Improved subtilisin YaB production in Bacillus subtilis using engineered synthetic expression control sequences

Alkaline elastase YaB, a favorable meat tenderizer, is an extracellular subtilisin-type protease produced by wild strain alkalophilic Bacillus YaB. The gene ale coding for subtilisin YaB with its own expression control sequence has been cloned and expressed in Bacillus subtilis, but at levels much lower than in the parental strain Bacillus YaB. This study investigates the influence of various expression control sequences including expression control sequences of cdd and veg from B. subtilis, a synthetic expression control sequence (SECS), and engineered synthetic expression control sequences (engineered SECSs) on the expression of subtilisin YaB in B. subtilis. The engineered SECSs were generated by using the Polymerase Chain Reaction; their UP element, Shine-Dargarno (SD) sequence, or both were different from those of the native SECS. The expression efficiencies of SECS and engineered SECSs were higher than those of expression control sequences of ale, cdd, and veg. Substitution of the SD sequence of SECS resulted in higher expression of subtilisin YaB than substitution of the UP element, whereas combined substitution of both gave the highest expression. These results demonstrate that engineering of SECSs is an approach for improving subtilisin YaB production in B. subtilis. Moreover, it is suggested that these enginnered SECSs could potentially be used to express homologous and heterologous proteins in B. subtilis at high level.     

Contribution of the microbial and meat endogenous enzymes to the free amino acid and amine contents of dry fermented sausages.

The role of the starter culture and meat endogenous enzymes on the free amino acid and amine contents of dry fermented sausages was studied. Five batches of sausages were prepared. The control batch was manufactured with aseptic ingredients without microbial inoculation. The other four experimental batches were manufactured with aseptic ingredients inoculated with Lactobacillus plantarum 4045 or Micrococcus-12 or L. plantarum 4045 and Micrococcus-12 or L. plantarum 4045 and Staphylococcus sp. Their effects on pH, a(w), myofibrillar proteins, and free amino acid and amine contents were studied. Sausages inoculated only with L. plantarum 4045 or with this starter combined with a Micrococcaceae had the lowest pH as a result of carbohydrate fermentation. In all batches similar patterns were observed for myofibrillar proteins and free amino acids which could indicate that meat endogenous proteases play an important role in proteolytic phenomena. No changes were observed in the amine fraction, indicating that the strains used as starter cultures did not show amino acid decarboxylase activity.     

Determination of nitrogen and protein content of meat and meat products

Chemical and instrumental methods for determination of nitrogen and protein are reviewed for their mode of action and utility in analysis of meat proteins and products. Although the Kjeldahl digestion method is satisfactory for determining total nitrogen, it is imprecise for determining total protein content. Presence of variable amounts of nonprotein nitrogenous components and of connective tissue proteins such as collagen and elastin produces error if the formula (N X 6.25) is used to calculate crude protein. Such fibrous proteins have higher nitrogen levels (over 18%) than other muscle proteins (about 16%), and a higher than actual protein value will be determined unless a lower conversion factor is used to correct for their content. To determine meat protein content more accurately, a combination of Kjeldahl determination with one or more additional tests to correct for nonprotein and fibrous protein content is recommended. The choice of the additional method(s) is based on the user's requirement for protein characterization, available time, type of meat product, and sample size.     

Tyrosinase-aided protein cross-linking: effects on gel formation of chicken breast myofibrils and texture and water-holding of chicken breast meat homogenate gels

