Immobilization of lipase by chemical modification of chitosan / 生物工程学报

Lipase (EC3.1.1.3) from Candida sp. 99-125 was immobilized on chitosan by chemical covalence. Lipase was first immobilized to chitosan beads by activating its hydroxyl groups with carbodiimide followed by cross-linking more lipase to the amino groups with glutaraldehyde. In this article, different factors that influenced the immobilization were investigated, and the optimum conditions were ascertained. Comparative studies of organic solvent and thermal stability between free lipase and immobilized lipase were conducted. Immobilization enhanced the lipase stability against changes of temperature and organic solvent. Immobilization lipase can be reused in the synthesis system of palmitate hexadecyl. Operational stability tests indicated that the immobilized lipase occurs after 16 consecutive batches, the conversion rate remained 85%. Such results revealed good potential for recycling under esterification system.

Progress in metabolism and crucial enzymes of glycerol conversion to 1,3-propanediol / 生物工程学报

1,3-propanediol production by microbial fermentation has become the research hot spot for its amiability with the environment. Here the molecular mechanism of glycerol bioconversion to 1,3-propanediol was outlined by elucidating the fermentation strains, metabolic pathways, regulon and key enzymes. Of enzymes, glycerol dehydrogenase, the velocity-limiting enzyme in glycerol reductive pathway, was emphatically discussed with regard to its molecular structure and reactivating factors. This paper aims to provide the basis for genetic modification of fermentation strains.

Immobilization of lipase on macroporous resin and its application in synthesis of biodiesel in low aqueous media / 生物工程学报

Lipase from Candida sp. 99-125 was immobilized by physical adsorption onto macroporous resins. The results showed that the nonpolar resin NKA was the best carrier used in low aqueous media. 98.98% of degree of immobilization can be achieved when the adsorption procedure was performed in the presence of heptane. The hydrolytic activity and the apparent activity recovery of lipase adsorbed on resin in heptane was 4.07 and 3.43 times higher than that of lipase adsorbed in sodium phosphate buffer, respectively. The catalytic properties of immobilized lipase for production of biodiesel in low aqueous media were studied. Immobilized lipase displayed the highest activity when the crude enzyme/resin weight ratio was 1.92:1 and the water content(water/oil weight ratio) was 15% at 40 degrees C under pH 7.4. As lipase was adsorbed on NKA in heptane to produce biodiesel, the batch conversion rate can reach 97.3% when a three-step methanolysis protocol was used. After 19 consecutive batches, the conversion rate remained 70.2%.

Preparation of uniform-sized chitosan microspheres and application as carriers for protein drugs / 生物工程学报

Chitosan microsphere has been wildly researched in controlled release of protein and peptide drug because of its excellent mucoadhesive and permeation enhancing effect across the biological surfaces. The control of the size and size distribution of microspheres is necessary in order to improve reproducibility, bioavailability, and repeatable release behavior. In this work, uniform-sized chitosan microspheres containing insulin were prepared by a novel membrane emulsification technique combined with glutaraldehyde crosslinking method. In order to prepare uniform-sized chitosn microspheres, it is necessary to modify hydrophilic membrane into hydrophobicity. It is found that there exists a linear relationship between the size of chitosan microspheres and pore size of the membrane used, so it is easy to control the size of microspheres by using membranes with different pore size. In this study, the effect of different amount of crosslinker and crosslinking time on microspheres' morphology, encapsulation efficiency (EE) and release profile of drug in vitro were investigated. It is shown that the morphology of microspheres is more smooth and spherical, and the release rate is slower with the increase of amount of glutaraldehyde and prolongation of crosslinking time. When the molar ratio of amino group of chitosan to aldehyde group of glutaraldehyde is 1:0.7, and crosslinking time is 1 h, the highest EE was obtained (about 65%). Date obtained suggest that chitosan microspheres prepared by this new method would be a promising system for controlled release of protein drugs.

