Biochemical characterisation of MX-4, a plant cysteine protease of broad specificity and high stability

A new proteinase, mexicain-IV (MX-4), has been purified to homogeneity from the latex fruits of Jacaratia mexicana (formely Pileus mexicanus), a plant member of the Caricaceae family. MX-4 shows a Mr of 23.7 kDa, pI of 9.3 and maximum proteolytic activity on casein and BAPNA at pH 8.0–8.5 and pH 7.0–7.5, respectively. The amino acid sequence of MX-4 and its reversible inhibition by HgCl₂ show that the proteinase belongs to the family of cysteine proteinases. This enzyme exhibits rather broad substrate specificity, although there seems to be a slight preference for cleavage of peptides having certain hydrophobic residues in the P2 position. Biochemical and circular dichroism studies revealed that this enzyme belongs to the α + β class of proteins, in agreement with the results obtained by X-ray crystallographic structure determination, and showed that MX-4 has a higher pH and thermal stability than other members of the Caricaceae family, including papain. These properties make this novel protease a suitable novel analytical tool for the proteomic analysis of peptide fragments of great potential interest in the food industry and other industries.     

Characterization and ecological implications of midgut proteolytic activity in larvalPieris rapae andTrichoplusia ni

In their larval luminal midgut fluid,Trichoplusia ni (Lepidoptera: Noctuidae) andPieris rapae (Lepidoptera: Pieridae) contain endopeptidases as their primary proteases. Neither species has detectable exopeptidase activity. Studies using enzyme-specific substrates and inhibitors demonstrate that the endopeptidases are serine proteinases (both trypsinlike and chymotrypsinlike) with histidine at the active site. Optimal pH for the tryptic and chymotryptic activity is 8.5 and 8.0, respectively, forT. ni. and 8.0 and 9.0, respectively, forP. rapae. The efficiency of proteolytic digestion (as measured by the rate of in vitro digestion of a standard protein by the midgut luminal fluid) is positively correlated with the larval dietary protein requirement and is significantly influenced by the ratios of tryptic to chymotryptic activity present in the gut lumen of these two species of Lepidoptera.     

Myofibril-Bound Serine Proteinase (MBP) and its Degradation of Myofibrillar Proteins

Proteolysis of a myofibril-bound serine proteinase (MBP) from carp Cyprinus carpio on myofibrillar proteins and their gel formation ability were investigated. MBP readily decomposed myosin heavy chain as indicated by SDS-PAGE. In the preparation of kamaboko, the gel formation ability was diminished by addition of MBP. The optimum degradation temperatures of MBP to myosin heavy chain in myofibril and kamaboko gel were 55°C and 60°C, respectively. The degradation effects of MBP on actin, α-actinin and tropomyosin were studied by the immunoblotting method. Because of its myofibril-bound and myofibrillar protein degradation characteristics, MBP was regarded as the proteinase most probably involved in the modori effect.     

Comparison of muscle proteolytic and lipolytic enzyme levels in raw hams from Iberian and White pigs

Muscle enzyme activities in Biceps femoris from Iberian and White crossbreeds were quantified in vitro to determine possible differences due to crossbreed and the different slaughtering age (12-month-old Iberian pigs and 6-month-old White pigs). Large differences were found between proteolytic and lipolytic enzyme levels from the skeletal muscle of Iberian pig breed and White pig crossbreed. In raw hams from Iberian pig, calpain and cathepsin (B, L and H) were significantly lower, while cathepsin D was significantly higher than in White pigs. Significantly lower dipeptidyl peptidase (II and IV), aminopeptidase B, leucyl and pyroglutamyl aminopeptidase activities were found in Iberian pig breed compared with White pig crossbreed. Significantly higher levels of dipeptidyl peptidase III and alanyl aminopeptidase and lower activities of acid lipase and neutral esterase were found in Iberian breed. It was concluded that lower muscle enzyme activities and in consequence slower proteolysis and lipolysis should be desirable to obtain higher quality in dry-cured products. © 1998 SCI     

Biochemical properties of two isoforms of trypsin purified from the Intestine of skipjack tuna (Katsuwonus pelamis)

Two trypsins (A and B) from the intestine of skipjack tuna (Katsuwonus pelamis) were purified by Sephacryl S-200, Sephadex G-50 and DEAE-cellulose with a 177- and 257-fold increase in specific activity and 23% and 21% recovery for trypsin A and B, respectively. Purified trypsins revealed a single band on native-PAGE. The molecular weights of both trypsins were 24 kDa as estimated by size exclusion chromatography and SDS–PAGE. Trypsin A and B exhibited the maximal activity at 55 °C and 60 °C, respectively, and had the same optimal pH at 9.0. Both trypsins were stable up to 50 °C and in the pH range from 6.0 to 11.0. Both trypsin A and B were stabilised by calcium ion. Activity of both trypsins continuously decreased with increasing NaCl concentration (0–30%) and were inhibited by the specific trypsin inhibitors – soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone. Apparent K_m and K_cat of trypsin A and B were 0.22–0.31 mM and 69.5–82.5 S⁻¹, respectively. The N-terminal amino acid sequences of the first 20 amino acids of trypsin A and B were IVGGYECQAHSQPPQVSLNA and IVGGYECQAHSQPPQVSLNS, respectively.     

