Effect of graphene oxide with different morphological characteristics on properties of immobilized enzyme in the covalent method.

Although graphene oxide (GO) has great potential in the field of immobilized enzyme catalysts, the detailed effects of GO with different morphological structures on immobilized enzyme are not well understood. GOs were prepared from 8000 mesh and nanoscale graphite at different reaction temperatures, and used as carriers to immobilize alpha-amylase by cross-linking method. The properties of GOs were characterized through Atomic force microscope, Fourier-transformed infrared, X-ray photoelectron spectroscopy, Raman and UV-Vis. Furthermore, the dosage of cross-linking agent, cross-linking time, optimum temperature/pH, thermal/pH/storage stability, reusability and kinetic parameters of immobilized enzymes were investigated. The results showed that the loading of alpha-amylase on GOs was 162.3-274.2 mg g-1. The reusability experiments revealed high activity maintenance of immobilized alpha-amylase even after seven reaction cycles. Moreover, the storage stability of immobilized enzyme improved via immobilization in comparison with free one and it maintained over 70% of their initial activity after 20 days storage at 4 °C.

[Sequence analysis of 16S rDNA and genes of soluble methane monooxygenase from Methylomonas sp. GYJ3].

Soluble methane monooxygenase (MMO) from methanotrophs is a member of binuclear iron-containing multicomponent oxygenases, which can catalyze bioconversion of methane to methanol at ambient temperature and regulate methane recycle in nature. The research focused mainly on the sequence analysis of 16S rDNA and sMMO genes from Methylomonas sp. GYJ3. With the aid of the information from GenBank, the PCR primers and the sequence primers were designed, obtained a 5690bp of sMMO fragment and a 1280bp of 16S rDNA. Sequence comparison for MMOX with counterpart of other five strains showed that from 78% to 99% identity in protein level and from 71 % to 97% identity in gene level, in the separate comparison of six components, only orfY component had a lower identical. The multiple alignment of MMOX amino acid sequence with other four strains showed that there is a high conservation, especially in two Fe binding regions. 16S rDNA phylogenetic analysis demonstrated that Methylomonas sp. GYJ3 is relative with gamma proteobacteria. Phylogenetic analysis of MMOX amino acid sequence showed that Methylomonas sp. GYJ3 is closer to Methylomonas sp. KSW III of type I methanotrophs. It was concluded that Methylomonas sp. GYJ3 is belong to the genus of type I methanotroph Methylomonas, and the result was a direct evidence for the sMMO can be expressed in type I methanotrophs. The theoretical pI of hydroxylase was 6.28 and the theoretical MW of hydroxylase was 248874.41Da.

[Methane monooxygenases hydroxylase from a type II methanotroph: purification and physical-chemical properties].

Methanotrophs can catalyze hydroxylate of methane and some hydrocarbon. Which play an important role in mitigating global warming and have also potential significance for industrial applications or bioremediation. A high activity of hydroxylase, a crucial component in sMMO, from Methylosinus trichosporium IMV 3011 has been purified to homologues by using chromatographic techniques. The molecular weight of the hydroxylase determined by gel filtration is 201.3 kD, and SDS-PAGE showed that hydroxylase consists of three subunits(alpha beta gamma) with molecular weights of 58kD, 36kD and 23kD respectively, drawing a comparison both methods indicated that the hydroxylase is a homodimer with an (alpha beta gamma)2 configuration. Purified hydroxylase has a pI at 5.2 judged by thin layer isoelectric focusing. The purified hydroxylase contains 3.02 mol of iron per mol of protein. The stability pH for the hydroxylase in solution is 5.8-8.0 and the stability temperature is below 35 degrees C. The cells form show a long, bent, and rod-shaped with even surface observed by scanning electron microscopy.