The effects of Trichoderma reesei tyrosinase-catalyzed cross-linking of isolated chicken breast myofibril proteins as a simplified model system were studied with special emphasis on the thermal stability and gel formation of myofibrillar proteins. In addition, tyrosinase-catalyzed cross-linking was utilized to modify the firmness, water-holding capacity (WHC), and microstructure of cooked chicken breast meat homogenate gels. According to SDS-PAGE, the myosin heavy chain (MHC) and troponin T were the most sensitive proteins to the action of tyrosinase, whereas actin was not affected to the same extent. Calorimetric enthalpy (DeltaH) of the major thermal transition associated with myosin denaturation was reduced and with actin denaturation increased in the presence of tyrosinase. Low-amplitude viscoelastic measurements at constant temperatures of 25 degrees C and 40 degrees C showed that tyrosinase substantially increased the storage modulus (G') of the 4% myofibrillar protein suspension in the 0.35 M NaCl concentration. The effect was the most pronounced with high-enzyme dosages and at 40 degrees C. Without tyrosinase, the G' increase was low. Tyrosinase increased the firmness of the cooked phosphate-free and low-meat chicken breast meat homogenate gels compared to the corresponding controls. Tyrosinase maintained gel firmness at the control level of the low-salt homogenate gel and weakened it when both salt and phosphate levels were low. Tyrosinase improved the WHC of the low-meat and low-salt homogenate gels and maintained it at the level of the corresponding controls of phosphate-free and low-salt/low-phosphate homogenate gels. Microstructural characterization showed that a collagen network was formed in the presence of tyrosinase. Keywords: Chicken myofibrillar proteins; protein modification; cross-linking; tyrosinase; gelation; thermal stability; texture; water-holding capacity; microstructure.     

Monitoring of denaturation processes in aged beef loin by Fourier transform infrared microspectroscopy

We present the results of a Fourier transform infrared (FT-IR) microspectroscopic study using conventional FT-IR microscopy and FT-IR imaging to detect the denaturation process during four different heating temperatures (raw, 45, 60, and 70 degrees C) spatially resolved in bovine cryosections from longissimus dorsi muscle. FT-IR imaging, employing a focal plane array detector, which allowed the simultaneous collection of spectra at 4096 pixels, enabled the investigation of the heat-induced changes in the two major meat constituents, i.e., myofibrillar and connective tissue proteins, spatially resolved. The infrared spectra of both compounds revealed that the major spectral changes involved an increase in beta-sheet and a decrease in alpha-helical structures, which appeared to be much more pronounced for the myofibers than for the connective tissue. These conformational changes could be correlated to the denaturation of the major meat proteins, such as myosin, actin, and collagen.     

Detection and localization of oxidized proteins in muscle cells by fluorescence microscopy

In meat, no detailed studies on the intracellular distribution of oxidized proteins during oxidative stress have been performed, to our knowledge. Therefore, we used fluorescence microscopy to detect and locate protein carbonyls, oxidation products of basic amino acids, generated in bovine M. Rectus abdominis during either exposition to a chemical free radical generating system, or refrigerated storage, or cooking. The technique consisted of an immunohistochemical detection of carbonyls by reaction with the specific probe DNPH (2,4-dinitrophenylhydrazine) followed by the sequential addition of a first antibody against DNPH-carbonylated proteins and a CY3-labeled secondary antibody. The fluorescence of the CY3 probe increased regularly with level of free radical generating system and storage time. Moreover, an important heterogeneity of carbonyl distribution was observed, with a higher oxidation level at the periphery than inside the muscle cells. Cooking induced fluorescence increase only at the periphery of cells. Specific coloration of collagen by Sirius red showed that collagen was not involved in fluorescence. We can deduce that accumulation of oxidized proteins observed in the cell periphery was linked to membrane protein oxidation and not to connective tissue oxidation. Biochemical assays were performed in parallel on membrane and myofibrillar proteins to provide complementary quantitative data on level of oxidized proteins.     

