RESUMEN
For applications in food industries, a fungal α-amylase from Malbranchea cinnamomea was engineered by directed evolution. Through two rounds of screening, a mutant α-amylase (mMcAmyA) was obtained with higher optimal temperature (70 °C, 5 °C increase) and better hydrolysis properties (18.6 % maltotriose yield, 2.5-fold increase) compared to the wild-type α-amylase (McAmyA). Site-directed mutations revealed that Threonine (Thr) 226 Serine (Ser) substitution was the main reason for the property evolution of mMcAmyA. Through high cell density fermentation, the highest expression level of Thr226Ser was 3951 U/mL. Thr226Ser was further used for bread baking with a dosage of 1000 U/kg flour, resulting in a 17.8 % increase in specific volume and a 35.6 % decrease in hardness compared to the control. The results were a significant improvement on those of McAmyA. Moreover, the mutant showed better anti-staling properties compared to McAmyA, as indicated by the improved sensory evaluation after 4 days of storage at 4 and 25 °C. These findings provide insights into the structure-function relationship of fungal α-amylase and introduce a potential candidate for bread-making industry.
Asunto(s)
Pan , alfa-Amilasas , alfa-Amilasas/genética , alfa-Amilasas/metabolismo , Hidrólisis , TrisacáridosRESUMEN
Functional oligosaccharides exert obesity-reducing effects by acting at various pathological sites responsible for the development of obesity. In this study, tamarind xyloglucan oligosaccharides (TXOS) were used to attenuate metabolic disorders via the gut-liver axis in mice with high-fat-diet (HFD)-induced obesity, as determined through LC/MS-MS and 16S rRNA sequencing technology. A TXOS dose equivalent to 0.39 g/kg/day in humans restored the gut microbiota in obese mice, which was in part supported by the key microflora, particularly Bifidobacterium pseudolongum. Moreover, TXOS reduced the abundance of opportunistic pathogen species, such as Klebsiella variicola and Romboutsia ilealis. The bodyweight and weight gain of TXOS-treated (4.8 g/kg per day) mice began to decrease at the 14th week, decreasing by 12.8% and 23.3%, respectively. Sixteen fatty acids were identified as potential biomarkers in the liver, and B. pseudolongum and caprylic acid were found to tightly regulate each other. This was associated with reduced inflammation in the liver, circulation, and adipose tissue and protection from metabolic disorders. The findings of this study indicate that TXOS can significantly increase the gut microbiota diversity of obese mice and restore the HFD-induced dysbiosis of gut microbiota.
RESUMEN
2'-Fucosyllactose (2'-FL) is known for its ability to provide various health benefits to infants, such as gut maturation, pathogen resistance, improved immunity, and nervous system development. However, the production of 2'-FL using α-L-fucosidases is hindered by the lack of low-cost natural fucosyl donors and high-efficiency α-L-fucosidases. In this work, a recombinant xyloglucanase from Rhizomucor miehei (RmXEG12A) was applied to produce xyloglucan-oligosaccharide (XyG-oligos) from apple pomace. Then, an α-L-fucosidase gene (PbFucB) was screened from the genomic DNA of Pedobacter sp. CAU209 and expressed in Escherichia coli. The capability of purified PbFucB to catalyze XyG-oligos and lactose to synthesize 2'-FL was further evaluated. The deduced amino acid sequence of PbFucB shared the highest identity (38.4%) with that of other reported α-L-fucosidases. PbFucB showed the highest activity at pH 5.5 and 35 °C. It catalyzed the hydrolysis of 4-nitrophenyl-α-L-fucopyranoside (pNP-Fuc, 20.3 U mg-1), 2'-FL (8.06 U mg-1), and XyG-oligos (0.43 U mg-1). Furthermore, PbFucB demonstrated a high enzymatic conversion rate in 2'-FL synthesis with pNP-Fuc or apple pomace-derived XyG-oligos as donors and lactose as acceptor. Under the optimized conditions, PbFucB converted 50% of pNP-Fuc or 31% of the L-fucosyl residue in XyG-oligos into 2'-FL. This work elucidated an α-L-fucosidase that mediates the fucosylation of lactose and provided an efficient enzymatic strategy to synthesize 2'-FL either from artificial pNP-Fuc or natural apple pomace-derived XyG-oligos. KEY POINTS: ⢠Xyloglucan-oligosaccharide (XyG-oligos) was produced from apple pomace by a xyloglucanase from Rhizomucor miehei. ⢠An α-L-fucosidase (PbFucB) from Pedobacter sp. CAU209 shared the highest identity (38.4%) with reported α-L-fucosidases. â¢PbFucB synthesized 2'-FL using apple pomace-derived XyG-oligos and lactose with a conversion ratio of 31%.
