Purification and Characterization of a Trypsin‐Like Protease from Flatfish (Paralichthys olivaceus) Intestine

A trypsin‐like protease was purified from the intestine of flatfish (Paralichthys olivaceus) by gel filtration and anion‐exchange chromatography. The molecular weight was estimated to be 29.6 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Flatfish protease had maximal activity at 70C and pH 7.5 using N‐α‐benzoyl‐dl ‐arginine‐ρ‐nitroanilide as substrate. It was stable to heat treatment up to 50C and to pH ranges between 7.0 and 10.0. It was activated by calcium ion and completely inhibited by mercury ion and known serine‐protease inhibitors, such as phenylmethylsulfonyl fluoride, tosyl lysine chloromethyl ketone and benzamidine.     

PURIFICATION AND CHARACTERIZATION OF BACILLUS SUBTILIS JM-3 PROTEASE FROM ANCHOVY SAUCE

Bacillus subtilis JM‐3 was isolated from anchovy sauce naturally fermented in an underground cellar at 15 ± 3C for 3 years. The activity of the B. subtilis protease was highest in the 40–60% ammonium sulfate fraction. The yield of the purified protease was 5.3%, and its purification ratio was 35.6 folds. The molecular weight of the B. subtilis protease was 17.1 kDa, and its K_m and V_maxvalues were 1.75 μg/mL and 318 μM 1/min, respectively. The optimal temperature for protease activity was 60C, but optimal stability temperature was 30C. The optimal pH for protease activity and stability was 5.5. Therefore, the B. subtilis JM‐3 protease was classified as an acid protease. The relative activities of the B. subtilis JM‐3 protease were 69, 21 and 1.3% at 10, 20 and 30% NaCl concentrations, respectively. The best substrate for the B. subtilis JM‐3 protease was benzyloxycarbonyl‐glycine‐p‐nitrophenyl ester followed by bovine serum albumin. p‐Toluene‐sulfonyl‐L‐lysine chloromethylketone was the strongest inhibitor followed by soybean trypsin inhibitor, but N‐ethylmaleimide did not inhibit this enzyme. The B. subtilis JM‐3 protease was therefore presumed to be a trypsin‐like serine protease.     

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     

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.     

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.     

Characterisation of wines and distilled spirits from melon (Cucumis melo L.)

Melon fermentation and distillation was studied with a view to produce melon spirits. Three different substrates were obtained from melon (Cucumis melo L.). Sugar concentration was around 60–70 g L^?1 and the pH between 4.6 and 5.2. The substrates were clarified and then fermented at one of two pH levels at 20 °C. A commercial Saccharomyces cerevisiae starter culture was used to obtain melon wines with an alcohol concentration of 3.8–5.8% (v/v). The melon wines were distilled in a distillation column to yield distillates with an alcohol content of 33–60% (v/v). The major volatiles in the melon wines and in the distillates were analysed by gas chromatography (GC). The results demonstrated that melon could be a good substrate of fermentation and distillation but also yielded significant differences in the volatiles analysed in the different melon wines and distillates obtained from the different substrates in the different conditions of the experiment. Fermentation pH greatly affected the methanol, acetaldehyde, and butanol contents and thus the final quality of the spirits produced.     

Coupled Neutrase–Catalyzed Plastein Reaction Mediated the ACE-Inhibitory Activity In Vitro of Casein Hydrolysates Prepared by Alcalase

Casein hydrolysates with a degree of hydrolysis of 13.5% were prepared by hydrolyzing casein with an alkaline protease Alcalase, and showed ACE-inhibition in vitro with an IC₅₀ value of 45.2 μg/mL. The hydrolysates were modified by plastein reaction catalyzed by a neutral protease Neutrase to reveal the impact of the coupled Neutrase-catalyzed plastein reaction on the ACE-inhibition of the casein hydrolysates. The effects of addition level of Neutrase, substrate concentration, reaction temperature, and time on the plastein reaction of the casein hydrolysates were studied with the varying amount of free amino groups of the modified hydrolysates as index. The results illustrated that the amount of free amino groups of the modified hydrolysates increased in all occasions, and the addition level of Neutrase, substrate concentration, and reaction time had a clear impact on the plastein reaction. Six modified hydrolysates were prepared at a substrate concentration of 40% (by weight), Neutrase addition level of 3 kU/g peptides, reaction temperature of 35°C, and different reaction time. The assay results highlighted that the coupled Neutrase-catalyzed plastein reaction improved the ACE-inhibition of six modified hydrolysates with IC₅₀ values ranging from 15.6 to 20.0 μg/mL. Size exclusion chromatography analysis showed that some plasteins with a molecular weight of about 68 kDa existed in the modified hydrolysates. The results also demonstrated that it was the coupled Neutrase-catalyzed plastein reaction but not further hydrolysis of casein hydrolysates that enhanced the ACE-inhibition of the modified casein hydrolysates.     

