Proteasome from rabbit skeletal muscle: Some properties and effects on muscle proteins

Rabbit proteasome, likely to be a 20S proteasome, was purified and its properties were investigated to clarify its contribution to proteolysis during meat conditioning. The purified enzyme migrated as a single band on non-denaturing polyacrylamide gel and dissociated to a number of subunits (20000–29000 Da) under denaturing conditions. The molecular mass of this enzyme was found to be 580 000–800 000 Da by Sephacryl S-300 column chromatography. The isoelectric point of this enzyme was 5.5. The optimum pH for hydrolysis of succinyl-Leu-Leu-Val-Tyr-(4-methylcoumaryl-7-amide) (Suc-LLVY-MCA) was 8. This enzyme was almost stable in the range of pH 5–9 and up to 60 °C at pH 7.2. The enzyme activity was inhibited by diisopropyl fluorophosphate (DFP) and chymostatin, but was not affected by EDTA, leupeptin, E-64, bestatin, monoiodoacetic acid or pepstatin. The enzyme was activated about 8-fold by 0.01% sodium dodecyl sulfate (SDS), but was not by ATP or CaCl₂. Remarkably, SDS increased the V_max value of the enzyme. Rabbit proteasome was shown to degrade myosin heavy chain, -actinin, actin, tropomyosin, troponins and myosin light chains in the presence of SDS. In the absence of SDS, no change in myofibrillar proteins was observed. This enzyme did not degrade any sarcoplasmic proteins regardless of the presence of SDS.     

ENZYMATIC CHARACTERISTICS OF TRYPSIN FROM PYLORIC CECA OF SPOTTED MACKEREL (SCOMBER AUSTRALASICUS)

Trypsin was purified from the pyloric ceca of spotted mackerel (Scomber australasicus) by gel filtration on Sephacryl S‐200 and Sephadex G‐50. The purification and yield were 20‐fold and 81%, respectively, as compared to those in the starting crude extract. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the molecular weight of the enzyme was estimated to be 24,000 Da by SDS–PAGE. The trypsin was stable at pH 5–11 for 30 min at 30C, and its maximal activity against N^α‐p‐tosyl‐L‐arginine methyl ester was pH 8.0. Trypsin was heat‐stable up to about 50C for 15 min at pH 8.0. Optimum temperature of the trypsin enzyme was 60C. The enzyme was stabilized by calcium ion. The purified trypsin was strongly inhibited by serine protease inhibitors such as N‐p‐tosyl‐L‐lysine chloromethyl ketone and soybean trypsin inhibitor, suggesting that it is a trypsin‐like serine protease. N‐Terminal amino acid sequence of spotted mackerel trypsin was IVGGYECTAHSQPHQVSLNS.     

Bovine muscle 20S proteasome: I. Simple purification procedure and enzymatic characterization in relation with postmortem conditions

Over the last decade, several sets of evidence support a possible contribution of the 20S proteasome to the meat tenderizing process. This assumption was emphasized by recent investigations demonstrating that the 20S proteasome was active in the absence of activators and exhibited endo- and exoproteolytic activities, a status often strongly debated before. In the present work, we developed a new rapid and simple purification procedure for muscle 20S proteasome and revisited the physicochemical properties of this complex in relation with the postmortem muscle environmental conditions, i.e. temperature, pH, osmolarity, etc. From a crude extract obtained from freshly excised muscle tissue, reasonable amounts of highly pure proteasome were prepared within a maximum of 4 days using only three chromatography steps. This purified proteasome was used to investigate the effect of pH, temperature, ionic strength and sodium dodecyl sulphate (SDS) on the major hydrolytic activities of this complex, i.e. trypsin-like (TL), chymotrypsin-like (CL) and peptidylglutamyl peptide hydrolase (PGPH) activities. Taken together, the data obtained suggest that the 20S proteasome constitutes a high hydrolytic potential in postmortem muscle conditions. To attest this finding, the 20S proteasome was further quantified by ELISA in at death and postmortem muscles including Longissimus, Rectus abdominis, Diaphragma pedialis and Tensor fascia latae bovine muscles. The primary conclusion was that time course changes in proteasome concentrations were not dependent on the kinetics of the pH fall. Secondly, the proteasome concentration in conditioned meat was in good agreement with previously reported proteolytic activity. Furthermore, the decrease in the muscle proteasome concentration can be considered as slow and this is particularly true in type 1 muscles for which the decrease in the amount of this complex did not exceed 7% during the first three days postmortem. This would suggest that the 20S proteasome was relatively stable during meat conditioning, a feature supporting a potential role in the meat tenderizing process.     