Asunto(s)
Malus , Pedobacter , Lactante , Humanos , alfa-L-Fucosidasa/genética , alfa-L-Fucosidasa/metabolismo , Malus/metabolismo , Lactosa/metabolismo , Oligosacáridos/metabolismoRESUMEN
The present supply of prebiotics is entirely inadequate to meet their demand. To produce novel prebiotics, a d-mannose isomerase (XpMIaseA) from Xanthomonas phaseoli was first produced in Komagataella phaffii (Pichia pastoris). XpMIaseA shared the highest amino acid sequence identity (58.0%) with the enzyme from Marinomonas mediterranea. Efficient secretory production of XpMIaseA (282.0 U mL-1) was achieved using high cell density fermentation. The optimal conditions of XpMIaseA were pH 7.5 and 55 °C. It showed a broad substrate specificity, which isomerized d-mannose, d-talose, mannobiose, epilactose, and mannotriose. XpMIaseA was employed to construct a one-pot three-enzyme system for the production of mannosyl-ß-(1 â 4)-fructose (MF) using mannan (5%, w/v) as the substrate. The equilibrium yield of MF was 58.2%. In in vitro fermentations, MF significantly stimulated (≤3.2-fold) the growth of 12 among 15 tested Bifidobacterium and Lactobacillus strains compared with fructo-oligosaccharides. Thus, the novel d-mannose isomerase provides a one-pot bioconversion strategy for efficiently producing novel prebiotics.
Asunto(s)
Mananos , Manosa , Isomerasas Aldosa-Cetosa , Fructosa , Mananos/metabolismo , Manosa/metabolismo , Oligosacáridos/química , XanthomonasRESUMEN
Partially hydrolyzed konjac powder (PHKP) can be used to increase the daily intake of dietary fibers of consumers. To produce PHKP by enzymatic hydrolysis, a novel ß-mannanase gene (McMan5B) from Malbranchea cinnamomea was expressed in Pichia pastoris. It showed a low identity of less than 52% with other GH family 5 ß-mannanases. Through high cell density fermentation, the highest ß-mannanase activity of 42200 U mL-1 was obtained. McMan5B showed the maximal activity at pH 7.5 and 75 °C, respectively. It exhibited excellent pH stability and thermostability. Due to the different residues (Phe214, Pro253, and His328) in catalytic groove and the change of ß2-α2 loop, McMan5B showed unique hydrolysis property as compared to other ß-mannanases. The enzyme was employed to hydrolyze konjac powder for controllable production of PHKP with a weight-average molecular weight of 22000 Da (average degree of polymerization 136). Furthermore, the influence of PHKP (1.0%-4.0%) on the qualities of steamed bread was evaluated. The steamed bread adding 3.0% PHKP had the maximum specific volume and the minimum hardness, which showed 11.0% increment and 25.4% decrement as compared to the control, respectively. Thus, a suitable ß-mannanase for PHKP controllable production and a fiber supplement for steamed bread preparation were provided in this study. KEY POINTS: ⢠A novel ß-mannanase gene (McMan5B) was cloned from Malbranchea cinnamomea and expressed in Pichia pastoris at high level. ⢠McMan5B hydrolyzed konjac powder to yield partially hydrolyzed konjac powder (PHKP) instead of manno-oligosaccharides. ⢠PHKP showed more positive effect on the quality of steamed bread than many other dietary fibers including konjac powder.
