A preliminary study of the protease activities in germinating brown rice (Oryza sativa L.)

BACKGROUND: Proteases hydrolyse storage proteins to provide precursors for perpetuating species. The aim of this study was to investigate and characterise different proteases in germinating brown rice. RESULTS: The protease activity of brown rice increased sevenfold during 7 days of germination. It was highest on day 6 when determined at pH 3.5. With casein as substrate the proteases showed two catalytic groups: acidic proteases with an optimal pH of 3.5 and alkaline proteases with an optimal pH of 8.0. The acidic protease activity was inhibited by Ba²⁺ and Pb²⁺ but stimulated by Zn²⁺, while the alkaline protease activity was inhibited by Ca²⁺ and Pb²⁺ but stimulated by Mg²⁺ and Zn²⁺. SDS‐gelatin‐PAGE assay showed two protease activity bands at pH 3.5, while two different bands with higher molecular weights were observed at pH 8.0. Inhibition assay revealed that pepstatin A and E‐64 inhibited 67.63 and 38.26% respectively of the protease activity at pH 3.5, indicating the presence of aspartic and cysteine proteases. Metalloproteases played a major role under alkaline conditions (88.37% inhibition with EDTA). CONCLUSION: Germinated brown rice proteases fall into different classes with different properties. This study is helpful for their further purification. Copyright © 2010 Society of Chemical Industry     

Purification and Characterization of a Halotolerant Alkaline Serine Protease from Penicillium citrinum YL-1 Isolated from Traditional Chinese Fish Sauce

A halotolerant alkaline serine protease from Penicillium citrinum YL-1 which was isolated from traditional Chinese fish sauce was purified by ammonium sulfate precipitation, dialysis, and DEAE 52-Cellulose column, thereby resulting in a 4.66-fold increase in specific activity (110.68 U/mg). The molecular weight (MW) was estimated to be 32.27 kDa using SDS-PAGE analysis. The protease exhibited optimal activity toward the substrate casein at pH 8.0 at 40°C and was stable at pH 6.0–8.0 and 4–30°C. Activity was inhibited by NaCl and retained at 28.3, 21.4 and 18.1% of the initial activity after incubation for 6 h at 20, 25 and 30% NaCl concentrations, respectively. The enzyme was stimulated by Mn²⁺ and inhibited by K⁺, Ca²⁺, Zn²⁺, Mg²⁺, Fe²⁺, and Fe³⁺. K_m and V_max of the protease for casein were 1.93 mg/ml and 56.81 μg/(min·ml), respectively. Protease activity was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), which confirmed the serine protease nature of the enzyme. The protease can hydrolyze tilapia protein in the absence or presence of NaCl (5–30%), thus suggesting that this protease is more halotolerant than the protease from other bacteria with high salinity resistance based on the current literature. These properties make the halotolerant alkaline serine protease a suitable candidate enzyme for fish protein hydrolysis during fish sauce fermentation.     

Production of a potential collagenolytic protease by nejayote fermentation with Aspergillus oryzae

Nejayote is the wastewater obtained from maize lime cooking. It was used for Aspergillus oryzae growth and enzyme production. Protein production and collagenolytic activity were evaluated to test the effects of pH, spore inoculum and nejayote content in a 3³ factorial design. No collagenolytic activity was observed in the supernatant of the fermentation without nejayote. Using nejayote at 5% increased 1.5 times the fungus growth in comparison with nejayote 1% and 3%; and the maximum collagenolytic activity was reached after 3 days at pH 8 (P < 0.05). After sequencing, a 32 kDa alkaline protease was identified. Along with the alkaline conditions produced by the inclusion of nejayote solids, this waste is an important source of carbohydrates that improved the growth of A. oryzae to produce a protease that has the potential to break collagen. This permits a prospective revalorisation of a waste material to get a product with multiple biotechnological uses.     

微生物碱性蛋白酶研究进展

本文主要综述了微生物碱性蛋白酶在国内外的应用研究和现状,对微生物碱性蛋白酶的产生菌,碱性蛋白酶的应用、基因结构及性质,高产工程茵的选育技术等方面的研究进展进行了概述,并根据当前应用研究状况对微生物碱性蛋白酶在今后的发展进行了展望.     

Improving the performance of industrial ethanol‐producing yeast by expressing the aspartyl protease on the cell surface

The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell‐surface anchored expressed in industrial ethanol‐producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild‐type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98–99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild‐type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity. Copyright © 2010 John Wiley & Sons, Ltd.     

