Optimization of extracellular thermotolerant alkaline protease produced by marine <Emphasis Type="Italic">Roseobacter</Emphasis> sp. (MMD040)

Marine endosymbiontic Roseobacter sp. (MMD040), which produced high yields of protease, was isolated from marine sponge Fasciospongia cavernosa, collected from the peninsular coast of India. Maximum production of enzyme was obtained in Luria-Bertani broth. Catabolite repression was observed when the medium was supplemented with readily available carbon sources. The optimum temperature and pH for the enzyme production was 37 degrees C and 7.0, respectively. The enzyme exhibited maximum activity in pH range of 6-9 with an optimum pH of 8.0 and retained nearly 92.5% activity at pH 9.0. The enzyme was stable at 40 degrees C and showed 89% activity at 50 degrees C. Based on the present findings, the enzyme was characterized as thermotolerant alkaline protease, which can be developed for industrial applications.     

云斑尖塘鳢消化酶活力的研究

本文采用酶学分析方法研究了云斑尖塘鳢在正常摄食状态与饥饿的状态下胃、肠及肝胰脏组织中蛋白酶、淀粉酶和脂肪酶的活性。结果显示,在30℃的条件下,正常摄食组样本在酸性条件下的蛋白酶活力表现为:胃后肠肝胰脏前肠,中性和碱性条件下:后肠肝胰脏前肠及胃;饥饿组样本仅有胃表现出较高的酸性蛋白酶活性,其他器官的蛋白酶活性均很低。在正常和饥饿实验组中肝胰脏的淀粉酶活性均高于其他器官,胃肠的淀粉酶活性均较低。正常摄食组中脂肪酶活力后肠肝胰脏;而在饥饿组中仅有肝胰脏检测到脂肪酶活性。结果表明,云斑尖塘鳢适度饥饿组较正常摄食组消化酶活性大幅降低;其高蛋白酶活力及中等脂肪酶活力与其肉食性相一致;此外云斑尖塘鳢也具备少量的淀粉消化能力。     

虎纹蛙消化道淀粉酶和脂肪酶活力研究

以酶学分析方法研究了虎纹蛙消化道淀粉酶和脂肪酶的分布以及pH和温度对这两种消化酶活力的影响。结果表明：在各自生理pH值条件下,虎纹蛙消化道不同部位淀粉酶活力大小顺序依次为前肠〉中肠〉后肠〉食道〉胃,胃和肠淀粉酶最适pH值分别为8．6和7．0,最适温度分别为35℃和40℃。脂肪酶活力大小顺序依次为中肠〉后肠〉前肠〉胃〉食道,各部位之间差异显著（P〈0．05）,胃和肠脂肪酶的最适pH值均为9．0,最适温度分别为50℃和55℃。     

Amylase activity in the alimentary tract and salivary glands of Periplaneta americana L

The optimum conditions for amylase activity and the distribution of the enzyme in the salivary glands and various gut regions were investigated. Maximum activity of the enzyme was observed at 7.0 pH and 50 degrees C temperature and the activity increased with increasing time period, and enzyme and substrate concentrations. Amylase from the salivary glands was found to be exceptionally potent and the enzyme concentration decreased from the anterior to the posterior part of the gut in well-fed cockroaches. The findings are discussed with regard to the source of amylase synthesis.     

Characteristics and identity of obligately aerobic spoilage yeasts from fish silage

Yarrowia (Candida) lipolytica was the predominant organism isolated from the surface film of growth derived from ground hake gurry to which only phosphoric acid was added to give a pH of 4.0. The optimum pH for the crude extracellular protease activity of two distinguishable strains of Y. lipolytica, designated CL1 and CL2, with casein as substrate was 7.0. The optimum temperature of the crude extracellular protease activity from both strains was 50 degrees C. The addition of 2.0% glucose to broth cultures resulted in a significant increase in final cell mass and extracellular protease activity but resulted in a reduction in the units of protease activity per mg of dry cell mass at initial pH values of 5.6 and 7.0 but not an initial pH of 8.0.     

Protease from a strain of bacteria E. coli A2, specifically cleaving actin

A novel bacterial protease specifically hydrolyzing actin with the formation of a stable fragment with Mr of 36 kDa was obtained. This protease was shown to be synthesized at the stationary phase of bacterial culture growth. The actin hydrolysis by bacterial protease was inhibited by o-phenanthroline, EDTA and p-chloromercuribenzoate but not by N-ethyl-maleimide, phenylmethylsulfonylfluoride, Leu-peptin, pepstatin and other serine proteinase inhibitors. The protease was stable within the pH range of 4.5-8.5 and had an activity optimum at pH 7.0-8.0. The protease activity was maintained for 40 min at 45 degrees C and for 30 min at 50 degrees C; at 65 degrees C the enzyme was fully inactivated by 5 min heating. The protease preparations causing quantitative conversion of actin into a 36 kDa fragment did not hydrolyze casein, albumin, ovalbumin, lysozyme, DNAase I, RNAase, myosin, alpha-actinin, tropomyosin and troponin. It was assumed that the protease under consideration is a neutral metalloprotease specifically hydrolyzing actin.     

