Phenoloxidase activity of acridid grasshoppers from the subfamilies melanoplinae and oedipodinae

• 1.1. Phenoloxidase activity and wound melanization was studied in five species of grasshoppers representing the subfamilies Melanoplinae and Oedipodinae.
• 2.2. Most of the phenoloxidase activity was detected in the plasma fraction of grasshopper whole-body homogenates and supernatant fractions of the hemolymph. The species representing the Oedipodinae had 20–50% higher percentage of the total phenoloxidase activity associated with particulate matter from a whole-body homogenate when compared to the Melanoplinae.
• 3.3. Phenoloxidase activity could not be detected in sclerotized cuticle of adult grasshoppers.
• 4.4. The phenoloxidase existed as a zymogen which could be activated by chymotrypsin and inhibited by KCN and NaCN while EDTA showed no effect. It had optimum activity at 37°C and pH 7.3.
• 5.5. These findings are discussed in relation to wound repair and immune responses to infection in grasshopper species.

     

Human mannosidosis--the enzyme defect

Normal human liver α-mannosidase exists in at least 3 forms, separable by DEAE cellulose chromatography. The A and B forms are most active at pH 4.4 while activity of form C is maximal at pH 6.0. In two cases of mannosidosis, examined by ion exchange chromatography and isoelectric focusing, both A and B forms were absent and the residual α-mannosidase activity was due to the presence of the C form in normal amounts.     

Main serum protein of rainbow trout (Salmo gairdnerii): its biological properties and significance

• 1.1. Main serum α₁-protein (α₁P) of rainbow trout was purified and its biochemical and physico-pathological properties were studied.
• 2.2. α₁P was suggested to be a primitive protein having both properties of albumin and AFP in serum proteins of mammals according to the following results.
• 3.3. Molecular weight (75,000), two kinds of molecules (pI 4.55 and 5.05) and amino acid composition.
• 4.4. Dye- or ConA binding activity.
• 5.5. Estrogen binding activity and inhibitory effect on lymphoblastoid-forming activity.
• 6.6. Possible osmotic regulator.
• 7.7. Significant elevation of blood α₁P level in the course of hepatoma induction.

     

The isolation and partial characterization of α1-proteinase inhibitor from the serum of the ostrich (struthio camelus)

• 1.1. Native and cleaved α₁-proteinase inhibitor was purified from ostrich serum using Sepharose-blue dextran chromatography, ammonium sulfate precipitation and ion exchange chromatography on DEAE-Toyopearl 650 M at pH 8.8 and 6.5.
• 2.2. Ostrich α₁PI displayed M_r values of 68,100 using gradient PAGE and 66,200 using Ferguson plots.
• 3.3. Isoelectric focusing of ostrich α₁-PI in the pH range 3–10 revealed pi values of 4.84 and 4.91, and in the pH range 4–6 the characteristic microheterogeneity observed for mammalian α₁-PIs was displayed.
• 4.4. The presence of sialic acid, hexoses and hexosamines was detected using chemical methods, but were found in much lower quantities as compared to α₁-PIs of other species.
• 5.5. Western blot analysis demonstrated a positive reaction between the native and cleaved ostrich α₁-PIs and the antibodies to the ostrich α₁-PIs raised in rabbits. No cross-reactivity was demonstrated by Western blot analysis between human α₁-PI and antibodies to ostrich α,-PI.
• 6.6. The inhibitory effect of α₁-PI on elastase and chymotrypsin was also investigated.

     

Purification and characterization of the amylolytic enzymes of Saccharomycopsis fibuligera

• 1.1. A complex of extracellular amylolytic enzymes produced by Saccharomycopsis fibuligera KZ, grown on fine fibre (waste product from corn starch production) and corn-steep liquor, has been studied.
• 2.2. α-Amylases and glucoamylases, as the main representatives of this complex, were separated by hydrophobic chromatography on Spheron 300 LC.
• 3.3. Individual isoenzymes of one type were separated on FPLC-Mono Q.
• 4.4. The relative molecular weight of α-amylases is 54,000, glucoamylases 62,000, maximal activity is reached by both enzymes between pH 5.0 and 6.2 at a temperature of 40–50°C.
• 5.5. Glucoamylases have a higher stability of the native structure than α-amylases, they retain 55% of their original activity, even after 10 min of incubation at 100°C.