PURIFICATION OF A PROTEASE FROM AN ALKALOPHlLIC BACILLUS SUBTILIS CHZ1 ISOLATED FROM A ZIMBABWEAN HOT SPRING

A proteolytic enzyme produced by Bacillus subtilis CHZ1 was purified using ammonium sulfate precipitation, gel filtration and cationic exchange on S‐Sepharose fast flow column chromatography. Production of the protease was higher when the Bacillus strain was cultured in a synthetic medium, M162, supplemented with 0.3% (w/v) organic compared to inorganic nitrogen sources. Enzyme production was growth dependent and production was highest when tryptone was used as the nitrogen source. When run on SDS‐PAGE gel, the purified enzyme gave a 35 kDa band, suggesting that it consisted of one polypeptide chain. High enzyme activity was observed in the pH range of 6–10 with a maximum value at pH 8.0 when 0.5% (w/v) azocasein was used as the substrate. Optimum temperature for protease activity was found to be 60–80C, and the enzyme had considerable thermal stability for 5.5 h retaining about 90% activity after 5.5 h. At 2.5 mM concentration, PMSF, Ag⁺ and Hg⁺ inhibited activity of the protease. Metal cofactors like Mn²⁺, Mg²⁺ and Fe²⁺ increased the enzyme activity by about 20%. Zn²⁺, Cu²⁺ and Ca²⁺ did not affect the enzyme's activity. The pH and thermal stability as well as high specific activity of this enzyme can be exploited for industrial applications.     

Purification and Characterization of Bacillus acidopullulyticus Pullulanase for Enzymatic Starch Modification

The pullulanase (EC 3.2.1.41) of Bacillus acidopullulyticus was purified using anion exchange chromatography and preparative isoelectric focusing. The purified preparation migrated as a single protein band upon SDS gel electrophoresis and its molecular weight was estimated to be 102,000. Two main activity bands having pl-values 5.0 and 5.2 were detected by analytical isoelectric focusing. The enzyme was purified 4-fold with the yield 38% by this two-step purification procedure. The purified pullulanase showed maximal activity at 50°C and pH 5 and was slightly activated by Ca₂₊. It was stable at 50°C but totally lost its activity at 60ºC in one hour. This purified pullulanase efficiently hydrolyzed the α-1,6-glucosidic bonds of pullulan and gelatinized starch.     

中性植酸酶产生菌的筛选、鉴定及酶学性质

从土壤中分离到一株高产中性植酸酶的菌株,酶活力达12.52U/mL。对其进行形态、生理、生化、分子生物学鉴定,初步鉴定为放射型根瘤杆菌(Agrobacterium radiobacter)。酶学性质研究结果表明：该酶的最适反应温度为45℃;最适反应pH 值为7.0;65℃处理60min 酶活力保持80% 左右,有一定耐热性;在pH5.5～8.0 之间,稳定性较好;Ba2+ 对酶活力有一定的激活作用,Fe2+、Mg2+、Zn2+ 和Cu2+ 对酶活力均有一定程度的抑制作用,其中Fe2+ 的抑制作用最强。     

法尔皮有孢汉生酵母(Hanseniaspora valbyensis)醇乙酰基转移酶的分离纯化及特性

乙酸苯乙酯是苹果酒里的重要香气物质之一,是在醇乙酰基转移酶(AATase)的催化下由乙酰辅酶A和苯乙醇缩合而成。从法尔皮有孢汉生酵母(Hanseniasporavalbyensis)中收集细胞膜片段,然后经过DEAESepharose、SephadexG-75、OctylSepharose三步层析分离得到纯酶。纯化后的酶经SDS-PAGE电泳,得到一条明显的条带,分子量在37kD左右。此酶的最适反应条件是在30℃、pH7.0,在10℃以下酶比较稳定,pH稳定在7.0～8.0。重金属离子Hg2+、Zn2+、Pb2+对酶活有明显的抑制,EDTA、Mn2+对酶活没有太大的影响,Mg2+能激活此酶。此酶也能催化其它重要乙酸酯的合成,但对乙酸苯乙酯的合成具有特异性。     