Asunto(s)
Amorphophallus , beta-Manosidasa , Amorphophallus/genética , Clonación Molecular , Concentración de Iones de Hidrógeno , Mananos/química , Onygenales , Pichia/genética , Polvos , beta-Manosidasa/química , beta-Manosidasa/genéticaRESUMEN
The xyloglucanase gene (RmXEG12A) from Rhizomucor miehei CAU432 was successfully expressed in Pichia pastoris. The highest xyloglucanase activity of 25,700 U mL-1 was secreted using high cell density fermentation. RmXEG12A was optimally active at pH 7.0 and 65 °C, respectively. The xyloglucanase exhibited the highest specific activity towards xyloglucan (7915.5 U mg-1). RmXEG12A was subjected to hydrolyze tamarind powder to produce xyloglucan oligosaccharides with the degree of polymerization (DP) 7-9. The hydrolysis ratio of xyloglucan in tamarind powder was 89.8%. Moreover, xyloglucan oligosaccharides (2.0%, w/w) improved the water holding capacity (WHC) of yoghurt by 1.1-fold and promoted the growth of Lactobacillus bulgaricus and Streptococcus thermophiles by 2.3 and 1.6-fold, respectively. Therefore, a suitable xyloglucanase for tamarind powder hydrolysis was expressed in P. pastoris at high level and xyloglucan oligosaccharides improved the quality of yoghurt.
Asunto(s)
Glucanos/biosíntesis , Glicósido Hidrolasas/metabolismo , Oligosacáridos/biosíntesis , Rhizomucor/enzimología , Saccharomycetales/metabolismo , Xilanos/biosíntesis , Yogur , Estabilidad de Enzimas , Glucanos/aislamiento & purificación , Glicósido Hidrolasas/aislamiento & purificación , Concentración de Iones de Hidrógeno , Hidrólisis , Lactobacillus delbrueckii/crecimiento & desarrollo , Peso Molecular , Oligosacáridos/aislamiento & purificación , Streptococcus/crecimiento & desarrollo , Tamarindus/química , Temperatura , Factores de Tiempo , Xilanos/aislamiento & purificaciónRESUMEN
ß-N-acetylhexosaminidases have attracted much attention in recent years due to their potential application in oligosaccharide production, in particular lacto-N-triose II (LNT2) and lacto-N-neotetraose (LNnT) synthesis, which can be further used as backbone precursors for human milk oligosaccharides. A novel ß-N-acetylhexosaminidase gene from Tyzzerella nexilis (TnHex189) was heterologously expressed in Bacillus subtilis. The highest ß-N-acetylhexosaminidase activity of 14.5 U mL-1 was obtained in a 5-L fermentor by fed-batch fermentation for 27 h. TnHex189 was optimally active at pH 5.0 and 45 °C. It efficiently synthesized LNT2 with a conversion ratio of 57.2% (4.7 g L-1). The synthesized LNT2 was further converted to LNnT by a reported ß-galactosidase (BgaD-D) in 8 h, with a conversion ratio of 17.3% (6.1 g L-1). These unique synthesis activities may make this enzyme a good candidate for the food industry.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clostridiales/enzimología , Trisacáridos/biosíntesis , beta-N-Acetilhexosaminidasas/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Clostridiales/genética , Estabilidad de Enzimas , Fermentación , Expresión Génica , Concentración de Iones de Hidrógeno , Oligosacáridos/metabolismo , beta-N-Acetilhexosaminidasas/química , beta-N-Acetilhexosaminidasas/genéticaRESUMEN
To produce manno-oligosaccharides from cassia gum, a mutated glycoside hydrolase family 134 ß-mannanase gene (mRmMan134A) from Rhizopus microsporus var. rhizopodiformis F518 was expressed in Pichia pastoris and a high expression level (3680â¯Uâ¯mL-1) was obtained through high cell density fermentation. mRmMan134A exhibited maximum activity at pH 5.5 and 50⯰C. It was then subjected to hydrolyze cassia gum with 70.6% of overall yield of manno-oligosaccharides. From the hydrolysate, seven components (F1-F7) were separated and identified as mannose, mannobiose, galactose, mannotriose, mannotetraose, 61-α-d-galactosyl-ß-d-mannobiose, and mannopentaose, respectively. According to in vitro fermentation, the manno-oligosaccharides were able to promote the growth of three Bifidobacterium strains and six Lactobaillus strains with 3.0-fold increment in culture absorbance, and these strains preferred manno-oligosaccharides with degree of polymerization (DP) 2-3 rather than those with DP 4-5. Novel manno-oligosaccharides from cassia gum with promising prebiotic activity were provided in the present study.