Effect of feeding pre-L-methionine on high-cell-density fermentation for S-adenosyl-L-methionine production / 生物工程学报

The yield of S-adenosyl-L-methionine (SAM) by saccharomyces cerevisiae fermentation was affected by the strategy of feeding L-methionine. The effects that feeding strategies and the amount of precursor L-methionine had on the production of SAM by saccharomyces cerevisiae G14 were investigated. The results showed that feeding L-methionine could obviously improve the accumulation of SAM, and both the biomass and SAM yield relied heavily on different feeding strategies. In our work, it was found that total amount of L-methionine added should be no less than 0.7g per 10 grams of dry cell weight. Five different feeding strategies had been investigated in our experiment, and such comparison indicated that favorable results could be achieved as the biomass reached the status of high cell density (120g/L). If 9 grams of the precursor L-methionine was introduced once and for all, the accumulation of SAM reached maximum of 4.31g/L at the 18th hour after addition; if the precursor amino acid was fed at a rate of 2g/h in 5 h, maximum yield of 4.98g/L was achieved at the 28th hour after feeding. Thus high cell density fermentation can be successfully applied to SAM production by Saccharomyces cerevisiae with the consequence of over 130g/L of biomass gained using the above two strategies.

Molecular cloning of an amidase gene from Nocardia sp. and its expressionin Escherichia coli / 生物工程学报

The amidase of Nocardia sp. is one of important industrial enzymes. Based on DNA and protein sequence alignment from different strains, a new gene of amidase was successfully cloned from Nocardia YS-2002, which is widely used for industrial production of acrylamide in China. DNA sequence analyses showed that the 1466bp cloned-fragment contains promoter, open reading frame and terminating-palindrome. Protein sequence alignment and phylogenetic tree analyses showed that the amidase coming from Nocardia sp. YS-2002 is a kind of specialamidase, without the typical conserved sequence of the amidases. Enzymatic characteristics predictions indicated that the molecular weight and pI of the new amidase is approximately 38.05 kD and 4.88, respectively, and it would be stable when heterogeneously expressed in E. coli. By inserting the ORF of the amidase into plasmid pET-28a(+), a recombinant strain, pEAB, was selected using E. coli BL21(DE3) as the host. SDS-PAGE analyses of both the whole cells and ultrasonic-treated cells confirmed the feasibility of the heterogeneous expression of amidase in the recombinant E. coli. But the activity of amidase in E. coli BL21(DE3) not more than 0.5 u/mg, because most of the enzymes expressed were formed as inclusion bodies.

Molecular Mechanism of Hyaluronan Biosynthesis in Streptococci and Construction of Engineering Strains / 中国生物工程杂志

Hyaluronic acid(HA)is a linear polysaccharide chain composed of alternating ?-1,4-glucuronic acid(GlcA)and ?-1,3-N-acetylglucosamine(GlcNAc)moieties.Construction of engineering strain has become the prevailing strategy for increasing yield and improving its quality,especially the molecular weight.Here the molecular mechanism of HA biosynthesis in Streptococcus strains was reviewed,involving fermentation strains,operon structure,crucial enzyme and construction of engineering strains.In addition,the prevalent problems in HA fermentation production were also discussed and the protocols were tentatively put forward for the upcoming research and industrial production.

Biological synthesis of L-ascorbyl palmitate / 生物工程学报

Biological synthesis of L-Ascorbyl Palmitate in organic system were studied in this text. The contradiction between conversion of vitamin C and concentration of L-Ascorbyl Palmitate were resolved. High conversion of vitamin C and concentration of L-Ascorbyl Palmitate were obtained by Novo435. A series of solvents(log P from -0.24 to 3.5 )were investigated for the reaction,and acetone was found to be the most suitable from the standpoint of the enzyme activity and solubility of L-ascorbic. And the equilibrium of the reaction was affected by the addition of the molecular sieves and temperature. Reaction carried out at 60 degrees C and with 20% 0.4nm molecular sieves is good for the enzyme to keep its activity and for making the equilibrium go to the product. With 1.094 g palmitic acid, 0.107 g vitamin C and 0.020 g Novo435, rotate rate of 200 r/min, the conversion of ascorbic reached 80% and the concentration of L-ascorbyl palmitate is 20 g/L after 48 h. Furthermore, reaction batch of Novo435 and substrates recycle were observed, the result indicated that Novo435 may used 4-5 times continuously with high conversion. And 6-O-unsaturated acyl L-ascorbates were synthesized through Novo435 condensation of ascorbic acid and various unsaturated fatty acids with high conversion in this text.

Study on the repeated batch fermentation of lipase by immobilized Rhizopus / 生物工程学报

Repeated batch fermentation with Rhizopus arrhizus immobilized by polyurethane was optimized in details including the time to replace, the volume of the replaced medium and the optimal composition of the medium to replace. Immobilized cells showed stability for repeated use. Immobilized cells could be used for 9 batches, lasting 140h in flask and 6 batches in 5L fermenter when the substitute culture medium consisted of (%): soybean flour 3.0, earthnut oil 0.5 and the liquid was replaced by 40% . The time for fermentation was reduced largely and the lipase productivity was 3 - 6 times as high as that in batch fermentation.