Vacuolar and extracellular maturation of Saccharomyces cerevisiae proteinase A

The vacuolar aspartyl protease proteinase A (PrA) of Saccharomyces cerevisiae is encoded as a preproenzyme by the PEP4 gene and transported to the vacuole via the secretory route. Upon arrival of the proenzyme proPrA to the vacuole, active mature 42 kDa PrA is generated by specific proteolysis involving the vacuolar endoprotease proteinase B (PrB). Vacuolar activation of proPrA can also take place in mutants lacking PrB activity (prb1). Here an active 43 kDa species termed pseudoPrA is formed, probably by an autocatalytic process. When the PEP4 gene is overexpressed in wild-type cells, mature PrA can be found in the growth medium. We have found that prb1 strains overexpressing PEP4 can form pseudoPrA extracellularly. N-terminal amino acid sequence determination of extracellular, as well as vacuolar pseudoPrA showed that it contains nine amino acids of the propeptide, indicating a cleavage between Phe67 and Ser68 of the preproenzyme. This cleavage site is in accordance with the known substrate preference for PrA, supporting the notion that pseudoPrA is formed by autoactivation. When a multicopy PEP4 transformant of a prb1 mutant was grown in the presence of the aspartyl protease inhibitor pepstatin A, a significant level of proPrA was found in the growth medium. Our analyses show that overexpression of PEP4 leads to the secretion of proPrA to the growth medium where the zymogen is converted to pseudoPrA or mature PrA in a manner similar to the vacuolar processing reactions. Amino acid sequencing of secreted proPrA confirmed the predicted cleavage by signal peptidase between Ala22 and Lys23 of the preproenzyme.     

EXTRACELLULAR PROTEASE ACTIVITY IN A STRAIN OF SACCHAROMYCES CEREVISIAE

The isolation and analysis of a strain of laboratory yeast with extracellular protease activity is described. The proteolytic activity found in culture supernatants exceeded the parental strain by at least an order of magnitude and apparently was due neither to cell lysis nor to increased cell wall permeability. The extracellular proteases were heterogeneous in composition, consisting of possibly 3 aberrantly secreted intracellular proteinases. This extracellular protease activity was conferred by a single recessive mutation (epr1.1) in a gene displaying classical Mendelian inheritance. EPR1 was tentatively assigned to chromosome XV with loose linkage to the HIS3 gene. Strains carrying the mutant epr1.1) allele also possessed increased levels of secreted invertase activity and it is proposed that EPR1 is closely involved in the intracellular protein translocation pathway of yeast.     

Characterization of a novel protease from Bacillus cereus and evaluation of an eco‐friendly hydrolysis of a brewery byproduct

Proteases and proteolytic enzymes constitute one of the most important groups of enzymes and are attracting worldwide attention in attempts to exploit their physiological and biotechnological applications. In this study, partial purifications and biochemical and antimicrobial characterizations of a protease from Bacillus cereus spp., originally isolated from fermented cabbage, were carried out. The crude extract obtained after purification, involving ammonium sulphate precipitation and dialysis, was designated as a partially purified protease (PPP). The obtained PPP had a specific activity of 0.395–2.539 U/g at 32 °C, with maximum activities for the fractions precipitated at 60 and 80% ammonium sulphate. The PPP activity ranged between 20 and 55 °C, with an optimum temperature at 40 °C. At 60 °C, the PPP retained more than 30% of its activity. The optimum pH for the PPP was achieved at pH 9, indicating the alkaline source of the enzyme. Protease production was specifically dependent on the calcium concentration in the culture medium. Also the robustness of the protease on brewer's spent grain hydrolysis was demonstrated. This suggests a potential eco‐friendly application of the enzyme. Finally, it was found that the PPP inhibited the growth of Escherichia coli O157:H7. This novel property of the PPP liberated by the B. cereus spp. could provide important future benefits to industry. Copyright © 2015 The Institute of Brewing & Distilling     

YEAST EXTRACELLULAR PROTEOLYTIC ENZYMES FOR CHILL-PROOFING BEER

Yeast belonging to various genera were screened for extracellular protease activity. Of the 119 strains examined the most prolific protease producers belonged to the genera Candida, Kluyveromyces and Hansenula. No strains of Sacharomyces were found to secrete protease. In general only those proteases secreted by the genus Candida possessed chill-proofing activity. However one strain of Debaryomyces and one of Pichia also produced proteases with chill-proofing activity.     