Asunto(s)
Cassia/metabolismo , Oligosacáridos/metabolismo , Prebióticos , beta-Manosidasa/metabolismo , Técnicas de Cultivo Celular por Lotes , Bifidobacterium/efectos de los fármacos , Bifidobacterium/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Hidrólisis , Manosa/metabolismo , Oligosacáridos/aislamiento & purificación , Oligosacáridos/farmacología , Rhizopus/enzimología , Temperatura , beta-Manosidasa/genéticaRESUMEN
A novel α-amylase gene (RmAmyA) from Rhizomucor miehei was cloned and expressed in Pichia pastoris. RmAmyA showed 70% amino acid identity with the α-amylase from Rhizomucor pusillus. A high α-amylase activity of 29,794.2â¯U/mL was found through high cell density fermentation. The molecular mass of RmAmyA was determined to be 49.9â¯kDa via SDS-PAGE. RmAmyA was optimally active at 75⯰C and pHâ¯6.0, and it did not require Ca2+ to improve its activity. It exhibited broad substrate specificity towards amylose, amylopectin, soluble starch, pullulan, and cyclodextrins. High level of maltose (54%, w/w) was produced after liquefied starch was hydrolysed with RmAmyA for 16â¯h. Moreover, the addition of RmAmyA into Chinese steamed bread resulted in 7.7% increment in the specific volume, and 17.2% and 11.5% reduction in the chewiness and hardness, respectively. These results indicate that RmAmyA might be a potential candidate for applications in the food industry.
Asunto(s)
Maltosa/metabolismo , Rhizomucor/enzimología , alfa-Amilasas/metabolismo , Pan/análisis , Industria de Alimentos , Concentración de Iones de Hidrógeno , Hidrólisis , Pichia/metabolismo , Almidón/metabolismo , Especificidad por Sustrato , Temperatura , alfa-Amilasas/química , alfa-Amilasas/genéticaRESUMEN
A novel α-amylase gene (TdAmyA) with an open reading frame of 1431â¯bp, deducing 476 amino acids, was cloned from the thermophilic fungus Thermomyces dupontii L18. The recombinant α-amylase was successfully over-expressed in Pichia pastoris. The highest α-amylase activity of 38,314â¯U/mL was obtained with protein content of 28.7â¯mg/mL after 168â¯h high-cell density fermentation. Molecular mass of purified TdAmyA was 61.2â¯kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 59.2â¯kDa by gel filtration. TdAmyA was a glycoprotein with 5.3% (w/w) of carbohydrate. TdAmyA exhibited maximal activity at 60⯰C and pHâ¯6.5, and was thermostable up to 55⯰C within pHâ¯4.5-10.0. It was more active towards linear starchy substrates than branched ones. The hydrolysis products were mainly comprised of maltose and maltotriose. TdAmyA produced the highest maltose content of 51.8% after 8â¯h hydrolysis. Thus, TdAmyA might be a candidate α-amylase for maltose syrup production.
Asunto(s)
Proteínas Fúngicas , Maltosa/química , Pichia , Almidón/química , alfa-Amilasas , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/sangre , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Trisacáridos/química , alfa-Amilasas/biosíntesis , alfa-Amilasas/química , alfa-Amilasas/genéticaRESUMEN
Mannan is one of the major constituent groups of hemicellulose, which is a renewable resource from higher plants. ß-Mannanases are enzymes capable of degrading lignocellulosic biomass. Here, an endo-ß-mannanase from Rhizopus microsporus (RmMan134A) was cloned and expressed. The recombinant RmMan134A showed maximal activity at pHâ¯5.0 and 50⯰C, and exhibited high specific activity towards locust bean gum (2337â¯U/mg). To gain insight into the substrate-binding mechanism of RmMan134A, four complex structures (RmMan134A-M3, RmMan134A-M4, RmMan134A-M5 and RmMan134A-M6) were further solved. These structures showed that there were at least seven subsites (-3 to +4) in the catalytic groove of RmMan134A. Mannose in the -1 subsite hydrogen bonded with His113 and Tyr131, revealing a unique conformation. Lys48 and Val159 formed steric hindrance, which impedes to bond with galactose branches. In addition, the various binding modes of RmMan134A-M5 indicated that subsites -2 to +2 are indispensable during the hydrolytic process. The structure of RmMan134A-M4 showed that mannotetrose only binds at subsites +1 to +4, and RmMan134A could therefore not hydrolyze mannan oligosaccharides with degree of polymerization ≤4. Through rational design, the specific activity and optimal conditions of RmMan134A were significantly improved. The purpose of this paper is to investigate the structure and function of fungal GH family 134 ß-1,4-mannanases, and substrate-binding mechanism of GH family 134 members.