重组大肠杆菌耐热α - 淀粉酶的分离纯化及其酶学性质研究

对重组大肠杆菌耐热α- 淀粉酶分离纯化及其酶学性质进行研究,结果表明：该酶分子量约为90kD,最适温度为60～70℃,最适pH 值为6.6,酶学动力学常数Km 值为143.52mmol/L;酶活力在pH5.4～7.8 较为稳定;4℃保存2 周酶活力仅下降一半,35℃保温3d 有50% 以上的酶活力,70℃以上酶失活很快;Mn2+ 对酶催化作用有较大的促进,K+、Ca2+ 有微弱的促进作用,Mg2+ 对催化反应无影响,Cu2+ 的抑制作用最强,其他金属离子Co2+、Zn2+、Fe2+ 对酶催化作用有不同程度的抑制作用;有机离子对酶催化作用均是抑制作用,其中抑制作用最强的是SDS。     

新型耐热耐酸普鲁兰酶的分离纯化与酶学特性分析

从泡盛曲霉(Aspergillus awamori)XF发酵液中分离纯化普鲁兰酶(PulXF)并研究其酶学性质。通过硫酸铵沉淀、阴离子交换层析、凝胶过滤层析和疏水层析,纯化得到一种电泳纯的PulXF。纯化倍数8.16倍,比活力为309.28 U/mg。SDS-PAGE检测得单条带,表明PulXF为单亚基蛋白,分子量63.7 kDa。在50~80 ℃,pH4.5~8.0范围内,酶能保持高活性,最适反应温度60 ℃,最适pH5.0。在较广的pH范围内(pH3.0~8.0),28 ℃下,经过24 h酶活能保持80%以上活性。K_m值和V_max分别为0.92 mg/mL和11.90 μmol/min。酶活测定及TLC分析显示PulXF对普鲁兰多糖有强水解活性,终产物麦芽三糖;对支链淀粉和可溶性淀粉有较弱活性,对直链淀粉、α-环糊精、β-环糊精和糖原无活性。表明从泡盛曲霉XF分离的PulXF属于I型普鲁兰酶。Ca2+、Zn2+和Mg2+能够促进酶的活性,其中Ca2+激活作用最强。而PulXF在5%浓度的SDS、CTAB、Tween 80和30%的乙醇中保持高稳定性。基于PulXF在苛刻环境下的高稳定性及高活性,很有希望在淀粉加工、食品饮料等领域以及需要在高温、高酸环境下使用的生物技术工业中使用。     

Purification and characterization of trypsin from the viscera of sardine (Sardina pilchardus)

Trypsin from the viscera of Sardina pilchardus was purified by fractionation with ammonium sulphate, heat treatment and Sephadex G-100 gel filtration with a ninefold increase in specific activity and 9% recovery. The molecular weight of the enzyme was estimated to be 25,000 Da on SDS–PAGE. This enzyme showed esterase-specific activity on Nα-benzoyl-l-arginine ethyl ester (BAEE). The purified enzyme was inhibited by benzamidine, a synthetic trypsin inhibitor, and phenylmethylsulphonyl fluoride (PMSF) a serine-protease inhibitor, but was not inhibited by the β-mercaptoethanol. The optimum pH and temperature for the enzyme activity were pH 8.0 and 60 °C, respectively. The relative activity at pH 9.0 was 95.5% and the enzyme showed pH stability between 6.0 and 9.0. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECQKYSQ. S. pilchardus trypsin, which showed high homology to other fish trypsins, had a charged Lys residue at position 9, where Pro or Ala are common in fish trypsins. The enzyme was strongly inhibited by Zn²⁺ and Cu²⁺.     

Purification and characterization of extracellular proteinase produced by Brevibacterium linens ATCC 9172

The micro-organism Brevibacterium linens ATCC 9172 produced five extracellular proteinases, as shown by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) with copolymerized gelatine. One of these was purified to homogeneity by ion-exchange chromatography and native preparative PAGE. The optimum pH and temperature for the proteinase were 8.0 and 50°C, respectively. The enzyme remained stable over a pH range from 6 to 10. The molecular weight estimated by SDS–PAGE was 56 kDa. Serine proteinase inhibitors 3,4-dichloroisocoumarin (3,4-DCI) and phenylmethylsulphonylfluoride (PMSF) inhibited, while Mg²⁺ and Ca²⁺ ions activated the proteinase.     