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     

CHARACTERIZATION OF AN 'ENDOPOLYGALACTURONASE' OF THE YEAST KLUYVEROMYCES MARXIANUS

Analytic isoelectric focusing showed that the ‘endopolygalacturonase’ from Kluyveromyces marxianus consists of 21 multiple enzyme components. Neither changes in cultivation conditions, enzyme substrate or reaction conditions resulted in any quantitative or qualitative differences in the enzyme patterns. Two of these multiple enzyme forms, the main band, (IP = pH 5.8) which accounted for approximately 95% of the total activity, and an ‘acid-band’ (IP = pH 2.7), were purified by means of preparative isoelectric focusing and their molecular weights and amino acid compositions were determined. The molecular weight of the main band was established as 76,000 daltons (2 subunits of 47,900 and 28,100), The molecular weight of the ‘acid-band’ was 32,200. Both amino acid analyses and molecular weight determinations suggested that the proteins are chemically and physically different. The pH optimum was 4 for the pure enzyme on different pectin substrates, e.g., high molecular weight pectic acid, low molecular weight pectic acid and highly methylated pectins B and C. The temperature optimum obtained for pure enzyme with high or low molecular-weight pectic acid as substrate was 40° C. V_max and Km-value determination at different pH values with low molecular weight pectic acid as a substrate was used to identify the catalytically active groups at the active site. They were tentatively identified as an unprotonated α-carboxyl group and a protonated carboxyl group of aspartic acid.     

Impact of the fungal protease produced by Fusarium culmorum on the protein quality and breadmaking properties of winter wheat

Samples artificially infected by Fusarium culmorum were analyzed to assess the effects of Fusarium infection on the protein quality of winter wheat (Triticum aestivum L.). The Fusarium infection did not noticeably influence either the crude protein content or the water absorption ability of the wheat flour. The protease activity found in the wheat flour was inversely correlated to the sedimentation value. In contrast, it was positively correlated to both the free amino acid content and the degree of infection as expressed in Fusarium protein equivalents (FPE) quantified by an enzyme-linked immunosorbent assay. A distinct reduction in the content of both total glutenin and high molecular weight glutenin subunits was detected in the seriously infected samples (FPE>20 µg g^–1). The infection with Fusarium impaired the dough quality and led to a deformed loaf shape. The fungal protease was active over both a wide range of temperatures (from 10 to 100 °C) and a wide range of pH values (from 4.5 to 8.5). The maximum protease activity was displayed at 50 °C in the pH range 6.0–8.0. This property indicates that the protease produced by F. culmorum may impair storage proteins throughout all the processing procedures associated with wheat flour, thereby causing weak dough properties and, consequently, unsatisfactory bread quality.     

Purification and properties of cysteine protease from rhizomes of Curcuma longa (Linn.)

BACKGROUND: Turmeric rhizome (Curcuma domestica Linn.) contains proteases and has proteolytic activity. Curcumin from turmeric rhizomes has been used for healing manu ailments, including cancer have been used for healing many ailments, including cancer. The purpose of this study was to purify turmeric protease and to research their biochemical characteristics. RESULTS: Cysteine protease from C. domestica has been purified to homogeneity using acetone precipitation followed by preparatory native polyacrylamide gel electrophoresis (PAGE). This protocol resulted in six fold purification with 28% final recovery. The purified turmeric protease showed a prominent single peak and band on high‐performance liquid chromatography and sodium dodecyl sulfate–PAGE, respectively, and an estimated molecular weight of 43 KDa, and exhibited optimal activity between 37 and 60 °C. The protease activity of the turmeric protease was significantly inhibited by iodoacetic acid. The turmeric protease had higher alanine and glutamate content and cleaved synthetic peptides N‐Cbz‐Ile‐Pro and N‐Cbz‐Phe‐Leu in a time‐dependent manner. Peptide mass fingerprint using matrix‐assisted laser desorption/ionization–time of flight mass spectroscopy revealed peptide matches to proteasome subunit alpha type 3 of Oryza sativa ssp. japonica (Rice). The turmeric protease showed antifungal activity at 10 µg mL^?1 towards pathogens Pythium aphanidermatum, Trichoderma viride and Fusarium sp. CONCLUSION: Cysteine addition significantly activated turmeric protease. The protease inhibition test suggested that turmeric protease belonged to the cysteine type. The biochemical characteristics of turmeric protease described in this paper can provide useful information for potential end uses of turmeric protease for pharmaceutical industry applications such as therapeutics. Copyright © 2009 Society of Chemical Industry     