Asunto(s)
Glicósidos/metabolismo , Mananos/metabolismo , Rhizopus/enzimología , beta-Manosidasa/química , beta-Manosidasa/metabolismo , Secuencia de Aminoácidos , Catálisis , Clonación Molecular , Cristalografía por Rayos X , Conformación Proteica , Homología de Secuencia , Especificidad por SustratoRESUMEN
An engineered ß-mannanase (mRmMan5A) from Rhizomucor miehei was successfully expressed in Pichia pastoris. Through high cell density fermentation, the expression level of mRmMan5A reached 79,680â¯Uâ¯mL-1. The mRmMan5A showed maximum activity at pH 4.5 and 65⯰C, and exhibited high specific activities towards mannans. To produce manno-oligosaccharides, palm kernel cake (PKC) was pretreated by steam explosion at 200⯰C for 7.5â¯min, and then hydrolyzed by mRmMan5A. As a result, the total manno-oligosaccharide yield reached 34.8â¯g/100â¯g dry PKC, indicating that 80.6% of total mannan in PKC was hydrolyzed. Moreover, the kilo-scale production of manno-oligosaccharides was carried out to verify the feasibility of mass production. A total of 261.3â¯g manno-oligosaccharides were produced from 1.0â¯kg of dry PKC. An effective ß-mannanase for the bioconversion of mannan-rich biomasses and an efficient method for the production of manno-oligosaccharides from PKC are provided in this paper.
Asunto(s)
Oligosacáridos , Pichia , beta-Manosidasa , Explosiones , Mananos , VaporRESUMEN
Partially hydrolyzed guar gum (PHGG), an important supplemental dietary fiber, has been used as food ingredient in many industries. In this study, a novel ß-mannanase gene (RmMan5A) from Rhizomucor miehei was successfully expressed in Pichia pastoris and subjected for PHGG production. Enzyme activity of fermentation supernatant reached 85,200UmL-1 after 168h high cell density fermentation. The purified RmMan5A exhibited the highest enzyme activity at pH 7.0 and 65°C. RmMan5A was then employed for guar gum hydrolysis and PHGG obtained demonstrated a weight-average molecular weight (Mw) of 2.5×104Da. Total dietary fiber accounted 90.6% of PHGG and 24.9% (w/w) of PHGG were identified as manno-oligosaccharides with degree of polymerization<7. PHGG was further fractionated (F1-F4) by gradual ethanol precipitation. PHGG F1 with an Mw value of 3.6×104Da and a mannose/galactose (M/G) ratio of 1.47 was precipitated initially, followed by PHGG F2 and F3 which showed lower Mw and higher M/G ratio. According to the structure analysis, the distribution of α-d-galactose of PHGG F1 was compact and regular, and that of other fractions was more random. A suitable ß-mannanase for PHGG production and some useful information of PHGG are provided in this paper.