Actinomucor elegans、Aspegillus oryzae和Rhizopus oligosporus产蛋白酶条件及蛋白酶性质的比较

比较了腐乳生产菌株Actinomucor elegans、豆酱和酱油生产菌株Aspegillus oryzae以及天培生产菌株Rhizopus oligosporus产生蛋白酶的条件和所产蛋白酶的性质。结果表明,不同的菌株产酶条件及蛋白酶的性质有较大的差异:少孢根霉主要产生酸性蛋白酶,在pH2.5-4.0的酸性介质中、32℃条件下培养时产酶能力较强,所分泌的蛋白酶系在pH5.0时酶活力最高,在pH5.0附近最稳定;米曲霉可以产生酸性、中性及碱性蛋白酶,所产生的蛋白酶活力显著高于少孢根霉和毛霉,米曲霉在酸性条件下产酸性蛋白酶能力强,在中性条件下产中性蛋白酶能力强,在碱性条件下产碱性蛋白酶能力强,在28-32℃时产酶能力强,所分泌的蛋白酶系在pH5.0-9.0的广泛范围内有很强的活力,在pH6.0-8.0的范围内稳定性强;毛霉可以产生酸性、中性及碱性蛋白酶,但酶活力明显低于米曲霉,毛霉在中性偏酸性(pH5.5)的介质中产酸性蛋白酶的能力较强,但介质的酸碱度对毛霉产中性及碱性蛋白酶没有影响,在28℃时产酸性、中性和碱性蛋白酶的能力都比较强,毛霉所分泌的蛋白酶系在pH5.0-9.0的广泛pH范围内有活力,在pH5.0-6.0时酶活力最高,在pH5.0-7.0时稳定强。     

高温变性豆粕发酵菌株的选育

以米曲霉CICC40186为出发菌株,经紫外诱变处理后,获得对高温变性豆粕分解能力强的突变菌株8421和8422。与出发菌株相比,两突变株所产酸性、中性和碱性三种蛋白酶分别提高了73.2%、91.2%,81.1%、83.7%,37.6%、32.9%;三氯乙酸(TCA)可溶性蛋白含量分别提高了约13.0%和13.2%。经6次传代,遗传性状稳定。     

Proteolysis characteristics of Actinomucor elegans and Rhizopus oligosporus extracellular proteases under acidic conditions

Enzymatic hydrolysis of soybean protein isolate by the extracellular proteases from Actinomucor elegans and Rhizopus oligosporus at pH 3.0, 3.5, 5.0, 5.5 and 6.0 was investigated. The activity of the A. elegans protease is lower than that of R. oligosporus, but both proteases exhibit considerable degradation of soybean protein at pH 5.5 and 6.0. The water‐soluble protein content and the degree of hydrolysis of the hydrolysates are increased significantly, and bitterness values are very low. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) reveals that these proteases have different cutting sites on peptide polymers. At pH 5.5, there is a lower content of total free amino acids (39.20 mg per 100 mL; 62.68% hydrophobic amino acids) in the R. oligosporus protease hydrolysate. In conclusion, treatment with R. oligosporus protease at pH 5.5 achieves efficient degradation of soybean protein, suggesting a promising industrial process for making bitterless protein hydrolysates.     

Silk Degumming and Utilization of Silk Sericin by Hydrolysis Using Alkaline Protease from Beauveria Sp. (MTCC 5184): A Green Approach

Conventionally, degumming is carried out at 90°C--110°C temperature by boiling the raw silk with Marseilles soap and sodium bicarbonate which eventually requires a lot of water and energy. In this study, degumming of Chinese bivoltine raw silk fibres with alkaline protease produced by Beauveria sp. (MTCC 5184) is studied. Complete degumming was obtained in 45 min with 75 units of enzyme per gram of silk. Degumming was found to be optimal at 50°C and pH 9.0. Scanning electron micrographs showed that the sericin deposits were removed and the obtained fibres were clean, separated, had smooth feel with shine as compared to untreated fibres. Sericin isolated from silk cocoon (by-product which goes waste) was hydrolyzed with the same alkaline protease obtained from Beauveria sp. to get small molecular weight peptides. These peptides can be utilized further for cosmetic, pharmaceutical, and various industrial applications.     