Characterisation of antioxidant peptides from enzymatic hydrolysate of golden melon seeds protein

The purpose of present research was to study novel antioxidant peptides from Golden melon seeds. Alkaline protease was used to hydrolyse the Golden melon seeds protein to obtain the hydrolysed peptides. These antioxidant peptides were purified and identified by ultrafiltration, gel filtration chromatography and RP-HPLC-ESI-MS/MS. Results showed that the peptide fraction (GMSHp3) with molecular weight (MW) <3 kDa obtained by ultrafiltration had the highest antioxidant capacity. This fraction was further purified via gel filtration chromatography into six sub-fractions, among which GMSHp3-3 exhibited the highest hydroxyl radical scavenging effect. Fraction GMSHp3-3 was further purified via RP-HPLC-ESI-MS/MS and sequenced as six potential antioxidant peptides with amino acids sequences of RMSFPVMCRN, LMRVLAQLG, ALAPLVALPAA, LVGKPAPD, LPAAHKA and AHAAGYGG, among which LMRVLAQLG, LPAAHKA and AHAAGYGG possessed effective ferric reducing power. These results indicated that novel antioxidant peptides from golden melon seeds protein hydrolysates might be potential antioxidant source of functional foods or nutraceutical supplements.     

Production of bioactive peptides from tomato seed isolate by Lactobacillus plantarum fermentation and enhancement of antioxidant activity

Tomato seed contains proteins of high nutritional value and nutraceutical properties, which can be recovered for application as food additives. In this study, we investigated the use of a Lactobacillus plantarum strain to obtain high-added-value peptides from the fermentation process using tomato seed meal extract as the substrate. Potentially tomato seed meal extract have antioxidant activity which is correlated to the amino acid structures, compositions and sequences. After 24 h of fermentation, the radical scavenging activity of the isolated extract was increased by 87%. The increase of antioxidant activity is potentially attributed to the production of different bioactive peptides bio transformed during fermentation. L. plantarum growth on tomato seed meal extract as substrate reduced content of crude and soluble proteins by 18.44% and 68.99%, respectively, after 24 h of fermentation. Gel filtration chromatography showed a depolymerization of high molecular weight of polymers. HPLC analysis showed a significant decrease in the concentration of total amino acids, especially glutamic acid and aspartic acid. FTIR results showed that the fermentation favors the production of new amides and aromatic compounds. The production of protease by L. plantarum was investigated and results showed that highest activity (401.45 U/ml) were obtained after 20 h of fermentation. Results confirmed that L. plantarum could degrade and convert tomato seed proteins into bioactive peptides that contributed positively to the improvement of antioxidant activity of the protein isolate.     

Purification and Partial Characterization of Trypsin from the Viscera of Tropical Sierra (Scomberomorus Sierra) from the Gulf of California

Trypsin from the viscera of sierra (Scomberomorus sierra) was purified by affinity chromatography on Sepharose‐4B coupled to soybean trypsin inhibitor and characterized with respect to its purity, sensitivity to temperature, pH and inhibition. Trypsin was purified from sierra viscera with 11.9‐fold and 29.7% yield. The enzyme had a molecular weight of 25.4 kDa estimated by SDS‐PAGE and two possible trypsin isoforms were observed in activity gels. Trypsin activity was strongly inhibited by soybean trypsin inhibitor and porcine trypsin inhibitor, showing a partial inhibition by a serine protease inhibitor. The optimal activity of the enzyme was observed at pH 9 and 60C with n‐α‐benzoyl‐dl‐arginine‐p‐nitroanilide as a substrate. The enzyme maintained more than 50% of its activity in temperatures up to 50C and within the pH range of 8–10 for a period of up to 2 h.     