Studies of enzymatic synthesis of biodiesel / 生物工程学报

Biodiesel, an alternative diesel fuel, fatty acid alkyl ester, is made from renewable biological sources such as vegetable oils and animal fats. Two processes for biodiesel synthesis, enzymatic lipase catalytic esterification from fatty acid and transesterification from oils and fats, was investigated. The effects of various lipases, enzyme amount and purity, solvent, water absorbent, inhibition of short chains alcohol, specificity of substrate, molar ratio of substrate on esterification were studied in detail. The esterification degree with the optimal parameter and process can reach up to 92%. The purity of biodiesel obtained by separation and purification is up to 98%, and the half-life of the immobilized lipase for the esterification process can be up to 360hr, Moreover, the preliminary studies of the transesterification including the amount of methanol and mode of adding methanol into reaction system were made. The transesterification degree with adding methanol stepwise can reach 83%.

Cloning and expression of spinach glycolate oxidase in Escherichia coli / 生物工程学报

The cDNA coding spinach glycolate oxidase (GO) was amplified by RT-PCR using the total RNA of spinach leaves as the template, and was cloned into cloning vector pMD18-T. After the DNA sequence was determined, the go gene was subcloned into E. coli expression vector pBV220, pET-22b(+), pTIG-Trx and pThioHisC. SDS-PAGE analysis revealed that the recombinant E. coli BL21 (DE3) (pTIG-Trx-GO) and E. coli BL21 (DE3) (pET-22b(+)-GO) expressed the predicted 38 kD glycolate oxidase, and the enzyme activity was also detected.

Optimal Study on the Fermentation Conditions of S-adenosyl-L-methionine / 微生物学通报

This paper focuses mainly on the study of optimal fermentation conditions of S-adenosyl-L-methionine.Effects of carbon sources,nitrogen sources,inorganic constituents,growth factors and adding time of L-methionine on the yield,the content and biomass of S-adenosyl-L-methionine are studied.And ingredients of the culture medium are also optimized by the method of uniform design.The final optimum culture medium contains: glucose 30 g,Yeast powder 11 g,(NH_4)_2SO_4 12 g,K_2HPO_4?3H_2O 5 g,KH_2PO_4 10 g,MnSO_4?H_2O 0.09 g,ZnSO_4?7H_2O 0.14 g,MgCl_20.5 g,CaCl_2 0.3 g,CuSO_4 0.005 g per liter. Using that optimum culture medium,the yield of S-adenosyl-L-methionine can reach 0.9 g/L in Erlenmeyer flask which is 30 % higher than before.Experiment on 5 L fermenter reveals that the accumulation of S-adenosyl-L-methionine can reach 2.66 g/L.Biomass is 23.4 g/L.

EFFECTS OF SOME SALT-TOLERANT FUNGI IN DISSOLBING PHOSPHATE ROCK UNDER DIFFERENT SALT AND ALKALINE / 微生物学通报

Four specific salt-tolerant fungi for dissolving phosphate rock (FM) w ere isolated from the coastal saline soils. It was found that the biomass and a b ility to release phosphate from the materials decreased with increasing of the N aCl content. During pH7.0～8.5, the biomass and ability of to release phospho rus from the materials of FM1 decreased sharply with pH rising, but the FM2 and FM4 decreased not considerably, optimal pH for FM3 was at pH8.5. At pH9.0, the bi oma ss and ability of to release phosphorus from the materials decreased quickly. F M2 was stable at alkaline and high salt content condition.

SCREENING FOR AND CHARACTERISTICS OF MICROBIAL FLOCCULANT / 微生物学通报

strain 49 #(BD-4),which had the highest flocculating activity among 752 strains,was picked out.The fermentation condi tions and the flocculating influence factors were studied.By testing,this cult ure broth had a much more ability to get rid of suspended materials in water and to decolourize the high-density dyestuff wastewater. In laboratory scale,bioflocculant was roughly extracted from culture broth by t he technological process,water extraction-organic solvent sediment-vacuum dry .The suitable work conditions of each step were set up through the tests.0 198 5g dry product was obtained from 100mL culture broth.