The role of peripheral interactions in chymosin specificity

Kinetic parameters for the splitting of model peptide substrates and chi-casein with chymosin have been interpreted on the basis of the three-dimensional structure of chymosin. Model peptide substrates contain a fragment of the chi-casein sequence in the region of the bond Phe-105--Met-106 splitted with the enzyme. It was shown that the possible reason of the enormous milk-clotting efficiency of chymosin may be partly associated with the electrostatic interaction of the positive charged segment 98-102 (His-Pro-His-Pro-His) of the substrate and outer loop of the enzyme which contains Glu-245, Asp-247, Asp-249, Asp-251.     

Partial purification of new milk-clotting enzyme produced by Nocardiopsis sp

Numerous attempts have been made to replace calf rennet with other milk clotting proteases because of limited supply and increasingly high prices. The aim of this work was to investigate the characteristic of the milk-clotting enzyme from Nocardiopsis sp. The partial purification extract was obtained by fractional precipitation with ammonium sulphate. Of the fractions obtained by precipitation, 40-60% possessed the milk-clotting activity (156.25 U/mg). The chromatography of 40-100% ammonium sulphate fraction in DEAE-cellulose yielded four fractions (F4, F5, F6, F7) with milk-clotting activity. The F5 yielded the best milk-clotting activity (20 U/ml). Both crude and partially purified extract were active at the range pH 4.5-11.0, however, optimum activity was displayed at pH 11.0 and pH 7.5, respectively. The milk-clotting activity was highest at 55 degrees C for both crude and partially purified extract. The crude and partial purification extract were inactivated at 65 and 75 degrees C after 30 min.     

Production and characterization of the milk-clotting protease of<Emphasis Type="Italic"> Myxococcus xanthus</Emphasis> strain 422

The cheese industry is seeking novel sources of enzymes for cheese production. Microbial rennets have several advantages over animal rennets. (1) They are easy to generate and purify and do not rely on the availability of animal material. (2) The production of microbial clotting enzymes may be improved by biotechnological techniques. In this work, the biochemical characterization of a novel milk-clotting extracellular enzyme from Myxococcus xanthus strain 422 and a preliminary evaluation of its cheese-producing ability are reported. Strain 422 was selected from four M. xanthus strains as the best producer of extracellular milk-clotting activity, based on both its enzyme yield and specific milk-clotting activity, which also afforded lower titration values than enzymes from the three other M. xanthus strains. The active milk-clotting enzyme from M. xanthus strain 422 is a true milk-clotting enzyme with a molecular mass of 40 kDa and a pI of 5.0. Highest milk-clotting activity was at pH 6 and 37 °C. The enzyme was completely inactivated by heating for 12 min at 65 °C. The crude enzyme preparation was resolved by anion-exchange chromatography into two active fractions that were tested in cheese production assays of compositional (dry matter, fat content, fat content/dry-matter ratio, and moisture-non-fat content) and physicochemical properties (firmness, tensile strength, pH and Aw) of the milk curds obtained. Purified protein fraction II exhibited a significantly higher milk-clotting ability than either protein fraction I or a total protein extract, underlining the potential usefulness of M. xanthus strain 422 as a source of rennet for cheese production.     

Immobilization of Bacillus licheniformis 5A1 milk-clotting enzyme and characterization of its enzyme properties

Milk-clotting enzyme from Bacillus licheniformis 5A1 was immobilized on Amberlite IR-120 by ionic binding. Almost all the enzyme activity was retained on the support. The immobilized milk-clotting enzyme was repeatedly used to produce cheese in a batch reactor. The production of cheese was repeated 5 times with no loss of activity. The specific activity calculated on a bound-protein basis was slightly higher than that of free enzyme. The free and immobilized enzyme were highly tolerant to repeated freezing and thawing. The optimum temperature for milk-clotting activity was 70 °C with the free enzyme whereas, it was ranged from 70 to 80 °C with the immobilized milk-clotting enzyme. The activation energy (E _A) of the immobilized milk-clotting enzyme was lower than the free enzyme (E _A = 1.59 and 1.99 Kcal mol⁻¹ respectively). The immobilized milk-clotting enzyme exhibited great thermal stability. The milk-clotting optimum pH was 7.0 for both free and immobilized enzyme. The Michaelis constant K _m of the immobilized milk-clotting enzyme was slightly lower than the free enzyme.     