酶脱支处理对玉米缓慢消化淀粉形成的影响

以玉米原淀粉为原料,研究普鲁兰酶脱支处理糊化后制备缓慢消化淀粉(slowly digestible starch,SDS)过程中各影响因素(温度、p H值、酶用量、贮藏及干燥条件)对SDS形成的影响。结果表明,在57.5℃、p H 4.9、酶用量60 U/g的条件下脱支8 h,然后煮沸灭酶30 min,再经4℃冷藏、60℃干燥后,可得SDS含量为31.09%的产品。原淀粉、酶脱支处理样品及脱支并去除快速消化淀粉样品的X射线衍射图谱表明,脱支处理后,玉米淀粉结晶结构由A型向B型转变。因此,通过酶脱支处理提高SDS含量的可能原因是形成了新的结晶结构,SDS含量与结晶的数量和质量有关。采用酶法制备SDS具有较好的工业化应用前景。     

Ammonia assimilation in Klebsiella pneumoniae F-5-2 that can utilize ammonium and nitrate ions simultaneously: purification and characterization of glutamate dehydrogenase and glutamine synthetase

The two ammonia-assimilating enzymes glutamate dehydrogenase (GDH; EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) were synthesized steadily during the cell growth of Klebsiella pneumoniae F-5-2 that can utilize NH4+ and NO3- simultaneously under aerobic conditions. The enzymes were purified to homogeneity from cell extracts and characterized. The molecular mass of the purified GDH was 300 kDa with six identical 52-kDa subunits. GDH showed its maximal activity (aminating) at pH 8.0 and was stable between pHs 5.5 and 11.5. The enzyme was NADP-specific and strongly inhibited by Ag+. It catalyzed the amination of 2-ketovalerate, 2-ketoadipate, and 2-ketobutyrate, in addition to 2-ketoglutarate. The purified GS has a molecular mass of 470 kDa with eight identical 60-kDa subunits. GS showed its maximal activity at pH 8.0 and was stable between pHs 6.0 and 7.0. The enzyme was strongly inhibited by Fe3+, Hg2+, and Cu2+.     

杨桃多酚氧化酶的部分纯化及其特性研究

杨桃多酚氧化酶粗酶液经过DEAE-Toyopearl650M离子交换柱层析,Butyl-Toyopearl650M疏水柱层析后,被纯化了21.6倍,回收率为45.2%。该酶能迅速地催化焦性没食子酸的酶促氧化反应,而对邻苯二酚、间苯二酚、对苯二酚和绿原酸则完全无催化活性。该酶对焦性没食子酸的Km值为7.92mmol/L,其最适pH为8.0,pH稳定性范围在pH4.0-11.0,最适温度为60℃,热稳定性相对较高,在≥90℃加热30min后仍残留约9%的酶活性。该酶的最佳抑制剂是抗坏血酸,其次是NaHSO3和盐酸-L-半胱氨酸,Cu2+、Zn2+、Ca2+等金属离子对该酶也有一定的抑制作用。     

蜡状芽孢杆菌Bacillus cereus SWWL6产耐有机溶剂脂肪酶发酵条件优化及酶学性质

以蜡状芽孢杆菌Bacillus cereus SWWL6为产耐有机溶剂脂肪酶的出发菌株,对发酵产酶条件进行优化并研究其酶学性质。通过四因素三水平L9(34)正交试验,确定产酶的最优发酵条件为：pH6.5、温度35℃、每250mL容量瓶中装液量55mL、接种量4%,此条件下酶活力达45.62U/mL。该酶最适pH值范围为7.0～8.0,碱性条件下稳定。最适温度为40℃,超过40℃,酶活力急剧下降。K+、Na+、Mg2+、Ba2+对该酶活性有激活作用,Al3+、Cu2+、Zn2+、Ca2+对该酶活性有一定的抑制作用,Fe2+对该酶活性的作用效果不明显。SDS、EDTA均对该酶活性有抑制作用,其中尤以SDS的抑制作用最强。该酶对所试有机溶剂甲醇、环乙烷、异丙醇、乙醇、甲苯、丙酮、异戊醇均有一定的耐受性。     

Heat inactivation and kinetics of polyphenoloxidase from palmito (Euterpe edulis)

