶活力为4280单位/克干重固定化酶。活力回收可达18～35%。3.偶联时硫酸铵的存在可稍微提高固定化核酸酶P_1的活力,但其稳定性却比对照的差。4.载体上苯胺基含量过多会不利于所固定化的酶显示活力,用α-萘酚封闭残留的苯胺基,可以明显增加固定化酶的稳定性。     

一种固定化酶的新载体——对氨基苯砜乙基(ABSE-)交联琼脂

1.8%琼脂经环氧氯丙烷交联后,在碱性条件下与对β-硫酸酯乙砜基苯胺(SESA)反应制得了对氨基苯磺酰乙基-(ABSE)-交联琼脂。醚化反应最适pH 是10。控制SESA 加入量制得含有107～1216微克分子苯胺基/克干重琼脂载体。2.ABSE-交联琼脂经重氮化后可在pH6.4～8.0偶联核酸酶P_1,每克琼脂可结合105～120毫克核酸酶P_1,固定化酶活力为4280单位/克干重固定化酶。活力回收可达18～35%。3.偶联时硫酸铵的存在可稍微提高固定化核酸酶P_1的活力,但其稳定性却比对照的差。4.载体上苯胺基含量过多会不利于所固定化的酶显示活力;用α-萘酚封闭残留的苯胺基,可以明显增加固定化酶的稳定性。     

蚕丝固定化脂肪酶的研究

研究了蚕丝固定化脂肪酶的工艺条件,并考察了固定化脂肪酶的稳定性。试验结果表明：蚕丝与对-β-硫酸酯乙砜基苯胺(SESA)进行反应的最适条件是PH=10．8,SESA：2．0g／g蚕丝,反应生成的对氨基苯磺酰乙基蚕丝(ABSE-蚕丝)经重氮化后与脂肪酶偶联的最适条件是：pH=7．5,偶联时间>10h。加酶量为168～308u／g蚕丝时,所得固定化脂肪酶活力为106～160u√g蚕丝．此时固定化冀的活力回收率较高(>52％)。固定化脂肪酶稳定性较高．其操作半衰期约为250h。     

用吸附-交联法在磁性胶体粒子上固载中性蛋白酶

利用吸附-交联法,在磁性胶体粒子表面固载 ASl.398 中性蛋白酶,可以制备出活性达 2500U/g 的磁性固定化中性蛋白酶.该固定化酶具有较好的耐热性和操作稳定性,最适作用 pH6.0—6.5,最适作用温度60℃.考察了交联剂用量、温度、pH及酶与载体比例对 AS1.398 中性蛋白酶固定化的影响.     

N-琥珀酰壳聚糖固定化中性蛋白酶的制备及性质研究

以N-琥珀酰壳聚糖为载体固定中性蛋白酶,研究了固定化酶的适宜温度、pH、热稳定性和酸碱稳定性等酶学性质,同时对固定化酶和游离酶的红外光谱图作了分析比较.结果表明:中性蛋白酶经固定化后,最适酶反应pH由7升至8,最适温度没有改变,仍为50℃,所得固定化酶具有较宽的酸碱稳定性范围,在pH 7～9都保持较高活力,并且同定化酶的热稳定性比游离酶有较大的提高.红外光谱分析表明,酶固定化前后的红外光谱图的部分特征峰有较大的差异.     

木霉GXC产β-葡聚糖酶条件和酶学性质

研究了木霉GXC产β-葡聚糖酶的条件.结果表明,最适产酶碳源为麸皮,氮源为硫酸铵;产酶的最适条件为初始pH为4.0～5.0,30℃培养44h.粗酶液经硫酸铵沉淀、Sephadex G-25、Sephadex G-100和DEAE-Sehadex A-50柱层析得到纯β-葡聚糖酶,SDS-PAGE凝胶电泳显示一条带,测得分子量为35kD.该酶最适反应pH5.0,最适反应温度为60℃,在40℃以下、pH4.0～5.0酶活力相对稳定.5.0mmol/L以下的Ca2+、Zn2+和Fe2+,以及10.0mmol/L以下的Co2+对酶活力有激活作用;而Cu2+和Fe3+具有抑制作用.     

氨基末端磁性载体固定化中性蛋白酶的研究

以氨基末端磁微粒为载体,用戊二醛作交联剂,通过共价交联结合法固定化AS1.398中性蛋白酶.可以制备出活力达45 000 U/g磁性固定化酶.探讨了该载体对中性蛋白酶的最适固定化条件,并对磁性固定化酶的热稳定性,储存稳定性、操作稳定性等进行了研究,确定了此载体对酶的固载能力大于200 mg/g（载体）,及固定化磁性酶最适pH为7.5, 最适温度为60℃等催化特性.     