牛凝乳酶原基因在乳酸乳球菌中的表达

【目的】利用乳酸乳球菌nisin诱导基因表达系统(the NIsin Controlled gene Expression system,NICE)表达牛凝乳酶原。【方法】从克隆载体pS19-PPC中获得牛凝乳酶原基因,将该基因与表达载体pNZ8148连接并电转化乳酸乳球菌NZ9000,转化子经酶切、PCR和测序鉴定后,用nisin进行诱导表达,表达产物利用SDS-PAGE和Western blot鉴定,表达产物纯化后检测凝乳活性。【结果】重组牛凝乳酶原与天然牛凝乳酶原比较,其分子量大小、免疫性质、生物活性和抑制剂敏感性没有发现显著差异,其凝乳活性可达2×103IMCU/mL。【结论】在乳酸乳球菌中表达了具有凝乳活性的牛凝乳酶原,同时乳酸乳球菌作为发酵剂和凝乳酶产生菌双重角色的实现,为奶酪加工提供了新思路和新方法。     

Identification of essential amino acid residues in the Sinorhizobium meliloti glucosyltransferase ExoM

ExoM is a beta(1-4)-glucosyltransferase involved in the assembly of the repeat unit of the exopolysaccharide succinoglycan from Sinorhizobium meliloti. By comparing the sequence of ExoM to those of other members of the Pfam Glyco Domain 2 family, most notably SpsA (Bacillus subtilis) for whom the three-dimensional structure has been resolved, three potentially important aspartic acid residues of ExoM were identified. Single substitutions of each of the Asp amino acids at positions 44, 96, and 187 with Ala resulted in the loss of mutant recombinant protein activity in vitro as well as the loss of succinoglycan production in an in vivo rescue assay. Mutants harboring Glu instead of Asp-44 or Asp-96 possessed no in vitro activity but could restore succinoglycan production in vivo. However, replacement of Asp-187 with Glu completely inactivated ExoM as judged by both the in vitro and in vivo assays. These results indicate that Asp-44, Asp-96, and Asp-187 are essential for the activity of ExoM. Furthermore, these data are consistent with the functions proposed for each of the analogous aspartic acids of SpsA based on the SpsA-UDP structure, namely, that Asp-44 and Asp-96 are involved in UDP substrate binding and that Asp-187 is the catalytic base in the glycosyltransferase reaction.     

Partial purification of pepsin from turkey proventriculus

In the present study, turkey proventriculus (stomach) was mixed with 10% NaCl (1:2 w/v) and extracted by centrifugation to produce crude extract. The partial purification of the extract was carried out by using Sephadex G-75 resin in gel filtration column chromatography. Crude extract (CE) and fractions obtained by gel filtration were analysed for milk-clotting activity (MCA), protein content, proteolytic activity (PA), and SDS-PAGE electrophoresis. The first 7 fractions did not have any activities; the fraction of 9, 10 and 11 had the most milk-clotting and proteolytic activities. Electrophoretic patterns proved that further purification steps should be applied for better results.     

江米酒中凝乳酶产生菌的分离及产酶条件的优化

江米酒是我国南方的一种传统食品, 江米酒中微生物所产凝乳酶具有凝乳作用。从江米酒中分离纯化得到4株菌, 经菌落分离计数和酪蛋白平板实验研究确定产凝乳酶的优势菌为其中的霉菌, 并对该霉菌产凝乳酶条件进行了初步优化, 结果表明: 2倍浓度的PDA培养基中添加5%葡萄糖而不添加氮源是霉菌产凝乳酶的最佳培养基, 在此条件下其所产凝乳酶活力可比优化前提高144%。     

Production of microbial rennin from Mucor miehei in a continuously fed fermenter

In this study, microbial production of rennin, a milk-clotting enzyme, from a commercial strain of Mucor miehei NRRL 3420 has been investigated in a continuously fed fermenter for prolonged times. The spherical film-type growth of the culture has been accomplished in the fermenter and the effects of medium pH, mixing and dilution rates, and feed -glucose concentration on milk-clotting activity have been elaborated. In the fermenter, optimum operational parameters have been determined as 400 rpm, 0.125 day⁻¹, and 7.5 g l⁻¹ for mixing rate, dilution rate, and feed -glucose concentration, respectively. Under these conditions, the fermenter operated 575 h continuously producing 1.24 IU ml⁻¹ maximum milk-clotting activity without concentration. In the fermenter sample at maximum milk clotting activity, the R factor and specific milk-clotting activity were determined as 1.55 × 10⁻³ IU PU⁻¹ and 5.28 IU mg⁻¹ medium protein, respectively, denoting competitive characteristics of a commercial rennet after concentration.     

Screening for Milk-clotting Enzyme from Basidiomycetes

Screening tests for aspartic proteinases with milk-clotting activity were done on basidiomycetes. Crude enzymes from 6 strains had a high ratio of milk-clotting activity to caseinolytic activity. These enzymes showed acidic pH optimum for proteolytic activity and were inhibited considerably by pepstatin, a specific aspartic proteinase inhibitor. Among them, the crude enzyme from Laetiporus sulphureus was more heat-labile than the other enzymes.     

Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115

Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.     

Demonstration of chymosin (EC 3.4.23.4) in the stomach of newborn pig.

The stomach of newborn pig contains a proteinase that is immunologically closely related to calf chymosin (rennin) (EC 3.4.23.4.). None of the pepsins from the stomach of adult pig is present in the newborn pig. Pig chymosin has optimal general proteolytic activity around pH 3.5. The ratio of milk-clotting activity to general proteolytic activity is about 30--70 times higher than that of pyloric and fundic pepsins.     

Production of intracellular milk-clotting enzyme in submerged cultures of Fusarium subglutinans.

Fusarium subglutinans (Wollenweber and Reinking) Nelson et al. was found to produce intracellular milk-clotting enzyme (MCE) with good milk-clotting activity (MCA). The crude activity of the produced enzyme was recorded as optimum at 55 degrees C and pH 4.5. The highest yield i.e. 78.43 SU/mg dry biomass was obtained after 4 days of rotary shaking at 30 degrees C when the fermentation medium containes wheat flour 2%, glucose 1% and (NH4)2SO4 0.1% with an initial pH value 6.0. Under these conditions, the maximum ratio of MCA to proteolytic activity (PA) amounting to 603.31 SU/PU mg(-1) was also achieved. Production of intracellular MCE by F. subglutinans was assumed to be active growth-associated type. This enzyme preparation was less active than the calf rennet, but was superior to those of Meito's and Pfizer's rennets.     

江米酒中凝乳酶产生菌的分离及产酶条件的优化

江米酒是我国南方的一种传统食品, 江米酒中微生物所产凝乳酶具有凝乳作用。从江米酒中分离纯化得到4株菌, 经菌落分离计数和酪蛋白平板实验研究确定产凝乳酶的优势菌为其中的霉菌, 并对该霉菌产凝乳酶条件进行了初步优化, 结果表明: 2倍浓度的PDA培养基中添加5%葡萄糖而不添加氮源是霉菌产凝乳酶的最佳培养基, 在此条件下其所产凝乳酶活力可比优化前提高144%。     

Strain variability of <Emphasis Type="Italic">Irpex Lacteus</Emphasis> basidiomycetes in the synthesis of specific milk-clotting proteinases

The ability to synthesize milk-clotting (rennet) proteinases was studied in eight strains of Irpex Lacteus basidiomycetes. It was found that the studied I. lacteus strains are characterized by different enzyme activities in their culture liquid. For I. lacteus strains 2425, 2426, and 2427, the maximum milk-clotting activity was detected during the exponential growth phase on the 15th day of cultivation on glucose–peptone medium. These I. lacteus strains are the most prospective milk-clotting enzyme producers for further research and practical application. I. lacteus strains 2421, 2422, 2423, 2424, and 2428 showed lower values of the enzyme activity and require additional research to determine the optimal culture conditions.     

Human lipoprotein lipase. Analysis of the catalytic triad by site-directed mutagenesis of Ser-132, Asp-156, and His-241.

Lipoprotein lipase (LPL) plays a central role in normal lipid metabolism as the key enzyme involved in the hydrolysis of triglycerides present in chylomicrons and very low density lipoproteins. LPL is a member of a family of hydrolytic enzymes that include hepatic lipase and pancreatic lipase. Based on primary sequence homology of LPL to pancreatic lipase, Ser-132, Asp-156, and His-241 have been proposed to be part of a domain required for normal enzymic activity. We have analyzed the role of these potential catalytic residues by site-directed mutagenesis and expression of the mutant LPL in human embryonic kidney-293 cells. Substitution of Ser-132, Asp-156, and His-241 by several different residues resulted in the expression of an enzyme that lacked both triolein and tributyrin esterase activities. Mutation of other conserved residues, including Ser-97, Ser-307, Asp-78, Asp-371, Asp-440, His-93, and His-439 resulted in the expression of active enzymes. Despite their effect on LPL activity, substitutions of Ser-132, Asp-156, and His-241 did not change either the heparin affinity or lipid binding properties of the mutant LPL. In summary, mutation of Ser-132, Asp-156, and His-241 specifically abolishes total hydrolytic activity without disrupting other important functional domains of LPL. These combined results strongly support the conclusion that Ser-132, Asp-156, and His-241 form the catalytic triad of LPL and are essential for LPL hydrolytic activity.     