Antioxidant and antibacterial activities of peptide fractions from flaxseed protein hydrolysed by protease from Bacillus altitudinis HK02

Flaxseed protein (FP) hydrolysates by crude protease of strain Bacillus altitudinis HK02 were further separated into five fractions by ultrafiltration membranes with a molecular weight cut‐off of 10, 5, 3 and 1 kDa for the analysis of antioxidant activities and antibacterial ability. The results demonstrated that the fraction of 1‐ to 3‐kDa peptides exhibited higher antioxidant activities on the free radical‐scavenging ability and the reducing power than those of Vit C, Vit E, BHA and other fractions. The fraction with a low molecular weight (<1 kDa) of peptides demonstrated the highest ferrous ion‐chelating ability and a higher inhibition of lipid peroxidation than BHA and other fractions. Moreover, it also exhibited the best growth inhibition of Pseudomonas aeruginosa and Escherichia coli. The results, including the concentration‐dependent effect of peptides fractions, demonstrated that it is feasible to derive functional ingredients of natural antioxidants along with antimicrobial activity from FPs hydrolysed by protease from B. altitudinis HK02.     

ACYL TRANSPEPTIDATION AND HYDROLYTIC REACTIONS CATALYZED BY ENDOTHIA PARASITICA PROTEASE WITH SMALL PEPTIDE SUBSTRATES

Endothia parasitica protease shows hysteretic behavior (a lag phase) when acting upon L-leucyl-L-leucine amide at pH 4.5. Initially, the products are L-leucyl-L-leucine, L-leucyl-L-leucyl-L-leucine and L-leucine amide with little formation of L-leucine and no ammonia. The relative concentration of L-leucyl-L-leucine is about one-fourth that of L-leucine amide. As the reaction continues the relative concentration of L-leucine increases slowly until it is equal to the concentration of L-leucyl-L-leucine near the end of the reaction with each being one-fourth that of L-leucine amide. Formation of these products can only be explained by a combination of transpeptidation and hydrolytic reactions. By itself, L-leucyl-L-leucine is cleaved very slowly but when mixed with L-leucyl-L-leucine amide the rate is several fold greater than the combined rates of the two alone. L-Leucyl-L-leucyl-L-leucine and L-leucyl-L-leucyl-L-leucine amide as substrates of E. parasitica protease give normal kinetic behavior with no evidence of formation of higher molecular weight compounds. Addition of as little as 1 μM L-leucyl-L-leucyl-L-leucine (K_m= 0.348 mM) to a reaction containing 1 mM L-leucyl-L-leucine amide increases the rate of reaction with a shortening of the lag phase. Addition of 100 μM L-leucyl-L-leucyl-L-leucine to such a reaction eliminates the hysteretic behavior. It is proposed that E. parasitica protease is initially in an inactive form which can be activated by substrates as small as L-leucyl-L-leucyl-L-leucine but not by L-leucyl-L-leucine amide. During the lag phase observed with substrates such as L-leucyl-L-leucine amide, the enzyme produces, via acyl transpeptidation, larger compounds which can rapidly activate the enzyme. While in the activated form, E. parasitica protease cleaves L-leucyl-L-leucine amide via normal kinetic behavior; however, both acyl transpeptidation and hydrolysis still occur as shown by the relative concentrations of L-leucine, L-leucyl-L-leucine and L-leucine amide of 1:1:4 near end of the reaction. E. parasitica protease cannot cleave carbobenzoxy-L-leucyl-L-leucine indicating that, at least with this substrate, the enzyme cannot perform amino transpeptidation reactions.     

Purification and characterization of Aspergillus oryzae LK-101 salt-tolerant acid protease isolated from soybean paste

A novel salt-tolerant acid protease was produced from Aspergillus oryzae LK-101 (AOLK-101). The AOLK-101 protease was purified to homogeneity by ammonium sulfate precipitation, DEAE-Sephadex A-50 and Sephadex G-100 chromatographies in order. The specific activity and the purification ratio of the purified protease were 2,301 unit/mg and 11.6 fold, respectively, with 25 kDa of molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrpphoresis (SDS-PAGE). Its optimal pH and temperature were pH 6.5 and 50°C, respectively. This protease was relatively stable at pH 4.5–7.5, below 40°C, and up to 10% salt concentration. The protease was moderately inhibited by Ag²⁺ and Zn²⁺, and strongly by ethylenediamide tetraacetic acid (EDTA) and phenylmethysulfonyl fluoride (PMSF), but activated by Cu²⁺ and Mn²⁺. Therefore, the AOLK-101 protease was a serine protease based on the influence of metal ions and inhibitors. K _m , V _max , k _cat , and k _cat /K _m values of AOLK-101 protease for hammastein milk casein were 1.04 mg/mL, 124.84 unit/L, 163.5/sec, and 3.9×10⁶/m·sec, respectively.     

