Abstract:Enzyme engineering is a combined technology of enzymology and engineering, which is becoming one of the major fields of modern biotechnology. In recent years, China has made some advances in enzyme engineering research. To promote enzyme engineering research in China, invited reviews and selected research articles were published in this special issue of “Enzyme Engineering”. The reviews and research articles focus on the fields of enzymatic conversion, therapeutic enzymes, enzymes as additives to animal feedstuff, enzymes for degradation of organic pollutes, and enzymes for biofuel and biorefinery.

Abstract:Enzymes play such a pivotal role in cellular metabolism that enzyme assays are important for bio-engineering, disease diagnoses and drug discovery. Among the reported methods, fluoremetry has attracted more and more attention due to its high sensitivity and possibility of continuous dynamic monitoring. The recent progresses and applications in enzyme assays using fluorescent probes were reviewed. Different methods were classified into direct fluorescence detection and indirect fluorescence detection according to their labeled substrates and detection mechanisms. Our writing purpose is to provide the readers with a flavor of the kinds of tools and strategies available in enzyme assays with fluorescent probes. Also, the research situation and prospects were discussed.

Abstract:Wild-type biocatalysts usually show high activity and selectivity towards their native substrates. Since non-native substrates are often used in synthetically useful biocatalytic transformations, it is necessary to engineer enzymes for improved activity, stability and selectivity (chemo-, regio- and stereoselectivity). Herein we give an overview of the recent advances in engineering the enantioselectivity of biocatalysts, with an aim to stimulate further development of this important field in China.

Abstract:With the rapid development of industrial biotechnology, breakthrough in enzymology and biocatalysis has been made in recent years, especially in areas of stability and activity of enzyme in nonaqueous media, screening, construction and modification of solvent-tolerant biocatalysts, as well as the development of green solvent with excellent biological and environmental compatibility. Recent trend and future focus include: in silico virtual screening and construction of solvent-tolerant biocatalysts based on bioinformatic technology, modification and construction of native solvent-tolerant biocatalysts, the development of environmental friendly green solvent such as ionic liquids.

Abstract:Yeast surface display involves that the exogenous protein, which was fused with the yeast outer shell cell wall protein, was genetically anchored on the yeast cell surface. It has been widely used in expression and screening of functional protein. Here, we focused on the construction of lipase-displaying systems and its application in enzymatic biosynthesis, such as fatty acid methyl esters, short-chain flavour esters and sugar esters applications, and so on.

Abstract:It is well known that non-aqueous enzymatic catalysis has emerged as an important area of enzyme engineering with the advantages of higher substrate solubility, increased stereoselectivity, modified substrate specificity and suppression of unwanted water-dependent side reactions. As a result, non-aqueous enzymatic catalysis has been applied in the biocatalytic synthesis of important pharmaceuticals and nutriceuticals. With the advancement of non-aqueous enzymatic catalysis in recent years, the efforts have been centered on the discovery and modification of solvent-tolerant biocatalysts for non-aqueous environments. Additionally, with the inevitable trends of green chemistry and sustainable development, green solvents have been utilized for increased number of enzymatic reactions to replace conventional organic solvents. In this review, modification, immobilization and mutagenesis of various enzymes for non-aqueous catalysis are discussed. Recent progress of non-aqueous enzymatic catalysis in solvent-free environments, reverse micelles, supercritical liquid and ionic liquid are also presented. In particular, while direct evolution, high-throughput screening and site-directed mutagenesis are combined as powerful tools for protein engineering, vapor/solid/ice water mixture, sticky solid-state liquid crystal and high density salt suspension are the future directions for solvent engineering in order to broaden the utility and elevate the efficiency of non-aqueous enzymatic catalysis.

Abstract:Nitriles are an important type of synthetic intermediates in the production of fine chemicals because of their easy preparations and versatile transformations. The traditional chemical conversion of nitriles to carboxylic acids and amides is feasible but it requires relatively harsh conditions of heat, acid or alkali. Nitrile converting enzymes (nitrilase, nitrile hydratase and amidase) which are used as biocatalyst for the production of fine chemicals have attracted substantial interest because of their ability to convert readily available nitriles into the corresponding higher value amides or acids under mild conditions with excellent chemo-, regio- and stereo-selectivities. Many nitrile converting enzymes have been explored and widely used for the production of fine chemicals. In this paper, various examples of biocatalytic synthesis of pharmaceuticals and their intermediates, agrochemicals and their intermediates, food and feed additives, and other fine chemicals are presented. In the near future, an increasing number of novel nitrile converting enzymes will be screened and their potential in the production of useful fine chemicals will be further exploited.