     

Purification and properties of a β-1,4-xylanase from a cellulolytic extreme thermophile expressed in Escherichia coli

• 1.1. An endoxylanase (EC 3.2.1.8) was purified from an Escherichia coli strain carrying a xylanase gene from the extreme thermophile “Caldocellum saccharolyticum strain Tp8T6.3.3.1. It was found to have an M_r of 42,000 and an isoelectric point of approx. 5.0.
• 2.2. The enzyme showed optimum activity at pH 5.0–7.7 and had an activation energy of 44 kJ mol⁻¹. It was stable at room temperature at pH 4.5–11.5 in the presence of 0.5 mg ml⁻¹ bovine serum albumin. The half-life of the enzyme at 75°C was 20 min at pH 6.0 in the presence of 0.5 mg ml⁻¹ bovine serum albumin.
• 3.3. The xylanase had highest activity on oat spelts xylan, releasing xylobiose and some xylotriose. The K_m for oat spelts xylan was 0.021% (w/v) at pH6.0.
• 4.4. The enzyme had high activity on sugar cane bagasse hemicelluloses A and B, lower activity on larchwood xylan and also hydrolysed carboxymethylcellulose, 4-methylumbelliferyl β-D-cellobioside and p-nitrophenyl β-D-cellobioside, but could not hydrolyse xylobiose.
• 5.5. It showed transferase activity on p-nitrophenyl β-D-xylopyranoside. Xylose did not inhibit the enzyme.

     

Chemical intermediates in α-methylnoradrenaline oxidation by tyrosinase—II. Kinetic study of process

• 1.1. The pathway for α-methylnoradrenaline oxidation to α-methylnoradrenochrome by tyrosinase. has been studied as a system of various chemical reactions coupled to an enzymatic reaction.
• 2.2. A theoretical and experimental kinetic approach was proposed for such a system, we named this type of mechanism as a mechanism enzymatic-chemical-chemical (E₂CC).
• 3.3. Rate constants for the implied chemical steps at different temperature and pH values, were evaluated from measurement of the lag period, arising from the accumulation of aminochroine, that took place when α-methylnoradrenaline was oxidized at acid pH.
• 4.4. The thermodynamic activation parameters of the chemical steps, the deprotonation and the internal cyclization of o-quinone into leukoaminochrome, were also calculated.

     

Activity and electrophoretic profiles of brain aldehyde dehydrogenases in mice genetically selected for their ethanol preference

The activities and electrophoretic profiles of NAD⁺-aldehyde dehydrogenases (EC 1.2.1.3, ALDHs) in the different brain regions from the mice of inbred C57BL 6J strain (alcohol preferring) and DBA/2 sirain (alcohol avoicling) were studied.

• 1.1. Kinetic studies on ALDH of the different brain regions revealed the existence of at least two ALDHs in both strains.
• 2.2. The ALDH activity in the cerebellum, which was measured spectrophotometrically with 0.1 and 5mM acetaldehyde as the substrates, was the highest of the three regions and it was about 2-fold higher than that in the cortex in both strains.
• 3.3. The ALDH activities of mice of DBA/2 strain were higher than those ol mice of C57BL/6J strain in all the regions. Only in the cortex the F₁ hybrids (intermediate alcohol preferring) between C57BL/6J and DBA/2 strains had intermediate ALDH activity between their parental strains.
• 4.4. Electrophoretic analysis of cortex ALDH revealed that strain differences were observed in the isozyme pattern between pH 7.2 and 7.8 with cither acetaldehyde or propionaldehyde as substrate. These findings suggest that the ALDH activity in the cerebral cortex could be related to alcohol preference.