春季树麻雀体内几种消化酶活性研究

应用酶学分析法测定了树麻雀Passer montanus春季的腺胃、肌胃、小肠、大肠、肝脏和胰脏组织中淀粉酶、蛋白酶和纤维素酶的活力.结果表明,不同组织中的消化酶活力差异显著,淀粉酶和蛋白酶以胰脏中活力最高,腺胃次之,纤维素酶均较低;同一组织中不同消化酶的活力差异显著,淀粉酶活性最高,蛋白酶次之,纤维素酶活力最低.这些差异提示消化酶活力大小与器官分化有关,并受食物组成的影响,因此产生了不同的酶活力分布.这是树麻雀长期适应东北地区寒冷环境的生存策略之一.     

Proteolytic activity of the enzyme complex of the mammalian pancreas in comparison with pancreatin

The proteolytic activity and thermal stability of the enzyme complex of cell suspension from pig and bovine pancreas glands was compared with those of pancreatin. The enzyme complex displayed the highest thermal stability and activity at 50 degrees C. The kinetic constants, energies of activation and inactivation of the enzyme complex, and pH optimum (7.0 +/- 0.1) at which this complex had the maximum proteolytic activity were determined. Pancreatin had a pH optimum of 8.0 +/- 0.1.     

In vitro evaluation of feed-grade enzyme activity at pH levels simulating various parts of the avian digestive tract

The activities of β-glucanase, xylanase, amylase, α-galactosidase and protease were measured at their published optimum pH levels and at pH levels of 3.0, 6.0, 6.5, 7.0 and 7.5 to simulate pH levels of the gizzard, the diet, the crop, and the proximal and distal parts of small intestine, respectively. The activity of β-glucanase was determined by measuring reducing sugars after incubation of β-glucan. Xylanase activity was assayed by measuring xylose after hydrolysis of xylan. The activity of amylase was measured through hydrolysis of soluble starch. The assay of α-galactosidase was based on a hydrolysis of p-nitrophenyl-α-d-galactoside followed by measurement of liberated p-nitrophenol. The activity of protease was assayed by measuring tyrosine after enzymatic hydrolysis of casein. β-Glucanase had high activity at pH levels of 3.0–7.0. Xylanase had no enzyme activity at pH 3.0, but had high activity at pH levels of 6.0–7.0. Amylase had high activity at pH levels of 6.0 and 6.5 but had no or very low activity at pH 3.0, 7.0 and 7.5. α-Galactosidase had high activity at pH 6, but not at other pH levels tested. Protease had either no or very low activity at all pH levels except at pH 3.0. These results suggest that the pH levels commonly found in the avian digestive tract may be a limiting factor for maximum activity of the exogenous enzymes, such as amylase, α-galactosidase and protease.     

Optimization, production, and partial characterization of an alkalophilic amylase produced by sponge associated marine bacterium Halobacterium salinarum MMD047

An endosymbiont Halobacterium salinarum MMD047, which could produce high yields of amylase, was isolated from marine sponge Fasciospongia cavernosa, collected from the peninsular coast of India. Maximum production of enzyme was obtained in minimal medium supplemented with 1% sucrose. The enzyme was found to be produced constitutively even in the absence of starch. The optimum temperature and pH for the enzyme production was 40°C and 8.0, respectively. The enzyme exhibited maximum activity in pH range of 6∼10 with an optimum pH of 9.0. The enzyme was stable at 40°C and the enzyme activity decreased dramatically above 50°C. Based on the present findings, the enzyme was characterized as relatively heat sensitive and alkalophilic amylase which can be developed for extensive industrial applications.     

An acidic protease from the grass carp intestine (Ctenopharyngodon idellus)

The acidic Protease was extracted from the intestine of the grass carp (Ctenopharyngodon idellus) by 0.1 M sodium phosphate buffer, pH 7.0 at 4 degrees C after neat intestine was defatted with acetone, and partially purified by ammonium sulfate precipitation, gel filtration chromatography and ionic exchange chromatography. SDS-PAGE electrophoresis showed that the enzyme was homogeneous with a relative molecular mass of 28,500. Substrate-PAGE at pH7.0 showed that the purified acidic protease has only an active component. Specificity and inhibiting assays showed that it should be a cathepsin D. The optimal pH and optimal temperature of the enzyme were pH2.5 and 37 degrees C, respectively. It retained only 20% of its initial activity after incubating at 50 degrees C for 30 min. The enzyme lost 81% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride (PMSF). Its V(max) and K(m) values were determined to be 3.57 mg/mL and 0.75 min(-1), respectively.     