Abstract:Industrial enzymes play dual roles for the production of chemicals and biochemicals, one is to act as direct catalyst for the reaction, the other is to participate in the fermentation process to convert substrates to fermentable sugars or to make it more efficient. The review briefs the applications of industrial enzymes for chemical productions, with emphasis on direct conversion of starch and their roles in bioethanol production process, also analyzes the benefits by using new enzymes and prospects for future development.

Abstract:We reviewed the microbial production of alkaline polygalacturonate lyase (PGL) and its application in the clean production of textile industry. Currently PGL is mainly produced by microbial fermentation and Bacillus sp. is an ideal wild strain for PGL production. Microbial PGL production was affected by many factors including the concentration and feeding mode of substrate, cell concentration, agitation speed, aeration rate, pH and temperature. Constructing the recombinant strain provided an effective alternative for PGL production, and the concentration of PGL produced by the recombinant Pichia pastoris reached 1305 U/mL in 10 m3 fermentor. The recombinant Pichia pastoris had the potential to reach the industrial production of PGL. PGL can be applied in bio-scouring process in the pre-treatment of cotton. Compared with the traditional alkaline cooking process, the application of PGL can protect fiber, improve the bio-scouring efficiency, decrease energy consumption and alleviate the environmental pollution. The future research focus will be the molecular directed evolution of PGL to make PGL more suitable for the application of PGL in bio-scouring process to realize the clean production of textile industry.

Abstract:Cutinase (EC 3.1.1.74) is a kind of hydrolase capable of catalyzing the cleavage of ester bonds of cutin to release fatty acids. Cutinase displayed hydrolytic activity not only toward cutin but also a variety of soluble synthetic esters, insoluble triglycerides and polyesters. Besides its hydrolytic activity, cutinase also showed synthetic activity and transester activity. Therefore, cutinase was evaluated as a versatile lipolytic enzyme used in food and chemical industry. Recently, it is found that cutinase has potential use in cotton bio-scouring and synthetic fibers modification. Cutinase is the most important enzyme in clean production of textile industry.

Abstract:There is a great diversity for cellulolytic microbes in nature and the strategies they use to digest cellulose. In addition to the cultured cellulolytic microbes, there are still a great number of microbes being not readily culturable in natural environments, which may represent great potential for identifying novel cellulases and their encoding genes. The rise of metagenomics and metaproteomics provides essential technologic tools to dig up these resources and significant progress has been made so far. This review gives an insight into some relative results that have arisen from the meta-genomic or proteomic analysis of definitive uncultured microbe communities. Their potential role in elucidating the process and mechanisms of cellulose degradation in natural environment from the point of “community system microbiology” is also discussed.

Abstract:Enzymes can degrade the anti-nutrient factors in feedstuff, increase nutrient digestibility, and reduce pollution to environment, and have been widely supplemented in animal feedstuff. However, the use of enzymes is limited because of their undesirable properties, such as thermoliability and susceptibility against protease digestions. And its commercialization is also limited by low production efficiency and high cost. Therefore, the focuses for future enzyme development will be: 1) to obtain novel enzymes with better properties by high-throughput screening of enzyme encoding genes, especially those from extreme and special environments; 2) to improve enzyme properties using directed mutagenesis and protein engineering methods; 3) to achieve high-level fermentation of enzymes by heterogonous expression and optimization of codons, vectors and fermentation conditions; 4) to determine the effect of enzymes to animals and utilize enzymes efficiently.

Abstract:With the development of the research on biotechnology and modern pharmacy, the application of enzyme drugs have grown rapidly and enzyme drugs have become an important branch of biopharmaceutics. In this article, some new varieties of therapeutic enzymes, enzyme targets, mechanisms and new technologies of application in therapeutic enzymes were reviewed, and the direction of development of therapeutic enzymes were discussed.

Abstract:Herb-glycosides are main active elements of Zhongcaoyao (Chinese traditional medicines, Chinese medical herbs). However, the herb-glycoside structures are not optimal active structure for the human bodies. After orally taken up, the herb-glycosides of Zhongcaoyao could be changed into other more active structures by the digestive system such as enzymes and intestinal microorganisms; then degraded and absorbed in the human body and play the real role of pharmic effect; but only a small amount could be changed and controlled by circadian state of the human body. If this biochange of herb-glycosides to more active structures in vivo was finished in vitro, it is very useful for the development of the Chinese traditional medicines, new plant medicines, health food, and function cosmetics. To biotransformate herb-glycosides to more active structure, this paper introduced the studies of author’s team on the new microorganism isolation of the special herb-glycosidases and enzyme fermentation, the special enzyme purification and characterization.