Asunto(s)
Galactanos/biosíntesis , Mananos/biosíntesis , Pichia/genética , Gomas de Plantas/biosíntesis , Rhizomucor/enzimología , beta-Manosidasa/genética , beta-Manosidasa/metabolismo , Fermentación , Galactanos/química , Expresión Génica , Concentración de Iones de Hidrógeno , Hidrólisis , Mananos/química , Peso Molecular , Gomas de Plantas/química , Rhizomucor/genética , TemperaturaRESUMEN
BACKGROUND: ß-Mannanase randomly cleaves the ß-1,4-linked mannan backbone of hemicellulose, which plays the most important role in the enzymatic degradation of mannan. Although the industrial applications of ß-mannanase have tremendously expanded in recent years, the wild-type ß-mannanases are still defective for some industries. The glycoside hydrolase (GH) family 5 ß-mannanase (RmMan5A) from Rhizomucor miehei shows many outstanding properties, such as high specific activity and hydrolysis property. However, owing to the low catalytic activity in acidic and thermophilic conditions, the application of RmMan5A to the biorefinery of mannan biomasses is severely limited. RESULTS: To overcome the limitation, RmMan5A was successfully engineered by directed evolution. Through two rounds of screening, a mutated ß-mannanase (mRmMan5A) with high catalytic activity in acidic and thermophilic conditions was obtained, and then characterized. The mutant displayed maximal activity at pH 4.5 and 65 °C, corresponding to acidic shift of 2.5 units in optimal pH and increase by 10 °C in optimal temperature. The catalytic efficiencies (kcat/Km) of mRmMan5A towards many mannan substrates were enhanced more than threefold in acidic and thermophilic conditions. Meanwhile, the high specific activity and excellent hydrolysis property of RmMan5A were inherited by the mutant mRmMan5A after directed evolution. According to the result of sequence analysis, three amino acid residues were substituted in mRmMan5A, namely Tyr233His, Lys264Met, and Asn343Ser. To identify the function of each substitution, four site-directed mutations (Tyr233His, Lys264Met, Asn343Ser, and Tyr233His/Lys264Met) were subsequently generated, and the substitutions at Tyr233 and Lys264 were found to be the main reason for the changes of mRmMan5A. CONCLUSIONS: Through directed evolution of RmMan5A, two key amino acid residues that controlled its catalytic efficiency under acidic and thermophilic conditions were identified. Information about the structure-function relationship of GH family 5 ß-mannanase was acquired, which could be used for modifying ß-mannanases to enhance the feasibility in industrial application, especially in biorefinery process. This is the first report on a ß-mannanase from zygomycete engineered by directed evolution.
RESUMEN
A novel endo-ß-1,3-1,4-glucanase gene (PbBglu16A) was cloned from Paenibacillus barengoltzii and heterogeneously expressed in Escherichia coli. The recombinant ß-1,3-1,4-glucanase (PbBglu16A) was purified to homogeneity with a recovery yield of 78.6% and a specific activity of 431.8Umg-1. The molecular mass of PbBglu16A was estimated to be 44.0kDa by SDS-PAGE. The optimal pH and temperature of PbBglu16A were 6.0 and 55°C, respectively. The enzyme was stable within pH 3.5-9.0 and up to 55°C. PbBglu16A exhibited high substrate specificity towards barley ß-glucan, oat ß-glucan and lichenin. PbBglu16A showed an endo-type cleavage pattern and hydrolyzed endogenous enzyme-deactivated oat bran into ß-gluco-oligosaccharides with a yield of 7.0%, which mainly consisted of trioligosaccharide and tetraoligosaccharide. Further, PbBglu16A could promote mashing with a reduced filtration time (14.0%) and viscosity (3.4%). Thus, PbBglu16A might be a promising candidate for the production of ß-gluco-oligosaccharides and in brewing industry.
Asunto(s)
Endo-1,3(4)-beta-Glucanasa/química , Paenibacillus/enzimología , beta-Glucanos/metabolismo , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Escherichia coli , Concentración de Iones de Hidrógeno , Especificidad por SustratoRESUMEN
In this study, a comparative analysis on the distribution of protease activities among 90 plant resources, including fruits and vegetables, has been performed. Protease activities of plant extracts were assayed at different pH values (pH 3.0, pH 7.5 and pH 10.5) using casein as a substrate. Ten fruits and thirteen vegetables show protease activities above 10U/g. Pineapple, fig and papaya, which are used for commercial protease production, exhibited high protease activities. Additionally, high protease activities were detected in kiwifruit (28.8U/g), broccoli (16.9U/g), ginger (16.6U/g), leek (32.7U/g) and red pepper (15.8U/g) at different pH values. SDS-PAGE and zymograms confirmed that various types of proteases existed in the five plant extracts and might be explored. Furthermore, five plant extracts were treated by different protease inhibitors. These results show that there are still many plant resources unexplored, which may be promising candidates for plant-derived protease production.