     

Changes in composition and proteolytic enzyme activities of artemia during early development

• 1.1. The proximate composition, total and free amino acids, and proteases of Artemia nauplii were determined during early development.
• 2.2. Moisture increased from 71.0% to 80.8%, crude protein decreased from 13.2% to 8.8%, crude fat and ash varied slightly.
• 3.3. The total amino acids decreased. Free amino acids changed in three patterns.
• 4.4. Trypsin, chymotrypsin, carboxypeptidase A, B and cathepsin B and C increased in activity. The activity of trypsin was lower, while cathepsin B and C were the highest.
• 5.5. The protease activities were maximal at pH 7.5 and 8.0, and at 45°C on casein.
• 6.6. The optimal pH for carboxypeptidase A was 4.0, for carboxypeptidase B was 4.5, for trypsin and chymotrypsin were 7.0–7.5. The protease(s) active at pH 9.0–9.5 were to be determined.

     

Control of mucin molecular forms expression by salivary protease: Differences with caries

• 1.1. A protease activity capable of degradation of the high mol. wt salivary mucus glycoprotein to a low mol. wt glycoprotein form was identified in human submandibular gland secretion.
• 2.2. The protease exhibited optimum activity at pH 7.0–7.4, and gave on SDS-PAGE under reducing conditions two major protein bands of 48 and 53 kDa. The enzyme showed susceptibility to PMSF, α₁antitrypsin, and egg white and soybean inhibitors, a characteristic typical to serine proteases.
• 3.3. The activity of the protease towards the high mol. wt mucus glycoprotein was found to be 3.8-fold higher in submandibular gland secretion of caries-resistant individuals than that of caries-susceptible. Furthermore, the enzyme from both groups displayed greater activity against the mucus glycoprotein of caries-resistant subjects.
• 4.4. Since the low mol. wt salivary mucus glycoprotein form is more efficient in bacterial clearance than the high mol. wt mucin, the enhanced expression of this indigenous salivary protease activity towards mucin may be the determining factor in the resistance to caries.

     

Purification,isolation and characterization of a phosphoglycolate phosphatase isoenzyme from human erythrocytes

• 1.1. Preparation, purification and characterization of a phosphoglycolate phosphatase (PGP)3 isoenzyme from human erythrocytes was achieved by DEAE-Sepharose CL.-6B chromatography and isoelectric focusing using carrier ampholytes. pH 4–6.
• 2.2. The isoenzyme has an isoelectric point of 5.00 ± 0.05 and could be purified 33.000 fold to a specific activity of 32.7 U/mg of protein. It represents the PGP phenotype 1 consisting of a single isoenzyme.
• 3.3. The enzyme is composed of two subunits (mol. wt 35,000) which are identical and not connected by SS-bridges.
• 4.4. At 4°C the isoenzyme is more stable in the pH range of 7–9 than at acid pH values.
• 5.5. Incubation at 30 and 40°C for 4 hr does not affect the activity of the isoenzyme.
• 6.6. It has a K_m-value of 0.28 mM for phosphoglycolate (PG) as substrate.

     

The major serum protein of fetal and newborn pigs: Biochemical properties and identification as a fetal form of α1-acid glycoprotein

• 1.I. The major protein component of fetal pig serum, has been immunologically identified as α₁-acid glycoprotein (orosomucoid).
• 2.2. Amino acid composition and total carbohydrate content (around 38% by weight) were similar in the adult and fetal forms of α₁-acid glycoprotein. These forms differ, however, in the proportion of individual monosaccharides.
• 3.3. Fucose, represented the 1.5% (by weight) in the fetal protein, and the 2.5% in its adult counterpart. The latter was more susceptible to ncuraminidase and also possesses a higher mannose/galactose ratio than the fetal form.
• 4.4. Insolubilized Concanavalin A (Con A) retained 80%, of the adult protein, whereas the fetal form was mostly Con A-non reactive. The proportion of this -non reactive fraction, as revealed by crossed immuno-affino-electrophoresis experiments, was age-dependent and varied from 62% at fetal age of 50–60 days to 80% at birth.