The active site topology of Aspergillus niger endopolygalacturonase II as studied by site-directed mutagenesis

Strictly conserved charged residues among polygalacturonases (Asp-180, Asp-201, Asp-202, His-223, Arg-256, and Lys-258) were subjected to site-directed mutagenesis in Aspergillus niger endopolygalacturonase II. Specific activity, product progression, and kinetic parameters (K(m) and V(max)) were determined on polygalacturonic acid for the purified mutated enzymes, and bond cleavage frequencies on oligogalacturonates were calculated. Depending on their specific activity, the mutated endopolygalacturonases II were grouped into three classes. The mutant enzymes displayed bond cleavage frequencies on penta- and/or hexagalacturonate different from the wild type endopolygalacturonase II. Based on the biochemical characterization of endopolygalacturonase II mutants together with the three-dimensional structure of the wild type enzyme, we suggest that the mutated residues are involved in either primarily substrate binding (Arg-256 and Lys-258) or maintaining the proper ionization state of a catalytic residue (His-223). The individual roles of Asp-180, Asp-201, and Asp-202 in catalysis are discussed. The active site topology is different from the one commonly found in inverting glycosyl hydrolases.     

Role of aspartic acid 121 in human pancreatic ribonuclease catalysis

Bovine pancreatic ribonuclease (RNase A) is one of the most well studied enzymes of the ribonuclease family, unlike its human counterpart, the human pancreatic ribonuclease (HPR), whose physiological role in the body is not clearly understood. Human pancreatic ribonuclease consists of 128 amino acids and the main residues located in the active site of RNase A are also conserved in HPR. In the current study, to investigate the role of Asp-121 in the catalytic activity of human pancreatic ribonuclease, several variants were generated in which Asp-121 was either mutated to an alanine or C-terminal residues beyond Asp-121, and Phe-120 were deleted. The HPR mutants were cloned, expressed in E. coli and purified to homogeneity, and functionally characterized. The mutation D121A in HPR significantly decreased the rate of the enzymatic reaction, however this decrease was not universally observed for all substrates studied. Removal of the seven C-terminal amino acid residues thereby exposing Asp-121 yielded an HPR mutant with enhanced activity, however a further deletion removing Asp-121 resulted in the complete inactivation of HPR. Our results indicate that Asp-121 is crucial for the catalytic activity of HPR and may be involved in the depolymerization activity of the enzyme.     

Production and characterization of a milk-clotting enzyme from Aspergillus oryzae MTCC 5341

Microbial milk-clotting enzymes are valued as calf rennet substitutes in the cheese industry. Aspergillus oryzae MTCC 5341 was identified to produce the highest milk-clotting activity during screening of 16 fungal strains. Solid state fermentation using wheat bran along with 4% defatted soy flour and 2% skim milk powder as substrate was optimal for growth of A. oryzae and production of the enzyme. Nearly 40,000 U/g bran of milk-clotting activity was present at the end of 120 h. The enzyme could be recovered by percolating the bran with 0.1 M sodium chloride for 60 min at 4°C. The decolorized enzyme preparation had high ratio of milk clotting to proteolytic activity. Affinity precipitation with alginate and subsequent elution with 0.5 M sodium chloride containing 0.2 M CaCl₂ resulted in an enzyme preparation with specific activity of 3,500 U/mg and 72% yield. Optimum pH and temperature for activity of the enzyme were characterized as 6.3 and 55°C, respectively. Milk-clotting enzyme showed differential degree of hydrolysis on casein components. High ratio of milk clotting to proteolytic activity coupled with low thermal stability strengthens the potential usefulness of milk-clotting enzyme of A. oryzae MTCC 5341 as a substitute for calf rennet in cheese manufacturing.     

Tissue-specific expression of multiple forms of cyprosin (aspartic proteinase) in flowers of Cynara cardunculus

Three heterodimeric aspartic proteinases (cyprosin 1, 2 and 3) with milk-clotting activity have previously been purified from flowers of Cynara cardunculua and partly characterized (U. Heimgartner et al. 1990, Phytochemistry 29: 1405–1410). These proteinases have now been further studied. Isoelectric focusing has revealed a micro-heterogeneity of the apparently pure cyprosins. Three isozymes with close isoelectric points around 4.0 have been found. Reversed-phase high performance liquid chromatography of electrophoretically purified large subunits of cyprosin has also shown a microheterogeneity. Peptide mapping of cyprosins 2 and 3 by trypsin or BrCN cleavage indicate that they are derived from common procyprosin(s). Studies on the organspecific accumulation of the enzyme were carried out using flower buds and flowers at different stages of development and styles and corollas from open flowers, leaves and seeds. Immunostained western blots revealed the presence of cyprosin in very young flowers in low amounts. The amount of enzyme increased towards later stages of development and it was mostly present in the violet parts of styles and corollas. The enzyme could not be detected in leaves or seeds. Proteolytic and milk-clotting activities correlate well with these findings. The enzyme was localized by immunolabelling in the epidermal cell layer of styles. Mature flowers collected at 8 different locations in Portugal showed some variation in proteolytic activity while the milk-clotting activity was essentially the same for all extracts.