Biochemical properties of recombinant leucine aminopeptidase II from Bacillus stearothermophilus and potential applications in the hydrolysis of Chinese anchovy (Engraulis japonicus) proteins

The effects of various factors on the activity and conformation of recombinant leucine aminopeptidase II (rLAP II) from Bacillus stearothermophilus and its potential utilization in the hydrolysis of anchovy proteins were investigated. The optimal temperature and pH of rLAP II were 55 °C and 8.0 in phosphate buffer, and its activity was strongly stimulated by Co(2+). Conformational studies indicated that maintaining the α-helical structure had a critical effect on rLAP II activity. rLAP II was used to hydrolyze anchovy proteins, and it exhibited high specificity for peptides with molecular weight between 6000 and 1000 Da and positive coordination with endogenous enzymes and commercial Flavourzyme. Its use will enhance protein hydrolysis in species of aquatic animals. rLAP II could potentially be used to remove bitterness in the protein hydrolysis industry.     

Enhancement of the catalytic activity of a 27 kDa subtilisin-like enzyme from Bacillus amyloliquefaciens CH51 by in vitro mutagenesis

AprE51 from Bacillus amyloliquefaciens CH51 is a 27 kDa subtilisin-like protease with fibrinolytic activity. To enhance the catalytic activity of AprE51, two residues, Gly-169 and Ser-101, which, according to the three-dimensional structural model of subtilisin, are located in the P1 substrate-binding site and S3 subsite, respectively, were mutated by site-directed mutagenesis. Results of the mutational analysis showed that substitution of alanine for Gly-169 increased the fibrinolytic activity 1.4-fold. All four Ser-101 mutations, that is, replacements with arginine, leucine, lysine, and tryptophan, also increased the fibrinolytic activity up to 3.9-fold. The S101W mutant with a bulky side chain was more active than mutants with a positively charged or nonpolar small side chains. The fibrinolytic activity of the S101W mutant was further increased by error-prone polymerase chain reaction. The AprE51-6 mutant (S101W/G169A/V192A) had stronger fibrinolytic activity than the S101W mutant. Purified AprE51-6 had a 2.5-fold higher k(cat) and a 2.3-fold lower K(m), which resulted in a 6-fold increase in catalytic efficiency (k(cat)/K(m)) relative to that of wild-type AprE51. In addition, AprE51-6 showed a relatively broader pH range and increased thermostability as compared to AprE51.     

Effect of meat cooking on physicochemical state and in vitro digestibility of myofibrillar proteins

The effect of meat cooking was measured on myofibrillar proteins from bovine M. Rectus abdominis. The heating treatment involved two temperatures (100 degrees C during 5, 15, 30, and 45 min and 270 degrees C during 1 min). Protein oxidation induced by cooking was evaluated by the level of carbonyl and free thiol groups. Structural modifications of proteins were assessed by the measurement of their surface hydrophobicity and by their aggregation state. With the aim of evaluating the impact of heat treatment on the digestive process, myofibrillar proteins were then exposed to proteases of the digestive tract (pepsin, trypsin, and alpha-chymotrypsin) in conditions of pH and temperature that simulate stomach and duodenal digestion. Meat cooking affected myofibrillar protein susceptibility to proteases, with increased or decreased rates, depending on the nature of the protease and the time/temperature parameters. Results showed a direct and quantitative relationship between protein carbonylation (p<0.01) and aggregation (p<0.05) induced by cooking and proteolytic susceptibility to pepsin. However, no such correlations have been observed with trypsin and alpha-chymotrypsin.     

Engineering of a broad specificity antibody for simultaneous detection of 13 sulfonamides at the maximum residue level

Sulfa antibiotics (sulfonamides) are a group of molecules sharing the p-aminobenzenesulfonamide moiety. Sulfonamides are used in veterinary and human medicine. Sometimes, the meat or milk of medicated animals is contaminated with residual sulfonamides. Current analytical methods for sulfonamides are unfit for screening of food, because they are either too laborious, insensitive, or specific for a few sulfa compounds only. A rapid immunoassay for detection of all sulfas in a single reaction would thus be useful. Previously, we used protein engineering to improve the broad specificity of sulfa antibody 27G3. In this study, we improved the best mutant of the previous studies with site-directed mutagenesis. The new mutants recognized different sulfonamides with affinities sufficient for detection of all 13 tested sulfonamides below the MRL level. We furthermore demonstrated the functionality of one mutant in some real sample matrices.     