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Frutas/enzimología , Péptido Hidrolasas/metabolismo , Verduras/enzimología , Actinidia/enzimología , Brassica/enzimología , Capsicum/enzimología , Caseínas/metabolismo , Electroforesis en Gel de Poliacrilamida , Zingiber officinale/enzimología , Concentración de Iones de Hidrógeno , Cebollas/enzimología , Extractos Vegetales/química , Inhibidores de Proteasas/metabolismoRESUMEN
Bacillus subtilis LM 4-2, a Gram-positive bacterium was isolated from a molybdenum mine in Luoyang city. Due to its strong resistance to molybdate and potential utilization in bioremediation of molybdate-polluted area, we describe the features of this organism, as well as its complete genome sequence and annotation. The genome was composed of a circular 4,069,266 bp chromosome with average GC content of 43.83 %, which included 4149 predicted ORFs and 116 RNA genes. Additionally, 687 transporter-coding and 116 redox protein-coding genes were identified in the strain LM 4-2 genome.
RESUMEN
Multiple proteases were optimized to hydrolyze the rice residue protein (RRP) to produce novel antioxidant peptides. An antioxidant peptide fraction (RRPB3) with IC50 of 0.25 mg/ml was purified from the RRP hydrolysate using membrane ultrafiltration followed by size exclusion chromatography and reversed-phase FPLC. RRPB3 was found to include four peptides (RRPB3 I-IV) and their amino acid sequences were RPNYTDA (835.9 Da), TSQLLSDQ (891.0 Da), TRTGDPFF (940.0 Da) and NFHPQ (641.7 Da), respectively. Furthermore, four peptides were chemically synthesized and their antioxidant activities were assessed by DPPH radical scavenging, ABTS radical scavenging assay and FRAP-Fe(3+) reducing assay, respectively. Both RRPB3 I and III showed synergistic antioxidant activity compared to each of them used alone. All four synthetic peptides showed excellent stability against simulated gastrointestinal proteases. Therefore, the peptides isolated from RRP may be used as potential antioxidants in the food and drug industries.
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Antioxidantes/química , Oryza/metabolismo , Péptido Hidrolasas/química , Péptidos/química , Hidrólisis , Oxidación-ReducciónRESUMEN
Purification and characterization of a chymosin from Rhizopus microsporus var. rhizopodiformis were investigated in the present study. A newly isolated R. microsporus var. rhizopodiformis F518 produced a high level of milk-clotting activity (1,001 SU/mL). A chymosin from the fungus was purified 3.66-fold with a recovery yield of 33.2 %. The enzyme appeared as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 37.0 kDa. It was optimally active at 60 °C and was stable up to 40 °C. The purified enzyme was an acid protease with an optimum pH of 5.2 and retained 80 % of residual activity within pH 2.0-8.0. The inhibition of 96 and 100 % by pepstatin A at 0.01 and 0.02 mM, respectively, revealed that the enzyme is an aspartic protease. Thus, high milk-clotting activity of the chymosin with good stability will strengthen the potential use of the chymosin as a substitute for calf rennet in cheese manufacturing.
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Quimosina/química , Quimosina/aislamiento & purificación , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Rhizopus/enzimología , Estabilidad de Enzimas , Calor , Concentración de Iones de HidrógenoRESUMEN
The family 10 xylanase (XynB) from Thermotoga maritima MSB8 is extremely thermostable and great potential in the applications of various fields of industry. The gene mxynB(64) was amplified by the method of PCR with the template of the genomic DNA of Thermotoga maritima MSB8, and cloned into the expression vectors of Escherichia coli and Pichia pastoris respectively. Xylanase B(40kD) was successfully expressed by the two heterologous protein expression systems with high-level production. The recombinant protein of XynB expressed in Pichia pastoris showed extreme thermostability and pH stability, which was optimally active at 90 degrees C and quite stable over the pH range of pH 5.0-10.8 at 70 degrees C. After incubation of the enzyme at 100 degrees C for 30 min, XynB retained 70% higher residual activity. The recombinant XynB expressed in Pichia pastoris is of great use in a variety of industrial and agricultural applications.