Low molecular weight serine protease from the viscera of sardinelle (Sardinella aurita) with collagenolytic activity: Purification and characterisation

A new low molecular weight (LMW) serine-protease from sardinelle (Sardinella aurita) viscera was purified using ammonium sulphate precipitation and Sephadex G-100 gel filtration, with a 3.82-fold increase in specific activity. The molecular weight of the enzyme was estimated to be 14.2 kDa by SDS-PAGE. The optimum pH and temperature for the enzyme activity were around pH 8.0 and 60 °C, respectively. The purified protease was strongly inhibited by phenylmethylsulphonyl fluoride, a serine-protease inhibitor, and soybean trypsin inhibitor. The N-terminal amino acid sequence of the first 10 amino acids of the purified protease was APVQPCVVVI. This sequence showed low homology with several peptidases, suggesting that the enzyme is a new protease. Interestingly, the protease was found to cleave collagen type I and hydrolyze succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpna), an amide substrate of chymotrypsin. Our findings indicate that the S. aurita protease is a new LMW enzyme with collagenolytic activity.     

Secretion and pH-dependent self-processing of the pro-form of the Yarrowia lipolytica acid extracellular protease

The secretion and maturation of the acid extracellular protease (AXP) of the yeast Yarrowia lipolytica have been characterized using antiserum raised against this enzyme. A 42 kDa pro-enzyme form of AXP was identified from lysates of radiolabelled Y. lipolytica cells and found to contain no N-linked carbohydrate moieties. Using pulse-chase immune precipitation it was demonstrated that the AXP precursor was secreted into the extracellular medium where, under conditions of low pH, it underwent autocatalytic activation forming the mature enzyme. Conversion of the AXP pro-form in the presence of the protease inhibitor pepstatin indicated that an intramolecularly-catalysed reaction mechanism was involved in AXP maturation. Further evidence supporting the role of autocatalytic processing came from the side-chain specificity of mature AXP towards the oxidized B-chain of insulin. © 1998 John Wiley & Sons, Ltd.     

Assessment of the production of Bacillus cereus protease and its effect on the quality of ultra-high temperature-sterilized whole milk

Bacillus cereus is one of the most important spoilage microorganisms in milk. The heat-resistant protease produced is the main factor that causes rotten, bitter off-flavors and age gelation during the shelf-life of milk. In this study, 55 strains of B. cereus were evaluated, of which 25 strains with protease production ability were used to investigate proteolytic activity and protease heat resistance. The results showed that B. cereus C58 had strong protease activity, and its protease also had the highest thermal stability after heat treatment of 70°C (30 min) and 100°C (10 min). The protease was identified as protease HhoA, with a molecular mass of 43.907 kDa. The protease activity of B. cereus C58 in UHT-sterilized whole milk (UHT milk) showed an increase with the growth of bacteria, especially during the logarithmic growth phase. In addition, the UHT milk incubated with protease from B. cereus C58 at 28°C (24 h) and 10°C (6 d) were used to evaluate the effects of protease on the quality of UHT milk, including protein hydrolysis and physical stability. The results showed that the hydrolysis of casein was κ-CN, β-CN, and α_S-CN successively, whereas whey protein was not hydrolyzed. The degree of protein hydrolysis, viscosity, and particle size of the UHT milk increased. The changes in protein and fat contents indicated that fat globules floated at 28°C and settled at 10°C, respectively. Meanwhile, confocal laser scanning microscopy images revealed that the protease caused the stability of UHT milk to decrease, thus forming age gelation.     

Partial purification and characterisation of cysteine protease in wheat germ

BACKGROUND: Proteases have become an essential part of the modern food and feed industry, being incorporated in a large and diversified range of products for human and animal consumption. The objective of this study was to purify and characterise a protease from wheat germ. RESULTS: After purification a single protease of molecular weight 61–63 kDa (determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis) was obtained. The purified protease had optimal activity at 50 °C and maintained its activity completely after incubation at 30 °C for 30 min, while over 47% of the activity was lost after incubation at 80 °C for 30 min. The purified protease had optimal activity and maintained maximum stability at pH 5.5, while the activity decreased after incubation for 30 min at other pH values. The protease was inhibited by Mg²⁺, Mn²⁺, Ba²⁺ and iodacetic acid and stimulated by Li⁺, Ca²⁺, Cu²⁺, β‐mercaptoethanol and dithiothreitol, while Zn²⁺, L ‐cysteine and glutathione had no significant effect on its activity. At pH 5.5 the enzyme had a K_m of 0.562 mg mL^?1 with casein as substrate and showed higher affinity to casein than to bovine serum albumin, ovalbumin and gelatin. CONCLUSION: The purified enzyme from wheat germ was identified as a cysteine protease. Copyright © 2011 Society of Chemical Industry     