Polyphenoloxidase (PPO, EC 1.14.18.1)was extracted from palmito (Euterpe edulis Mart) using 0.1 M phosphate buffer, pH 7.5. Partial purification of the enzyme was achieved by a combination of (NH₄)₂SO₄precipitation (35–90% saturation) and Sephadex G-25 and DEAE-cellulose chromatography. The purified preparation gave five protein bands on polyacrylamide gel electrophoresis, three of them with PPO activity. The K_mvalues for chlorogenic acid, caffeic acid, catechol, 4-methylcatechol and catechin were 0.57, 0.59, 1.1, 2.0 and 6.25 mM , respectively. PPO has a molecular weight of 51 000 Da, maximum activity at pH 5.6 with chlorogenic acid as substrate, and was stable between pH 5.0 and 8.0. The enzyme was heat stable at 50–60°C and inactivated at 75°C. The heat stability of palmito PPO was found to be pH dependent; at 50°C and pH 4.0 the enzyme was fully inactivated after 30 min. The pH/activity studies showed two groups with pK values c 4.6 and 6.7 involved in PPO catalysis.     

Properties of an alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix K1

A NAD+-dependent medium-chain alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix K1 was expressed in Escherichia coli and purified. The recombinant enzyme was a homotetramer of molecular mass 1.6 x 10(2) kDa. The optimum pH for the oxidative reaction was around 10.5 and that for the reductive reaction was around 8.0. The enzyme had a broad substrate specificity including aliphatic and aromatic alcohols, aliphatic and aromatic ketones, and benzylaldehyde. This enzyme produced (S)-alcohols from the corresponding ketones. The enzyme was thermophilic and the catalytic activity increased up to 95 degrees C. It maintained 24% of the original catalytic activity after incubation for 30 min at 98 degrees C, indicating that this enzyme is highly thermostable.     

Isolation and partial characterisation of bovine rumen myosin

Myosin was isolated from rumen muscle and purified by DEAE Sephadex A-50 chromatography. The purified myosin gave only two bands on SDS gel electrophoresis, one corresponding to the heavy chain of 210 000 D and the other corresponding to a light chain of 17 000 D. The pH optimum of the rumen myosin ATPase activity was found to be at 7·6; and at pH 9·1, there was no detectable activity. The ATPase activity of the rumen myosin was found to be lower than that of the skeletal myosin. Since it is known that the light chains are located on the globular head of the myosin molecule, where the ATPase activity is found, the lower rumen myosin ATPase activity may be due to the absence of certain light chains that are commonly found in the skeletal myosin. The rumen myosin also had a lower basic amino acid content and a lower ratio of basic to acidic amino acids than the corresponding skeletal muscle counterpart.     

Characterization of polyphenol oxidase from Napoleon grape

Polyphenol oxidase (PPO) from Napoleon grape was isolated using a two-phase partitioning approach with Triton X-114. The enzyme was purified in a latent form and could be optimally activated by the presence of 0.2% of sodium dodecyl sulphate (SDS) at pH 6.0. In the absence of SDS, the enzyme showed maximum activity at acid pH (3.0). The enzyme was kinetically characterized at pH 3.0 and pH 6.0 in the presence of 0.2% of SDS, using 4-tert-butylcatechol (TBC) as a substrate. The V_m/K_M ratio showed that Napoleon grape PPO presents greater affinity for TBC at acid pH (0.1 min⁻¹) that at pH 6.0 in the presence of SDS (0.02 min⁻¹). The enzyme was highly heat stable, 80% of activity remaining at 70 °C. Selected inhibitors were also studied, tropolone being the most active with a K_i value of 27 μM at acid pH and pH 6.0 in the presence of 0.2% SDS.     

Glutathione Peroxidase of Fish

Glutathione peroxidase (GSH-Px) activity was detected in the muscle and skin tissues from several fish species. The muscle GSH-Px showed an optimum pH at 8.0 for salmon and 8.5 for carp. Stability of salmon muscle enzyme was enhanced in the presence of reduced glutathione (GSH), but considerably decreased in the presence of tert-butylhydroperoxide. When salmon fillets were stored at -50°C, the GSH-Px activity increased gradually during storage. Fish muscle GSH-Px shows potential for preventing oxidative deterioration in muscle during storage and processing.