Stabilization of ficin extract by immobilization on glyoxyl agarose. Preliminary characterization of the biocatalyst performance in hydrolysis of proteins

A protein extract containing ficin was immobilized on glyoxyl agarose at pH 10 and 25 °C. The free enzyme remained fully active after 24 h at pH 10. However the enzyme immobilized on the support retained only 30% of the activity after this time using a small substrate. After checking the stability of ficin preparations obtained after different enzyme-support multi-interaction times, it was found that it reached a maximum at 3 h (40-folds more stable than the free enzyme at pH 5). The immobilized enzyme was active in a wide range of pH (e.g., retained double activity at pH 10 than the free enzyme) and temperatures (e.g., at 80 °C retained three-folds more activity than the free enzyme). The activity versus casein almost matched the results using the small substrate (60%) at 55 °C. However, in the presence of 2 M of urea, it became three times more active than the free enzyme. The immobilized enzyme could be reused five cycles at 55 °C without losing activity.     

无花果蛋白酶在阴离子交换树脂上的固定化

无花果蛋白酶通过８％戊二醛活化载体,共价结合到聚苯乙烯阴离子交换树脂ＧＭ２０１上,固定化作用在ｐＨ７．７,酶浓度０．８ｍｇ／ｇ树脂,４℃下进行６ｈ。得到的固定化酶表观Ｋ_ｍ值（酪蛋白,１．１１×１０~（－４）ｍｏｌ／Ｌ）小于溶液酶Ｋ_ｍ值（１．９６×１０~（－４）ｍｏｌ／Ｌ）;固定化酶活性在ｐＨ６～８保持稳定,溶液酶最适ｐＨ为７．２;固定化酶最适温度由溶液酶的５０～６０℃移至３７℃;固定化酶２５℃保持７ｄ,重复水解酪蛋白７次后,保留８３．３％活性。固定化酶对酪蛋白水解度达４７．５％,对大豆球蛋白达１１．６％。     

Immobilization of urease on vermiculite

Urease (EC 3.5.1.5) of high activity was obtained when the enzyme was immobilized on vermiculite crosslinked with 2.5% glutaraldehyde in chilled EDTA-phosphate buffer (pH 5.5). The highest activity of the immobilized enzyme was at 65°C and pH 6.5 while the optimum temperature for free urease was found to be 25°C. The thermal stability of immobilized urease was observed to be much better than that of the free urease. When stored at 4°C, urease immobilized on vermiculite retained 69 to 81% of its activity after 60 days and 61 to 75% of its original activity was retained after 4 repeated uses.     

Thiol-dependent aminopeptidase (350,000 mol wt) as a marker of epidermal contamination in sweat

L-Leucine 2-naphthylamide (Leu-NA) hydrolytic activity is increased 20-fold in eccrine sweat collected by simple scraping (SS) compared with sweat collected over the white petrolatum (Vaseline) barrier (clean sweat, CS) [Am. J. Physiol. 250 (Regulatory Integrative Comp. Physiol. 19): R691-R698, 1986]. Sephadex G-200 chromatography of SS but not that of CS showed a single peak of Leu-NA hydrolytic activity (at pH 8) at 350,000 mol wt. An enzyme with similar molecular weight was eluted from tape-stripped stratum corneum and from stripped skin in situ. Anion-exchange FPLC of the 350,000 fractions yielded a single Leu-NA hydrolase peak at pH 8 (pool IV), which also showed hydrolytic activity for benzoyl-L-arginine-2-naphthylamide (BANA). Both Leu-NA and BANA hydrolytic activities of pool IV were thiol dependent, inhibited by heavy metals, and activated by ethylenediaminetetraacetic acid. The pool IV enzyme also hydrolyzed L-lysine- and L-arginine-2-naphthylamide. The most prominent BANA hydrolase activity was seen in both SS and CS at pH 5.0 at 33,000, which was not associated with Leu-NA hydrolytic activity. Diethylaminoethyl cellulose chromatography of the 33,000 fractions yielded three peaks of BANA hydrolytic activity in SS but only one in CS, suggesting that this thiol-dependent BANA hydrolyzing enzyme in CS may be of sweat gland origin. We conclude that the 350,000 thiol-dependent Leu-NA-hydrolyzing aminopeptidase is one of the most prominent epidermal contaminants and thus is a useful marker of epidermal contamination in sweat samples.     

Preparation and properties of glucose isomerase of Streptomyces kanamyceticus cells entrapped in calcium alginate

Summary The properties of glucose isomerase in native, heat-treated and immobilized cells of Streptomyces kanamyceticus after heat and mineral treatment have been compared. The optimum pH for glucose isomerase in native cells was shifted from 8.2 to 8.6 by heat treatment and immobilization. There is no change in the optimum temperature (90°C) for activity of the enzyme by the above treatment. Heat-treated cells and immobilized cells show greater pH and thermal stability of the enzyme. The Km values of the enzyme of native cells, heat-treated cells and immobilized heat-mineral-treated cells are 208 mM, 212 mM and 166 mM respectively; Mg⁺⁺ and Co⁺⁺ enhance the activity of isomerase in all cases.     

Studies on the properties of immobilized urokinase: effects of pH and temperature

Human urokinase was immobilized on an ethylene vinyl acetate copolymer surface. Soluble urokinase showed its maximum activity at pH 8.5, while the immobilized enzyme was most active at pH 9.0. Apparently, the shift in optimal pH was due to the polyanionic nature of the carrier surface on which the enzyme was immobilized. Optimal temperatures of soluble urokinase and immobilized enzyme were identical, i.e., 37 degrees C. The stability of immobilized enzyme against thermal degradation was several times higher than that of the soluble enzyme. Its stability at higher temperatures is one of the main reasons for the clinical use of immobilized urokinase as an antithrombotic material.     