Increased effectiveness of the Trichoderma harzianum isolate T‐78 against Fusarium wilt on melon plants under nursery conditions

BACKGROUND: The use of isolates of the genus Trichoderma to control Fusarium wilt in melon plants is one of the most recent and effective alternatives to chemical treatments. In this work we have studied the immobilization of the isolate Trichoderma harzianum T‐78 on different carriers as an efficient method to control vascular Fusarium wilt of melon in nurseries. Different formulations were developed: liquids (spore suspension, guar gum and carboxymethylcellulose) and solids (bentonite, vermiculite and wheat bran). RESULTS: The introduction of F. oxysporum resulted in a significant decrease in seedling fresh weight. The treatments which gave a lesser reduction in weight and showing a greater biocontrol effect were the liquid conidial suspension and the solid treatments with bentonite and superficial vermiculite. Microbiological analyses revealed that the conidial suspension and all the solid treatments, except wheat bran, significantly decreased F. oxysporum populations. Of all the treatments assayed, bentonite produced the greatest decline in the F. oxysporum population. CONCLUSIONS: The most effective treatments against Fusarium wilt on melon plants were the solid treatments bentonite and superficial vermiculite. These two treatments gave the greatest plant weight, the lowest percentage of infected plants and the greatest T. harzianum population throughout the assay. Copyright © 2009 Society of Chemical Industry     

Cloning,nucleotide sequence and functions of XPR6, which codes for a dibasic processing endoprotease from the yeast Yarrowia lipolytica

Yarrowia lipolytica DO613, carrying the xpr6-13 mutation, secretes an inactive precursor of alkaline extracellular protease that has not been cleaved after the Lys-Arg at the end of the pro-region. Compared to wild type, DO613 membrane preparations had significantly reduced ability to cleave after Lys-Arg of an artificial substrate. The XPR6 gene was cloned by complementation by screening for restoration of production of alkaline protease activity. Sequencing of a 3735 base pair SalI-SphI XPR6 fragment revealed a large open reading frame with a coding capacity of 976 amino acids (molecular weight, 110 016). The deduced amino acid sequence had significant homology to Saccharomyces cerevisiae Kex2p, a processing endoprotease that cleaves after pairs of basic amino acids. Disruption of the XPR6 gene was not lethal, but it resulted in several phenotypic changes. First, essentially no mature alkaline extracellular protease was produced indicating that the low levels produced by strains carrying previously isolated xpr6 alleles were due to leaky mutations. Second, mating type B strains carrying the disrupted XPR6 gene did not mate, but mating type A strains did. Third, the XPR6 disruption strains grew poorly on rich media at pH 5·5 and above. Cells remained physically attached after budding and continued to bud forming large dog balloon-like structures. In addition, these structures aggregated forming visible clumps in liquid culture. These growth aberrations were largely eliminated by growing cells in medium at pH 4. Fourth, no mycelial forms were observed regardless of the pH.     

酶水解制备具有潜在免调节活性大豆肽的研究

由于已有的一些研究报道初步表明带正电荷的小分子肽具有免疫调节作用,本研究分别应用Alcalase、Flavourzyme、Protease A和Peptidase R四种商业酶,单独使用或将它们进行组合,分别以大豆分离蛋白（SPI）、可溶性大豆蛋白（SSP）和不溶性大豆蛋白（InSP）为底物,在不同的酶-底物比、底物浓度、pH和温度条件下进行水解大豆蛋白,测定了水解度、水解产物的分子量分布和带正电荷肽的相对含量.结果表明,Alcalase在相同条件下可以获得较高的水解度和较大的水解蛋白平均分子量;以InSP作为底物的水解产物中带正电荷肽的相对含量最高.水解条件对水解度有较明显的影响,但是平均分子量和正电荷肽相对含量的影响较弱。