     

Highly selective and efficient biotransformation of linarin to produce tilianin by naringinase

Objectives

To develop a practical method to prepare tilianin by highly selective and efficient hydrolysis of the C-7 rhamnosyl group from linarin.

Results

Naringinase was utilized to selectively catalyze the formation of tilianin using linarin as the starting material. The reaction conditions, including temperature, pH, metal ions, substrate concentration and enzyme concentration, were optimized. At 60 °C, naringinase showed enhanced α-l-rhamnosidase activity while the β-d-glucosidase activity was abrogated. The addition of Mg²⁺, Fe²⁺ and Co²⁺ was also beneficial for selective biotransformation of linarin to tilianin. Under the optimized conditions (pH 7.0 at 60 °C), linarin could be nearly completely transformed to tilianin with excellent selectivity (>98.9 %), while that of the by-product acacetin was less than 1.1 %. In addition, the structure of target product tilianin was fully characterized by HR-MS and ¹H-NMR.

Conclusion

A highly selective and efficient biotransformation of linarin to tilianin was developed by the proper control of incubation temperature, which enhanced the α-l-rhamnosidase activity of naringinase and blocked its β-d-glucosidase activity.

     

Differentiation between carrier-bound forms of non-suppressible insulin-like activity (NSILA) in serum

• 1.1. Gel-permeation chromatography of serum on Sephacryl S-300 at pH 7.4 has shown that NSILA was detected over a range of MW 50,000–400,000 with a peak at about MW 200,000.
• 2.2. When fractions from the above chromatography were rechromatographed on Sephadex G-75 at pH 2.4 major amounts of acid-stable NSILA were found in a fraction of MW 200,000–600,000 (77% of the fraction NSILA or 28% of total serum NSILA).
• 3.3. Further evidence was obtained for the presence of an active acid-dissociable complex in serum. This was present in both the MW 100,000–200,000 and 35,000–100,000 fractions and corresponded to 37% of total serum NSILA.
• 4.4. Con-A Sepharose affinity chromatography of the serum fractions from Sephacryl S-300 chromatography, followed by Sephadex G-75 chromatography under acid conditions, showed that the acid-stable complex was consistently found in weakly bound materials. The active acid-dissociable complex was found in the bound fractions, especially in the Sephacryl S-300 pool of MW 35,000–100,000.
• 5.5. Low MW NSILA (<15,000) was also released on acid treatment from an otherwise inactive high MW complex(es) of MW 35,000–600,000. This complex was not bound by Con-A Sepharose.

     

Constituents of the Essential Oil of Chrysanthemum japonense var. debile

One hundred fifty strains of actinomycetes were isolated from soils on plate cultures containing beet arabinan as the sole carbon source. About one-third of the culture fluids were found to have arabinosidase activity. A wild-type strain, Streptomyces sp. No. 17-1, was selected as the best producer of arabinosidase. The highest enzymatic activity was obtained in the culture fluid when the initial pH was adjusted to 9.0. An α-l-arabinofuranosidase was highly purified from the culture filtrate of No. 17-1 by combining column chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and isoelectric focusing. The molecular weight of the purified enzyme was estimated to be about 92, 000, and its isoelectric point was pH 4.4. The enzymatic activity was maximum at pH 6.0 and was completely inhibited by Hg²⁺. The apparent Km value of the enzyme for p-nitrophenyl-α-l-arabinofuranoside was determined to be 3.6 mM.     