Characteristics of the amylase of Arthrobacter psychrolactophilus

Properties of the extracellular amylase produced by the psychrotrophic bacterium, Arthrobacter psychrolactophilus, were determined for crude preparations and purified enzyme. The hydrolysis of soluble starch by concentrated crude preparations was found to be a nonlinear function of time at 30 and 40 °C. Concentrates of supernatant fractions incubated without substrate exhibited poor stability at 30, 40, or 50 °C, with 87% inactivation after 21 h at 30 °C, 45% inactivation after 40 min at 40 °C and 90% inactivation after 10 min at 50 °C. Proteases known to be present in crude preparations had a temperature optimum of 50 °C, but accounted for a small fraction of thermal instability. Inactivation at 30, 40, or 50 °C was not slowed by adding 20 mg/ml bovine serum albumin or protease inhibitor cocktail to the preparations or the assays to protect against proteases. Purified amylase preparations were almost as thermally sensitive in the absence of substrate as crude preparations. The temperature optimum of the amylase in short incubations with Sigma Infinity Amylase Reagent was about 50 °C, and the amylase required Ca⁺² for activity. The optimal pH for activity was 5.0–9.0 on soluble starch (30 °C), and the amylase exhibited a K _m with 4-nitrophenyl-α-D-maltoheptaoside-4,6-O-ethylidene of 120 μM at 22 °C. The amylase in crude concentrates initially hydrolyzed raw starch at 30 °C at about the same rate as an equal number of units of barley α-amylase, but lost most of its activity after only a few hours.     

Purification and Characterization of Protease and Chitinase from Bacillus cereus TKU006 and Conversion of Marine Wastes by These Enzymes

A chitinase- and protease-producing bacterium was isolated and identified as Bacillus cereus TKU006. The better condition on our tests for protease and chitinase production was found when the culture was shaken at 25 degrees C for 2 days in 25 mL of medium containing 2% shrimp shell powder (w/v), 0.1% K(2)HPO(4), and 0.05% MgSO(4).7H(2)O. The molecular masses of TKU006 protease and chitinase determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were approximately 39 and 35 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU006 protease and chitinase were 9, 50 degrees C, 3-11, 50 degrees C and 5, 40 degrees C, 3-11, 60 degrees C, respectively. TKU006 protease was inhibited completely by EDTA, indicating that the TKU006 protease was a metalloprotease. The TKU006 protease and chitinase retained 61%, 60%, 73%, and 100% and 60%, 60%, 71%, and 96% of its original activity in the presence of 2% Tween 20, 2% Tween 40, 2% Triton X-100, and 1 mM SDS, respectively. The antioxidant activity of TKU006 culture supernatant was determined through the scavenging ability on DPPH with 70% per milliliter. In conclusion, the novelties of the TKU006 protease and chitinase include its high stability to the surfactants and pH. Besides, with this method, we have shown that marine wastes can be utilized to generate a high-value-added product and have revealed its hidden potential in the production of functional foods.     

Characteristics and identity of obligately aerobic spoilage yeasts from fish silage

R.E. LEVIN AND R. WITKOWSKI. 1991. Yarrowia (Canadida) lipolytica was the predominant organism isolated from the surface film of growth derived from ground hake gurry to which only phosphoric acid was added to give a pH of 4.0. The optimum pH for the crude extracellular protease activity of two distinguishable strains of Y. lipolytica , designated CL1 and CL2, with casin as substrate was 7.0. The optimum temperature of the crude extracellular protease activity from both strains was 50.C. The addition of 2.0% glucose to broth cultures resulted in a significant increase in final cell mass and extracellular protease activity but resulted in a reduction in the units of protease activity per mg of dry cell mass at initial pH values of 5.6 and 7.0 but not an initial pH of 8.0     

土壤中高产蛋白酶菌株产酶条件及酶学性质

【背景】微生物蛋白酶已经成为工业用蛋白酶的主要来源,筛选具有特殊环境适应性的微生物成为生物酶资源的开发热点。【目的】通过对青藏高原土壤微生物产蛋白酶菌株的筛选、优化及相关特性研究,寻找新的蛋白酶资源,为高原菌种资源利用提供科学依据。【方法】采用形态学和分子生物学对筛选菌株进行菌种鉴定,利用单因素试验和正交试验对菌株进行发酵条件优化及酶学性质的探究。【结果】筛选出一株高产蛋白酶菌株XC2,经鉴定菌株XC2为枯草芽孢杆菌(Bacillus subtilis)。XC2最优产酶条件:可溶性淀粉4.0%,牛肉膏1.0%,K~+0.6%,培养温度34°C、初始pH 7.0、接种量2.0%的条件下200 r/min振荡培养13 h,所产蛋白酶活力最高为638.5 U/mL。XC2所产蛋白酶最适反应温度60°C,最适pH9.0;40-50°C、pH8.0-10.0条件下酶活稳定性较高;Mn~(2+)对酶活力有明显激活作用,而Zn~(2+)、Cu~(2+)、Fe~(2+)、Fe3+对酶活力有明显抑制作用。【结论】枯草芽孢杆菌XC2有较强的产碱性蛋白酶的能力,具有较好的应用前景。     

