Characterization of a β-Glucosidase from an Edible Mushroom,Lycoperdon Pyriforme

A β-glucosidase from Lycoperdon pyriforme, a wild edible mushroom, was characterized biochemically. The enzyme showed a maximum activity at pH 4.0 and 50°C when p-nitrophenyl-β-D-glucoside was used as a substrate. K_m and V_max values were calculated as 0.81 mM and 1.62 U/mg protein, respectively. The enzyme activity was conserved about 85% over a broad range of pH (3.0–9.0) at 4°C after 24 h incubation. The activity was fully retained after 60 min incubation at 20–40°C. Na⁺, Li⁺, Mg²⁺, Mn²⁺, Zn²⁺, Co²⁺, Ca²⁺, and Cu²⁺ did not affect the enzyme activity and 0.25% sodium dodecylsulfate inhibited the enzyme activity approximately 76%. Ethylenediamine tetra-acetic acid, phenylmethanesulfonylfluoride, and dithiothreitol showed no or a little negative effect on the enzyme activity. The resistance of the enzyme to some metal ions, chemicals, and ethanol along with the pH stability, can make it attractive for future applications in industry.     

Purification and characterization of β-agarase from Paenibacillus sp.

β-Agarase produced by Paenibacillus sp. WL (agarase WL) was purified using a combination of ammonium sulfate precipitation, DEAE-ion exchange, and gel-filtration chromatography. The purity of the agarase was increased by 11.9× with a recovery of 5.1% and a specific activity of 4,670.1 U/mg of protein. The molecular mass of the purified agarase was approximately 30 kDa (SDS-PAGE). The agarase was stable at temperature below 50°C and the favorable agar-hydrolysis activity was at 40°C. The agarase was active in the range of pH 5.0 to 8.0, and the optimal agar-hydrolysis pH value was approximately 6.0. Metal ions normally found in seawater (Na⁺, K⁺, Ca²⁺, Mg²⁺, and Al³⁺) could activate agarase WL. The Michaelis-Menten constant K _m and maximal reaction velocity V _max of purified agarase WL were 3.22 mg/mL and 41.5 μg/mL·min, respectively. The agarase WL was highly agar specific.     

Purification and Characterization of Intracellular α-galactosidase from Rhizopus sp. AOI

根霉Rhizopus sp．A01的菌丝体破碎液依次经过三相分离、SephaαexG-100凝胶过滤获得了电泳纯的α-半乳糖苷酶,纯化了54．8倍,总酶活回收率达到27．3％,在SDS-PAGE上显示相对分子质量为85．6ku的单-条带,凝胶过滤表明该酶表观相对分子质量为302ku。该酶水解对硝基苯-α—D-吡喃半乳糖苷的最适pH值为4．5,最适温度为55％,表观Kn值为（0．242±0．027）mmol／L,表观Kcat／Km值为4．089×10^5L／（mol·s）;对蜜二糖和棉子糖有弱的水解作用,水解速度依次为138．3μmol／（h·mg）、19．7μmol／（h·mg）。水解活性受Fe^2＋和Fe^2＋的显著激活,但受Mn^2＋、Cu^2＋、Hg^＋和Mg^2＋等离子的强烈抑制。该酶活性在pH4．0～8．2保持稳定,在50℃时保温90min,残余酶活达到了48％。     

Digestion of residual β-cyclodextrin in treated egg using glucoamylase from a mutant strain of Aspergillus niger (CFTRI 1105)

Glucoamylase enzyme isolated from the cultures of a mutant strain of Aspergillus niger (CFTRI 1105) was found to digest β-cyclodextrin in standard 1% solutions as well as in β-cyclodextrin-treated egg yolk and whole egg samples. About 70–95% of β-cyclodextrin in samples was digested with both dilute (reducing sugar activity 870 units) and concentrated (34,800 units) enzyme solutions at 40°C and 70°C for incubation periods of 15–360 min.     