Study of the thermal denaturation of ribonuclease A by differential thermal analysis and susceptibility to proteolysis

1. The thermally induced change in conformation of ribonuclease A in solution was investigated by differential thermal analysis and the susceptibility of the enzyme to proteolytic digestion by ficin. 2. A transition with a mid-point of 60.5°C at pH4.2 was observed directly by differential thermal analysis and shown to be a property of the native structure. 3. At pH4.2 ribonuclease A is susceptible to ficin digestion at 60°C but not at 18°C. 4. Chromatographic analysis of the digestion products reveals that transient active intermediates are produced during the digestion. 5. Three of these intermediates were purified and partially characterized. 6. The nature of those sections of the ribonuclease molecule that are involved in the thermal transition is discussed.     

Immobilization of the exo-maltohexaohydrolase by the irradiation method

Exomaltohexaohydrolase (E.C.3.2.1.98) was immobilized by radiocopolymerization of some synthetic monomers which were mixed in various combinations. Irradiation was carried out while the mixture of monomers and enzymes was frozen in petroleum ether-dry-ice bath. Recovery of the immobilized enzyme was 44-75%.The optimum pH of the enzyme slightly shifted to the acidic side. The pH stability was improved remarkably by immobilization. The enzyme was stable retaining more than 90% of its original activity in the range pH 4-11. The optimum reaction temperature of the enzyme increased about 2 degrees C. Heat stability was also improved by immobilization, and that the enzyme retained about 40% of its original activity after treatment at 75 degrees C for 15 min. The immobilized enzyme was stable to the repeated use of 20 cycles. The K(m) value of the enzyme for short-chain amylose was almost the same as that of native enzyme. When soluble starch was used as the substrate, the K(m), value of the enzyme was three times as large as that of native enzyme. Effects of various metal ions and inhibitors on the immobilized enzyme were also studied compared to the native enzyme.     

磁性固定化胰蛋白酶的催化特性及应用的研究

详细研究了磁性固定化胰蛋白酶的催化特性,并与溶液酶进行比较,发现胰蛋白酶经固定化后最适pH值向碱性方向移动了1．0个pH单位,最适温度提高了5℃,K值略有增大。对该固化酶的热稳定性和操作稳定性也进行了研究,结果表明,胰蛋白酶经固定化后热稳定性明显提高,操作稳定性也得到了一定的改善,经3次重复使用后,活性保持43．8％,对啤酒澄清和裸皮软化显示较好的应用前景。     

Enzymes in polyelectrolyte complexes. The effect of phase transition on thermal stability

Penicillin amidase, alpha-chymotrypsin and urease have been immobilized in water-soluble nonstoichiometric polyelectrolyte complexes (N-PEC). N-PEC are formed by modified poly(N-ethyl-4-vinyl-pyridinium bromide) (polycation) and excess poly(methylacrylic acid) (polyanion). N-PEC are a new class of polymers capable, characteristically, of phase transitions solution in equilibrium precipitate induced by slight change in pH or ionic strength. Neither the chemical structure of the carrier nor the number of cross-linkages between an enzyme and a carrier change on phase transition. That gives an unique opportunity to elucidate the difference between enzymes immobilized on water-soluble and water-insoluble supports. A detailed study of the phase transition effect on thermal stability of the enzymes and protein-protein interactions has been carried out. The following effects were found. Pronounced thermal stabilization of penicillin amidase and urease may be achieved on two conditions: the enzyme is in the precipitate; (b) the enzyme is linked to the N-PEC nucleus. Then the thermal stability of N-PEC-bound penicillin amidase increases 7-fold at pH 5.7, 60 degrees C, and 300-fold at pH 3.1, 25 degrees C, compared to the native enzyme. For urease, the thermal stabilization increases 20-fold at pH 5.0, 70 degrees C. The localization of enzyme on N-PEC has been established by titration of alpha-chymotrypsin bound to a polycation or polyanion with basic pancreatic trypsin inhibitor. Both in solution (pH 6.1) and in N-PEC precipitate (pH 5.7), an alpha-chymotrypsin molecule bound to a polyanion is fully exposed to the solution. If the enzyme is bound to a polycation, only 20% of alpha-chymotrypsin molecules in the precipitate and 40% in solution retain their ability for protein-protein interactions. This means that a polycation-bound enzyme is localized in the hydrophobic nucleus of the complex, whereas the polyanion-bound enzyme sits on the hydrophilic shell of the complex. On pH-induced phase transition (pH decreases from 6.1 to 5.7), there occurs a stepwise decrease in penicillin amidase activity which is due to a 9.8-fold increase in the Km for 2-nitro-4-phenylacetamidobenzoic acid. Change of the catalytic activity and thermal stability of N-PEC-bound penicillin amidase is fully reversible and reproducible. Such soluble-insoluble immobilized enzymes with controllable thermal stability and activity may be used for simulating events in vivo and in biotechnology.