Biosynthesis and properties of an extracellular metalloprotease from the Antarctic marine bacterium Sphingomonas paucimobilis

An extracellular protease from the marine bacterium Sphingomonas paucimobilis, strain 116, isolated from the stomach of Antarctic krill, Euphausia superba Dana, was purified and characterized. The excretion of protease was maximal at temperatures from 5 to 10°C, i.e. below the temperature optimum for the strain growth (15°C). The highly purified enzyme was a metalloprotease [sensivity to ethylenediaminetetraacetic acid (EDTA)] and showed maximal activity against proteins at 20–30°C and pH 6.5–7.0, and towards N-benzoyl-tyrosine ethyl ester (BzTyrOEt) at pH 8.0. At 0°C the enzyme retained as much as 47% of maximal activity in hydrolysis of urea denatured haemoglobin (Hb) (at pH 7.0), and at −5 and −10°C, 37 and 30%, respectively. The metalloprotease was stable up to 30°C for 15 min and up to 20°C for 60 min. These results indicate that the proteinase from S. paucimobilis 116 is a cold-adapted enzyme.     

pH值对中国龙虾消化酶活力的影响

采用酶学分析方法研究了pH对中国龙虾胃蛋白酶、类胰蛋白酶、淀粉酶、纤维素酶和脂肪酶活力的影响。结果表明,在设定的pH范围内,中国龙虾各消化酶的活力均随着pH的升高呈现先升后降的变化趋势。其中,胃、肠、肝胰腺内胃蛋白酶最适pH均为2.2,类胰蛋白酶最适pH分别为8.8-9.2、8.4、8.8,淀粉酶最适pH分别为7.0、7.0、7.4,纤维素酶最适pH分别为4.2、4.2-4.6、5.4,脂肪酶最适pH分别为7.2-7.6、7.2、6.8-7.2。同时测得中国龙虾胃、肠、肝胰腺内的生理pH分别为5.33、6.93、6.60。中国龙虾的消化酶活力存在器官特异性。在最适pH下,胃蛋白酶活力顺序为胃>肠>肝胰腺,类胰蛋白酶、纤维素酶、脂肪酶的活力顺序均为肝胰腺>肠>胃,淀粉酶的活力顺序为肠>肝胰腺>胃。     

An extracellular--pepstatin insensitive acid protease produced by Thermoplasma volcanium

In this study, some parameters for the production and caseinolytic activity of an extracellular thermostable acid protease from a thermoacidophilic archaeon Thermoplasma volcanium were determined. The highest level of growth and enzyme production were detected at pH 3.0 over an incubation period of 192 h at 60 degrees C. The pH optimum for the acid protease activity was 3.0 and the enzyme was fairly stable over a broad pH range (pH 3.0-8.0). The temperature for maximum activity of the enzyme was 55 degrees C and activity remained stable between 50 degrees C and 70 degrees C. These features could be of relevance for various biotechnological applications of this enzyme. Serine-(PMSF), cysteine-(DTT), metallo-(EDTA) and aspartate-(pepstatin) protease inhibitors did not inhibit the caseinolytic activity of the enzyme. Therefore, Tp. volcanium acid protease could be a member of the pepstatin-insensitive carboxyl proteinases.     

Digestive enzymes along the alimentary tract of the parrotfish Sparisoma cretense

The digestive enzyme profile (total protease, total carbohydrase, alpha-amylase and alpha-glucosidase activity and capacity) along the digestive tract was investigated in vitro at different temperature levels (5, 18, 25 and 37° C) for the only Mediterranean species of parrotfish Sparisoma cretense . Sparisoma cretense (collected from the wild from south-eastern Greece) exhibited very high potential for carbohydrate digestion, as well as relatively increased potential for protein digestion. Total protease activity and capacity was increased especially at neutral and alkaline pH levels and was similar in the anterior and posterior intestine. Total carbohydrase, alpha-amylase and alpha-glucosidase values were quite elevated at 37° C but relatively low at 5, 18 and 25° C. Total carbohydrase and alpha-amylase capacity in the posterior intestine became increasingly important with elevated temperature, while the opposite was apparent for α-glucosidase. These results suggest that, apart from the expected high potential for carbohydrate digestion exhibited, S. cretense also has the elevated capacity for protein digestion, although it lacks a specialized stomach and pyloric caeca.