Characterization of a β-glucosidase isolated from an alpeorujo strain of Candida adriatica

A β-glucosidase-producing strain, Candida adriatica CECT13142, was isolated from olive oil wastes (alpeorujo) and identified by PCR/restriction fragment length polymorphism of the rDNA internal transcribed spacer and sequence analysis of the D1/D2 region of the 26S rRNA gene techniques. The enzyme was purified by sequential chromatography on DEAE-cellulose and Sephadex G-100. The relative molecular mass of the enzyme was estimated to be 50 kDa by SDS-PAGE. The hydrolytic activity of the β-glucosidase had an optimum pH of 8.2 and an optimum temperature of 40°C. The enzyme displayed high substrate specificity and high catalytic efficiency (K_m 0.85 mM, V_max 12.5 U/g of cells) for p-nitrophenyl-β-D-glucopyranoside. Although β-glucosidases have been purified and characterized from several other organisms, the C. adriatica β-glucosidase is able to have optimal activity at alkaline pH.     

Production of Cell Membrane-Bound α- and β-Glucosidase by Lactobacillus acidophilus

Hydrolytic enzymes, viz. α- and β-glucosidase, were produced from indigenous isolate, Lactobacillus acidophilus, isolated from fermented Eleusine coracana. Production of these enzymes was enhanced by optimizing media using one factor at a time followed by response surface methodology. The optimized media resulted in a 2.5- and 2.1-fold increase in α- and β-glucosidase production compared with their production in basal MRS medium. Localization studies indicated 80% of the total activity to be present in the cell membrane-bound fraction. Lack of sufficient release of these enzymes using various physical, chemical, and enzymatic methods confirmed their unique characteristic of being tightly cell membrane bound. Enzyme characterization revealed that both α- and β-glucosidase exhibited optimum catalytic activity at 50 °C and pH 6.0 and 5.0, respectively. K _m and V _max of α-glucosidase were 4.31 mM and 149 μmol min⁻¹ mL⁻¹ for p-nitrophenyl-α-d-glucopyranoside as substrate and 3.8 mM and 120 μmol min⁻¹ mL⁻¹ for β-glucosidase using p-nitrophenyl-β-d-glucopyranoside as the substrate.     

Purification of β-carotene 15,15′-monooxygenase from pig intestine and its enzymatic hydrolysis of pigment in soybean oil

β-Carotene 15,15′-monooxygenase was isolated and purified from the intestinal mucosa of pigs, and then, it was applied to hydrolyse the pigment in soybean oil, and thus, vitamin A fortified soybean oil was obtained. The pig intestinal mucosal protein solution was purified to a specific activity of 2.487 × 10⁻⁴ U mg⁻¹, maximum reaction velocity (V_max) of 8.42 × 10⁻⁹ mol/h and Michaelis constant (K_m) of 2.03 × 10⁻⁵ m . The protein solution had a molecular mass of 156 kDa by gel filtration. The sodium deoxycholate concentrations, optimum pH, Tween 40 amount and enzyme amount for vitamin A production in soybean oil were determined to be 6.0 mm , 8.0, 3.0% (w/v) and 0.2 U/mL enzyme, respectively. Under these conditions, β-carotene 15,15′-monooxygenase produced 14.65 mg/L vitamin A after 20 h, with a conversion yield of β-carotene of 33.29% (w/w). Therefore, the nutrients in soybean oil were improved.     

Prebiotic effects of structurally identified galacto-oligosaccharides produced by β-galactosidase from Aspergillus oryzae

Novel galacto-oligosaccharides were produced by β-galactosidase from Aspergillus oryzae using lactose, and their structural characteristics and prebiotic effects were examined. Highly purified oligosaccharide fraction (HP) was prepared from a crude one (low purified, LP) by gel-filtration on Biogel P-2 column, which was further purified into S1 and S2 fractions by prep-HPLC. S1 and S2 were comprised of galactose (Gal) and glucose (Glc) in the ratio of 2 to 1. ESI-MS-MS and methylation analysis indicated that S1 and S2 were trisaccharides with structures of β-d-Galp-(1,6)-β-d-Galp-(1,4)-β-d-Glcp and β-d-Galp-(1,3)-β-d-Galp-(1,4)-β-d-Glcp, respectively. Herein, LP and HP were used as the carbon sources for determining the prebiotic activity score of probiotics including Lactobacillus and Bifidobacterium species. LP and HP at 1% and 2% (w/v) were observed at all positive scores on several probiotics, especially, B. infantis ATCC 15697 at the 2% level of HP (p<0.05). Consequently, structurally identified trisaccharides of HP can significantly enhance the growth of B. infantis.     