壳聚糖固定瑞士乳杆菌蛋白酶的条件研究

以壳聚糖为载体,戊二醛为交联剂,采用交联-吸附法对瑞士乳杆菌蛋白酶的固定化条件进行研究。在单因素试验基础上,以固定化酶活力为主要指标,研究凝结液、壳聚糖质量浓度、酶用量、交联时间、戊二醛质量浓度对瑞士乳杆菌蛋白酶固定化的影响。运用响应面对固定化条件进行优化,确定瑞士乳杆菌蛋白酶的最优固定条件：凝结液为4g/100mL NaOH-甲醇(体积比3:1)、壳聚糖质量浓度2.89g/100mL、酶用量2.95mg、交联时间1h、戊二醛质量浓度0.40g/100mL,此时固定化酶活力为28.67U。     

蛋白酶A对纯生啤酒泡持性的影响研究

该文研究蛋白酶A活力对纯生啤酒泡持性的影响,同时建立初始成品纯生啤酒及过滤前发酵液蛋白酶A活力限量值。结果表明,随着贮存时间的延长,纯生啤酒蛋白酶A活力和泡持值均呈下降趋势,最终残留蛋白酶A活力是影响其货架期泡持值的主要因素。纯生啤酒泡持值与蛋白酶A活力呈显著负相关（P＜0.01）。为了保证成品纯生啤酒在货架期内泡沫稳定性,发酵液出罐滤酒前的蛋白酶A活力应＜24×10-5 U/mL,相应成品纯生啤酒初始蛋白酶A活力应＜15×10-5 U/mL。该内控标准能为纯生啤酒生产企业控制泡持性提供指导。     

响应面法优化预酶解提取米渣蛋白的研究

为改善米渣中蛋白质的提取效果,采用中性蛋白酶酶解、碱溶两步法提取米蛋白.固定碱提取工艺,通过单因素实验,确定了预酶解的基本条件;并以pH和加酶量和反应时间为自变量,蛋白质提取率和质量分数为响应值,采用Box Behnken试验设计方法,对预酶解条件进行响应面（RSM）优化,确定了预酶解的最优工艺.最优条件下得到米渣蛋白质的提取率为78.27%,质量分数为72.86%,较直接碱提取,两者均有了一定的提高,且以蛋白质提取率提升幅度大,且优化条件下的实验结果与RSM回归方程预测值吻合良好.     

芽孢杆菌的产酶特性及其对抗生素的耐受性

以4株枯草芽孢杆菌（Bacillus subtilis）和2株地衣芽孢杆菌（Bacillus licheniformis）为研究对象,通过测定其成胞率、中性蛋白酶活性及对24种抗生素的耐受性,从中筛选优良芽孢杆菌。结果表明,从6株芽孢杆菌中筛选得到3株成胞率和产中性蛋白酶均较好的芽孢杆菌,分别为枯草芽孢杆菌BLCC1-0552、BLCC1-0615和地衣芽孢杆菌BLCC1-0441,液体发酵24 h后,成胞率较高,均达到98%;中性蛋白酶活性分别为90.58 U/mL、100.32 U/mL和24.72 U/mL。其中,枯草芽孢杆菌BLCC1-0615固体发酵玉米-豆粕型常规饲料产中性蛋白酶活力最高,发酵48 h时达到2 154.49 U/g。3株芽孢杆菌对抗生素的耐受性不一致,其中枯草芽孢杆菌BLCC1-0615对24种抗生素中的17种表现敏感,敏感率最高为70.83%。因此,确定优良菌株为枯草芽孢杆菌BLCC1-0615,其成胞率、产中性蛋白酶能力好及对抗生素耐受性较低,具有作为饲料添加剂应用于畜禽饲料中的潜力。     

Degradation of myofibrillar proteins by a myofibril-bound serine proteinase in the skeletal muscle of crucian carp (Carasius auratus)

A myofibril-bound serine proteinase (MBSP) in the skeletal muscle of crucian carp (Carasius auratus) was identified. Hydrolysis of myofibrillar proteins by the endogenous MBSP was studied. Myosin heavy chain (MHC) was degraded markedly when crucian carp myofibril was incubated at 55 °C, as shown by SDS-PAGE. Prolonged incubation of myofibril at 55 °C also caused the obvious degradation of tropomyosin, while the decomposition of other myofibrillar proteins, such as α-actinin and actin, was slight, as detected by Western blotting. The results suggest the existence of an endogenous myofibril-associated proteinase in crucian carp myofibril, which efficiently cleaves MHC and tropomyosin. Serine proteinase inhibitors (Lima bean trypsin inhibitor, PMSF and benzamidine) greatly suppressed the degradation of MHC, caused by the enzyme, while inhibitors for cysteine, metallo-, and aspartic proteinases showed only partial or incomplete inhibitory effects, indicating that the endogenous proteinase is a serine proteinase. Substrate specificity analysis, using partially purified crucian carp MBSP, suggested that the enzyme is a trypsin-like serine proteinase.