Purification and identification of barley (Hordeum vulgare L.) proteins that inhibit the alkaline serine proteinases of Fusarium culmorum

It has been proposed that microbial proteinase inhibitors, which are present in abundance in cereal grains, protect the seed against plant pathogens. So far, however, very little is known about the interactions of those inhibitors with the proteinases of phytopathogenic microbes. The increased alkaline proteinase activities of Fusarium head blight (FHB) diseased wheat and barley grain imply that the Fusarium fungi synthesize those enzymes during the colonization of the kernel. To study which barley proteins can inhibit Fusarium proteinases, and hence, possibly protect the seed from FHB, the proteins of a grain extract have been separated and tested for their abilities to inhibit two alkaline serine proteinases that we previously isolated from F. culmorum. The proteins were separated by size exclusion, ion exchange, and reversed-phase-HPLC chromatographies. The purified inhibitors were identified by their molecular masses and N-terminal amino acid sequences. The proteins that inhibited the subtilisin-like Fusarium proteinase were the chymotrypsin/subtilisin (CI) inhibitors 1A, 1B, and 2A and the barley alpha-amylase/subtilisin inhibitor (BASI). Only one of the purified proteins inhibited the trypsin-like proteinase, the barley Bowman-Birk inhibitor (BBBI). No novel inhibitors were detected.     

蛋白质组学在生鲜肉肉色变化机制研究中的应用

肉色是影响消费者购买欲望的最直观因素。宰后生鲜肉肉色的变化机制和肉色改善一直是肉类科学领域研究的热点。肉色体系复杂,其稳定性受动物宰前因素、从肌肉向可食用肉转化过程和宰后商业流通链条件等因素的影响。目前,仍有许多影响途径和作用机制不够清楚。研究者已采用蛋白质组学技术发现了许多与肉色相关的蛋白标志物和可能影响途径。该文基于近年的研究,综述了不同处理条件下与肉色相关的差异蛋白,主要包括结构性蛋白中的肌动蛋白、肌球蛋白和肌联蛋白,以及肌浆蛋白中的伴侣蛋白、热休克蛋白、代谢酶类和氧化还原酶类等,分析了差异蛋白表达对肉色的影响及其表达调控肉色体系的可能机制,为今后肉色变化机制的研究和肉色稳定性的调控提供了思路。     

Vacuolar invertases in sweet potato: molecular cloning, characterization, and analysis of gene expression

Two cDNAs (Ib beta fruct2 and Ib beta fruct3) encoding vacuolar invertases were cloned from sweet potato leaves, expressed in Pichia pastoris, and the recombinant proteins were purified by ammonium sulfate fractionation and chromatography on Ni-NTA agarose. The deduced amino acid sequences encoded by the cDNAs contained characteristic conserved elements of vacuolar invertases, including the sequence R[G/A/P]xxxGVS[E/D/M]K[S/T/A/R], located in the prepeptide region, Wxxx[M/I/V]LxWQ, located around the starting site of the mature protein, and an intact beta-fructosidase motif. The pH optimum, the substrate specificity, and the apparent K(m) values for sucrose exhibited by the recombinant proteins were similar to those of vacuolar invertases purified from sweet potato leaves and cell suspensions, thus confirming that the proteins encoded by Ib beta fruct2 and Ib beta fruct3 are vacuolar invertases. Moreover, northern analysis revealed that the expression of the two genes was differentially regulated. With the exception of mature leaves and sprouting storage roots, Ib beta fruct2 mRNA is widely expressed among the tissues of the sweet potato and is more abundant in young sink tissues. By contrast, Ib beta fruct3 mRNA was only detected in shoots and in young and mature leaves. It appears, therefore, that these two vacuolar invertases play different physiological roles during the development of the sweet potato plant.     