用固定化Aspergillus niger分生孢子生产柠檬酸

据日本发酵上工业Vol45No51987年报导;将黑曲Aspergillus niger的分生孢子悬浊液(2.32×10~8/ml)添加固定在3%的海藻酸钙凝胶上,然后将固定化凝胶上的黑曲分生孢子放入含砂糖14%的生产培养基中,     

Engineering the optimum pH of β-galactosidase from Aspergillus oryzae for efficient hydrolysis of lactose

β-Galactosidase (lacA) from Aspergillus oryzae is widely used in the dairy industry. Its acidic pH optimum and severe product inhibition limit its application for lactose hydrolysis in milk. In the present study, structure-based sequence alignment was conducted to determine the candidate mutations to shift the pH optimum of lacA to the neutral range. The Y138F and Y364F mutants shifted the pH optimum of lacA from 4.5 to 5.5 and 6.0, respectively. The acid dissociation constant (pKa) values of catalytic acid/base residues with upwards shift were consistent with the increased pH optimum. All variants in the present study also alleviated galactose inhibition to various extents. Molecular dynamics demonstrated that the less rigid tertiary structures and lower galactose-binding free energy of Y138F and Y364F might facilitate the release of the end product. Both Y138F and Y364F mutants exhibited better hydrolytic ability than lacA in milk lactose hydrolysis. The higher pH optimum and lower galactose inhibition of Y138F and Y364F may explain their superiority over lacA. The Y138F and Y364F mutants in the present study showed potential in producing low-lactose milk, and our studies provide a novel strategy for engineering the pH optimum of glycoside hydrolase.     

Isolation and Purification of Cellulase Produced by Mixed. Fermentation of AspergiUus niger Ⅲ and Trichoderca viride Ⅰ and Their Enzymatic Properties

对黑曲霉Ⅲ与绿色木霉Ⅰ混合发酵所产纤维素酶进行了分离纯化,并对其酶学性质进行了研究。通过硫酸铵分级沉淀、Sephadex G-100凝胶柱层析,得到5个洗脱峰,其中峰2含内切葡聚糖苷酶和外切葡聚糖苷酶,且内切葡聚糖苷酶活最高;峰3含内切酶、外切酶及β-葡萄糖苷酶,酶系较全面;峰5含内切酶和外切酶;峰1和峰4没有纤维素酶活性。采用DEAF FF弱阴离子交换柱层析对峰2进行分离纯化,从中分离纯化得到1种内切葡聚糖苷酶组分,经SDS-PAGE电泳分析,其分子量为61.5 KD。酶学性质研究结果表明,CMC酶活在pH4.0～6.0的条件下,可保持初始酶活的70%～80%,最适酶反应pH值为5.0;温度在30～50℃范围内,酶活较高,最适酶反应温度为50℃,若超过60℃,酶活迅速下降。     

Purification and characterization of a β-glucosidase capable of hydrolyzing soybean isoflavone glycosides from Pichia guilliermondii K123-1

A β-glucosidase, efficiently hydrolyzing isoflavone glycoside to isoflavone aglycone, was purified from Pichia guilliermondii K123-1, isolated from Korean soybean paste by ammonium sulfate precipitation, ion exchange column chromatography, gel filtration, and fast protein liquid chromatogram (FPLC). The molecular mass of purified enzyme was estimated to be 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). The optimum temperature for enzyme activity was 45°c and it decreased dramatically above 50°c. The maximal activity was at pH 4.5 and more than 80% of the activity was retained for 24 hr in the pH range from 4.0 to 8.0 at 4°C. The N-terminal amino acid sequence of the enzyme was determined to be GLNWDYDNDK. Based on its substrate specificity and catalytic properties, the activity of the purified β-glucosidase was more effective when the sugar moiety of the glycoside was glucose and the size of the aglycone similar to that of the isoflavones. The purified β-glucosidase efficiently converts genistin and daidzin to genistein and daidzein 1.96 and 1.75 times more than almond meal β-glucosidase.