Impact of buffering capacity on the acidification of wort by brewing‐relevant lactic acid bacteria

Acidified wort produced biologically using lactic acid bacteria (LAB) has application during sour beer production and in breweries adhering to the German purity law (Reinheitsgebot ). LAB cultures, however, suffer from end product inhibition and low pH, leading to inefficient lactic acid (LA) yields. Three brewing‐relevant LAB (Pediococcus acidilactici AB39, Lactobacillus amylovorus FST2.11 and Lactobacillus plantarum FST1.7) were examined during batch fermentation of wort possessing increasing buffering capacities (BC). Bacterial growth was progressively impaired when exposed to higher LA concentrations, ceasing in the pH range of 2.9–3.4. The proteolytic rest (50°C) during mashing was found to be a major factor improving the BC of wort. Both a longer mashing profile and the addition of an external protease increased the BC (1.21 and 1.24, respectively) compared with a control wort (1.18), and a positive, linear correlation (R ² = 0.957) between free amino nitrogen and BC was established. Higher levels of BC led to significant greater LA concentration (up to +24%) after 48 h of fermentation, reaching a maximal value of 11.3 g/L. Even higher LA (maximum 12.8 g/L) could be obtained when external buffers were added to wort, while depletion of micronutrient(s) (monosaccharides, amino acids and/or other unidentified compounds) was suggested as the cause of LAB growth cessation. Overall, a significant improvement in LA production during batch fermentation of wort is possible when BC is improved through mashing and/or inclusion of additives (protease and/or external buffers), with further potential for optimization when strain‐dependent nutritional requirements, e.g. sugar and amino acids, are considered. Copyright © 2017 The Institute of Brewing & Distilling     

地衣芽孢杆菌2709 蛋白酶分离纯化研究

对产自地衣芽孢杆菌(B. licheniformis)2709 的碱性蛋白酶通过乙醇沉淀、盐析、DEAE 阴离子交换层析、凝胶层析4 步纯化,最终获得电泳纯的酶。以去酰胺度及酶比活力为指标,对碱性蛋白酶分离纯化条件进行优化。结果发现：提纯酶的比活力达61069U/mg,纯化倍数为38.7,活性回收率为19.3%,去酰胺度为20.9%。并研究该酶的基本酶学特性,结果发现：该酶最适作用pH 值为10.0,最适反应温度为50℃。40℃保温2h 后该酶保持80% 以上的活力,在pH8～11 之间有较高的pH 值稳定性。     

Purification and characterisation of an acid protease from the Aspergillus hennebergii HX08 and its potential in traditional fermentation

A protease AP3 from Aspergillus hennebergii HX08 was purified by ammonium‐sulphate precipitation, followed by anion‐exchange chromatography and gel filtration. The molecular weight of acid protease AP3 was 33 kDa (SDS‐PAGE and MALDI‐TOF‐MS). The protease AP3 was identified as an acid protease with MALDI‐TOF/TOF tandem MS. Its optimal temperature and pH were 60°C and 4.0, respectively. Its K _max and V _max were 57.92 mg/mL and 32.57 U/mL, respectively. The enzyme was active over a broad pH and temperature range (pH 3.0–5.0 and 30–60°C), and exhibited high activity and stability in 2–12% (v /v) ethanol solvent. Subsequent studies suggest that the enzyme presents a relatively high substrate affinity to wheat protein (98% of total activity). Its application to solid‐state fermentation of wheat flour with Saccharomyces cerevisiae could increase the hydrolysis degree of wheat protein (28.26%) and amino acid nitrogen concentration of fermented grains (34.21%). Additionally, enhanced S. cerevisiae biomass (37.09%) and alcohol concentration (38.29%) were also observed during the process. Volatile compounds analysis of fermented grains by headspace solid‐phase micro‐extraction and GC‐MS revealed more flavour compounds. These results suggest its potential in food and alcohol industries. Copyright © 2017 The Institute of Brewing & Distilling