Dynamic MRI and thermal simulation to interpret deformation and water transfer in meat during heating

Understanding and controlling structural and physical changes in meat during cooking is of prime importance. Nuclear magnetic resonance imaging (MRI) is a noninvasive, nondestructive tool that can be used to characterize certain properties and structures both locally and dynamically. Here we show the possibilities offered by MRI for the in situ dynamic imaging of the connective network during the cooking of meat to monitor deformations between 20 and 75 °C. A novel device was used to heat the sample in an MR imager. An MRI sequence was developed to contrast the connective tissue and the muscle fibers during heating. The temperature distribution in the sample was numerically simulated to link structural modifications and water transfer to temperature values. The contraction of myofibrillar and collagen networks was observed at 42 °C, and water began to migrate toward the interfascicular space at 40 °C. These observations are consistent with literature results obtained using destructive and/or nonlocalized methods. This new approach allows the simultaneous monitoring of local deformation and water transfer, changes in muscle structure and thermal history.     

超高压前处理对虾仁冻藏期品质的影响

为研究超高压辅助脱壳所得到的中华管鞭虾虾仁在冻藏期的品质变化,以虾仁色泽、质构、肌原纤维蛋白相对含量、表面疏水性、总巯基相对含量和Ca²⁺-ATPase活性为指标,探讨超高压前处理对虾仁品质的影响。结果表明,冻藏6个月后虾仁L^*值、a^*值和b^*值均有所变化,与对照组相比,超高压前处理对虾仁L^*值的影响不显著,但能延缓冻藏后期a^*值的增加,对保持虾肉色泽的稳定有一定作用;超高压前处理对虾仁的咀嚼性和弹性影响不显著,但能增加虾仁的硬度。超高压前处理使冻藏初期虾仁肌原纤维蛋白的表面疏水性增加,而对其溶解性、巯基含量及Ca²⁺-ATPase活性的影响不显著;冻藏后期由于虾仁蛋白冷冻变性,致使肌原纤维蛋白溶解性、巯基含量及Ca²⁺-ATPase活性下降。综上所述,超高压辅助脱壳在一定程度上有利于保持冻虾仁的色泽和硬度。本研究结果为利用超高压技术辅助脱除中华管鞭虾虾壳,生产冷冻虾仁提供了新思路。     

Purification and substrate specificity of transglutaminases from blood and Streptoverticillium mobaraense.

A procedure for a fast and simple purification of bovine plasma transglutaminase was developed, which resulted in a homogeneous enzyme preparation. Two different procedures were developed for the purification of pig erythrocyte transglutaminase, both of which resulted in partial purification. Both enzymes were used in cross-linking reactions of alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin, casein, hemoglobin, glycinin, and myosin. The substrate specificity was compared to that of bacterial transglutaminase isolated from Streptoverticillium mobaraense. The bacterial transglutaminase caused cross-linking of a wider range of proteins and, thus, exhibited a lower substrate specificity than the blood transglutaminases. In addition, differences exist in the necessity of the addition of reducing agents. These differences allow specific applications of blood and bacterial transglutaminases at protein cross-linking in single or complex protein systems.     

Altering the substrate specificity of Candida rugosa LIP4 by engineering the substrate-binding sites

Candida rugosa (formerly Candida cylindracea) lipase (CRL) is an important industrial enzyme that is widely used in biotechnological applications such as the production of fatty acids and the synthesis of various esters. CRL comprises at least seven isozymes (LIP1-LIP7), which share a similar amino acid sequence but with different specificities for substrates. Previously, LIP4 was reported to have higher esterase activity toward long acyl-chain ester and lower lipase activity toward triglycerides. A296 and V344 of LIP4 were predicted to play decisive roles in its substrate specificity. In this study, site-specific saturation mutagenesis has been employed to study the substrate specificity of LIP4. Point mutations were separately introduced into A296 and V344 positions using degenerate primer sets containing 32 codons to generate two libraries of variants. LIP4 variants were heterologously expressed in the yeast Pichia pastoris. A specific plate assay was used to identify lipase-producing P. pastoris clones in a medium containing tributyrin. LIP4 variants with high activity toward short fatty acyl-chain triglyceride (tributyrin) were screened. Specificity analysis and biochemical characterization indicated that the recombinant variants A296I, V344Q, and V344H had properties remarkably different from those of wild-type LIP4. All three variant enzymes had significantly higher specific activities toward tributyrin than LIP4. In addition to short-chain triglyceride, A296I and V344Q also improved hydrolytic activities of triglycerides toward medium- and long-chain triglycerides tested. The results suggested that A296 played an important role in lipase activity and high-temperature dependence of LIP4, whereas it had no effect on the chain-length specificity in lipolytic reaction. The V344 residue had a significant effect on the substrate chain-length specificity of LIP4.     

