Purification and characterization of an iron-activated alkaline phosphatase produced by Rhizopus microsporus var. microsporus under submerged fermentation using

Current researches have been carried out to find microorganisms that can produce enzymes for differentbiotechnological purposes. Among the enzymes, the microbial phosphatases, responsible for hydrolyzingphosphoric acid anhydrides and esters, have been often employed in different sectors such as molecularbiology experiments and clinical diagnosis. This work aims to purify and characterize the alkaline phosphataseproduced by Rhizopus microsporus var. microsporus under submerged fermentation. This enzyme was purified9.9-fold with 13% recovery. The molecular mass for the glycoprotein was 123 kDa estimated with gel filtrationand 128 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that it is a monomericenzyme. Optimal temperature and pH for the alkaline phosphatase was 45°C and 8.5, respectively, with halflife (t50) of 40 minutes at 50°C. Under alkaline pH, the phosphatase activity was above 50% for 24 hours.FeCl3 increased the phosphatase activity. Alkaline phosphatase hydrolyzed different substrates, especiallyp-nitrophenylphosphate, with Km of 0.45 and 0.38 mmol l−1, in presence and absence of FeCl3, respectively.Thus, alkaline phosphatase from R. microsporus var. microsporus was characterized, highlighting importantcharacteristics and, thereby, making possible a future application.

Efficient immobilization of agarase using carboxyl-functionalized magnetic nanoparticles as support

Background: A simple and efficient strategy for agarase immobilization was developed with carboxyl-functionalized magnetic nanoparticles (CMNPs) as support. The CMNPs and immobilized agarase (agarase-CMNPs) were characterized by transmission electron microscopy, dynamic light scattering, vibrating sample magnetometry, scanning electron microscopy, X-ray diffraction, thermogravimetric analysis, and zeta-potential analysis. The hydrolyzed products were separated and detected by ESI-TOF-MS. Results: The agarase-CMNPs exhibited a regular spherical shape with a mean diameter of 12 nm, whereas their average size in the aqueous solution was 43.7 nm as measured by dynamic light scattering. These results indicated that agarase-CMNPs had water swelling properties. Saturation magnetizations were 44 and 29 emu/g for the carriers and agarase-CMNPs, respectively. Thus, the particles had superparamagnetic characteristics, and agarase was successfully immobilized onto the supports. Agaro-oligosaccharides were prepared with agar as substrate using agarase-CMNPs as biocatalyst. The catalytic activity of agarase-CMNPs was unchanged after six reuses. The ESI-TOF mass spectrogram showed that the major products hydrolyzed by agarase-CMNPs after six recycle uses were neoagarotetraose, neoagarohexaose, and neoagarooctaose. Meanwhile, the end-products after 90 min of enzymatic treatment by agarase-CMNPs were neoagarobiose and neoagarotetraose. Conclusions: The enhanced agarase properties upon immobilization suggested that CMNPs can be effective carriers for agarase immobilization. Agarase-CMNPs can be remarkably used in developing systems for repeated batch production of agar-derived oligosaccharides.

Xylanase and beta-xylosidase from Penicillium janczewskii: Purification, characterization and hydrolysis of substrates

Background: Xylanases and β-D-xylosidases are the most important enzymes responsible for the degradation of xylan, the second main constituent of plant cell walls. Results: In this study, the main extracellular xylanase (XYL I) and p-xylosidase (BXYL I) from the fungus Penicillium janczewskii were purified, characterized and applied for the hydrolysis of different substrates. Their molecular weights under denaturing and non-denaturing conditions were, respectively, 30.4 and 23.6 kDa for XYL I, and 100 and 200 kDa for BXYL I, indicating that the latter is homodimeric. XYL I is highly glycosylated (78%) with optimal activity in pH 6.0 at 65°C, while BXYL I presented lower sugar content (10.5%) and optimal activity in pH 5.0 at 75°C. The half-lives of XYL I at 55, 60 and 65°C were 125,16 and 6 min, respectively. At 60°C, BXYL I retained almost 100% of the activity after 6 h. NH4+,Na+, DTT and β-mercaptoethanol stimulated XYL I, while activation of BXYL I was not observed. Interestingly, XYL I was only partially inhibited by Hg2+, while BXYL I was completely inhibited. Xylobiose, xylotriose and larger xylooligosaccharides were the main products from xylan hydrolysis by XYL I. BXYL I hydrolyzed xylobiose and larger xylooligosaccharides with no activity against xylans. Conclusion: The enzymes act synergistically in the degradation of xylans, and present industrial characteristics especially in relation to optimal activity at high temperatures, prolonged stability of BXYL I at 60°C, and stability of XYL I in wide pH range.

Concentration, characterization and application of lipases from Sporidiobolus pararoseus strain

Lipases produced by a newly isolated Sporidiobolus pararoseus strain have potential catalytic ability for esterification reactions. After production, the enzymatic extracts (conventional crude and precipitated, 'CC' and 'CP', and industrial crude and precipitated, 'IC' e 'IP') were partially characterized. The enzymes presented, in general, higher specificity for short chain alcohols and fatty acids. The precipitated extract showed a good thermal stability, higher than that for crude enzymatic extracts. The 'CC' and 'CP' enzymes presented high activities after exposure to pH 6.5 and 40 ºC. On the other hand, the 'IC' and 'IP' extracts kept their activities in a wide range of pH memory but presented preference for higher reaction temperatures. Preliminary studies of application of the crude lipase extract in the enzymatic production of geranyl propionate using geraniol and propionic acid as substrates in solvent-free system led to a reaction conversion of 42 ± 1.5%.

Screening of alginate producing strain and optimization of fermentation condition / 中国海洋药物

Objective A strain with high alginase activity was screened and isolated by decomposing sodium alginate from the decaying parts of brown alga Laminaria japonica and Undaria pinnatifida,in order to produce alginase.Methods The strain s4 with high alginase activity was chosen by filtration.The alginase producing media was optimized and the alginase was produced and its characterization was investigated.Results The optimum fermentation conditions for alginate lyase producing as follows: media AlgNa 1.2%,NH4Cl 0.9%,NaCl 1.5%,pH=7.5,and temperature 25℃.Conclusion The alginase produced by strain s4 showed high alginase activity and good stability.