Purification of endo- and exolaminarinase and partial characterization of the exoacting form from the copepod acartia clausi (Giesbrecht, 1889)

• 1.1. The copepod Acartia clausi exhibited two laminarinases (exo- and endo-acting forms) purified by gel chromatography followed by affinity chromatography. Specific antibodies have been raised against the purified exolaminarinase antigen.
• 2.2. A single band of protein appeared on a polyacrylamide disc gel electrophoresis; its mol. wt is 21,000.
• 3.3. Biochemical properties of the purified enzyme showed a maximum activity at pH 5.2 and a temperature of 40°C with laminarin as substrate. The thermal stability of the enzyme and the effect of various cations on its activity were examined. The enzyme hydrolyses specifically the β(1–3) linked polysaccharides and had no activity against the α(1–4) or β(1–4) disaccharides or polysaccharides.
• 4.4. The kinetic parameters V_m and K_m vary with the temperature; the affinity constant (K_a) was maximum between 25–30°C. The Arrhenius plot defined two values of energy of activation: 7980 cal/mole and 17,506 cal/mole.
• 5.5. From the purification scheme the exoacting form appears to be largely dominant over the endoacting form.

     

Purification and Properties of α-d-Galactosidase Produced by Corticium rolfsii

An α-d-galactosidase was purified from the culture filtrate of Corticium rolfsii IFO 6146 by a combination of QAE-Sephadex A-50 and SE-Sephadex C-50 chromatography. The purified enzyme was demonstrated to be free of other possibly interfering glycosidases and glycanases. The maximum activity of the enzyme towards p-nitrophenyl α-d-galactopyrano-side was found to be at pH 2.5 to 4.5, and the enzyme was fairly active at pH 1.1 to 2.0. The enzyme was stable over a pH range 4.0 to 7.0 at 5°C for 72 hr and relatively unstable at pH 1.1 to 2.0 as compared with endo-polygalacturonase, α-l-arabinofuranosidase and β-d-galactosidase produced by C. rolfsii. The enzymic activity was completely inhibited by Hg²⁺ and Ag⁺ ions, respectively. Km values were determined to be 0.16 × 10^?3 m for p-nitrophenyl α-d-galactopyranoside and 0.26 × 10^?3m for o-nitrophenyl α-d-galactopyranoside. The values of V_max were also determined to be 26.6 μmoles and 28.6 μmoles per min per mg for p- and o-nitrophenyl α-d-galactopyranoside, respectively.     

Solvent production from starch: effect of pH on α-amylase and glucoamylase localization and synthesis in synthetic medium

Summary The fermentation of starch by Clostridium acetobutylicum ATCC 824 has been reviewed in an optimised synthetic medium. A progressive increase of pH from 4.4 to 5.2 led to a higher production of extracellular -amylase whereas glucoamylase was poorly affected. A portion of these enzymes was cell-associated and on increasing the pH from 4.4 to 5.8 a decrease was noted in cell-bound enzymes. The association was higher for the glucoamylase than for the -amylase. The highest rate of starch consumption was at pH 5.2 whereas due to the earlier shift to solvent production at low pH, the highest solvent production was at pH 4.4. This study suggested that the level of -amylase and then the rate of starch hydrolysis was the limiting step of sugar catabolism in C. acetobutylicum ATCC 824. Correspondence to: P. Soucaille     

A new assay of X-prolyl dipeptidyl-aminopeptidase activity in human serum with glycylproline p-phenylazoanilide as substrate

Summary A new assay procedure for X-prolyl dipeptidyl-aminopeptidase activity in human serum was developed with glycylproline p-phenylazoanilide tosylate as substrate. p-Phenylazoaniline liberated by the enzyme reaction was measured photometrically at 493 nm after stopping the reaction with acid. This assay was simple and sensitive, and less than 50 l of human serum was required for the assay. Km value was 2.5 mm and the optimum pH was 8.7. After disc gel electrophoresis of human serum, the enzyme activity could be distinctly observed as a reddish band on the gel when the gel was incubated with this substrate.