Purification and characterization of proteases from Bacillus amyloliquefaciens isolated from traditional soybean fermentation starter

Bacillus amyloliquefaciens FSE-68 isolated from meju, a Korean soybean fermentation starter, was identified on the basis of biophysical tests and 16S rRNA gene sequence. A neutral metalloprotease (NPR68) and an alkaline serine-protease (APR68) were purified by ammonium sulfate precipitation and cation exchange chromatography and identified on the basis of their activities at different pH values and the selective protease inhibitors. The molecular weights of NPR68 and APR68 measured with ESI-MS were 32743 (+/- 0.8) and 27443 (+/- 0.5) Da, respectively. Against oxidized insulin chains, the NPR68 has a cleavage preference at the site where leucine is located as a P1' residue followed by phenylalanine, and the APR68 has broad specificity and favors leucine at the P1 site. These results indicate that the proteases are natural variants of subtilisin and bacillolysin.     

Ferrochelatase catalyzes the formation of Zn-protoporphyrin of dry-cured ham via the conversion reaction from heme in meat

Ferrochelatase (FECH), the enzyme at the last step of the heme-biosynthetic pathway, is involved in the formation of Zn-protoporphyrin via an iron-removal reaction of heme. To improve the efficacy of the formation of Zn-protoporphyrin from heme, the use of recombinant FECHs from porcine, yeast, and bacteria was examined. Incubation of FECH with myoglobin in the presence of ascorbic acid and cysteine resulted in the efficient conversion of myoglobin-heme to Zn-protoporphyrin. Exogenously added recombinant yeast FECH facilitates the production of Zn-protoporphyrin from myoglobin-heme and heme in meat, via the replacement of iron in the protoporphyrin ring by zinc ions. A large amount of Zn-protoporphyrin was also generated by the catalysis of FECH using an intact piece of meat as a substrate. These findings can open up possible approaches for the generation of a nontoxic bright pigment, Zn-protoporphyrin, to shorten the incubation time required to produce dry-cured ham.     

Particulate metmyoglobin reducing activity and its relationship with meat color

There is controversy about the effect of storage time on metmyoglobin (MetMb) reducing activity in the sarcoplasmic fraction of meat and the role of this reducing activity in maintaining the color of red meat. The presence of metmyoglobin reducing activity in the myofibrillar fraction of muscle extracts was investigated as a possible reason for this controversy. NADH-dependent MetMb reducing activity was found in the particulate fraction of meat following sedimentation of a meat homogenate at 35 000g. There was 5.8 times more MetMb reducing activity in the particulate fraction compared with that of the sarcoplasmic (supernatant) fraction. The presence of metmyoglobin reducing activity in the myofibrils (MMRA), defined as the activity in the sediment after centrifugation of a meat homogenate at 2 000g, was confirmed. The myofibrillar fraction contained 63% of the total MetMb reducing activity available in the meat. The relationship between muscle MetMb reducing activities and meat color parameters was investigated in a beef patties system. The particulate MetMb reducing activity (PMRA) in beef patties was found to be a good indicator of the total metmyoglobin reducing activity in meat and was positively correlated with the color parameters of the patties, suggesting that PMRA may be associated with red meat color of meat patties.