Abstract:With the rapid development of socialization and industrialization, more and more pollutes were produced and discharged into natural environment. It is harmful to human health and life. These pollutes included refractory degradation organic compounds like PAHs, RDX, HMX, CL-20, PCBs and alkanes and their relative substances. Various compounds exist in nature with long life span. They are the most hazardous than other organics. The impact of pollutes can be treated by microorganisms. Result showed that it is an effective way for bioremediation of these pollutes with microbial metabolism or cometabolism. A few key enzymes, mainly oxidative and reductive enzymes, connected with the first step of initial degradation. Normally, enzymes grouped with other active fraction on the cell membrane are composed of one oxidative and reductive system for substrates oxidation. The metabolic intermediates can be used with TCA by microorganisms. The pathways of metabolism and the key enzymes were summarized. The further research topics should be focused on microorganism screen and its relative enzyme, pathway and mechanism of metabolism or cometabolism for such compounds degradation, and the result was hoped for the environmental protection.

Abstract:Sponges (Porifera) are the oldest living metazoan in the world, among which most of them (Demospongia) can produce silicic skeleton from orthosilicic acid in the seawater under the natural environmental conditions. These biosilicic materials exhibit good mechanical and optical properties as well as good biocompatibility. During the biosilicification process of sponges, a protein, named as silicatein, plays an important role and has attracted great attention from biologist, chemists and material scientists. This mini review highlights the discovery of silicateins and its function as both an enzymatic catalyst and an organic template for biosilicification. The studies since 1999 were briefly introduced on the application of silicatein as a biocatalyst and template for synthesis of silica-based and other inorganic materials. It is expected to stimulate the interests in the related researches in China.

Abstract:This article reviews the recent studies on h2o2 adaptation of Saccharomyces cerevisiae. When the cell exposed in the h2o2 sub-lethal doses, the plasma membrane permeability decreased, meanwhile the plasma membrane fluidity is minished. These changes resulted in a gradient across the plasma membrane, which conferring a higher resistance to oxidative stress. Recent work has also shown that the yeast cells adapted to H2O2 would lead to several changes in the expression of genes coding the key enzymes involved in the biosynthesis of lipid profile and in the organization of lipid microdomains of the plasma membrane, which finally decreased its’ permeability and fluidity. The reorganization of the plasma membrane might be the major mechanism of the h2o2 adaptation. Once the yeast cells adapted to the external h2o2, changes in plasma occurred. The h2o2 dependent signaling pathways in the plasma membrane might be activated by high levels of h2o2. But the details of the signaling events should still be further studies.

Abstract:Directed evolution strategy (error-prone PCR) was conducted to improve the activity of lipase from Rhizopus chinensis CCTCC M201021. Through two rounds of ep-PCR and pNPP top agar screening, two optimum mutant strains 1-11 and 2-28 were obtained with 2 and 4 fold of enzyme activity higher than that of parent strain, respectively. DNA sequencing of mutant lipase 2-28 revealed four amino acid substitutions: A129S, K161R, A230T, K322R. According to the simulated protein structure of Rhizopus chinensis lipase, A129S, K161R, A230T were located on the surface of the protein. A230T substitution improved the stability of the α-helix loop. K322R, near the catalytic center of lipase, located at a loop, formed a salt-bridge with a nearby aspartic acid (negative charged). Electrostatic force pulled the loop to the opposite direction of the substrate channel and made it easier for substrate to enter the lipase catalytic domain. Purified lipase was characterized and the result showed that Km of 2-28 lipase decreased by 10% compared with Km of the parent lipase, and Kcat was 2.75 fold improved than that of the original lipase.

Abstract:Firstly, We used error-prone PCR to induce mutations on Armillariella tabescens MAN47 β-mannanase gene, Secondly, we cloned the mutated fragments into secreted expression vector pYCα, Then the recombinant plasmids were transformed into Saccharomyces cerevisiae BJ5465 after amplified and extracted in DH5α cells. Through three cycles of error-prone PCR we built a mutant database, Then we screened one optimum (named M262) from about 104 mutants. The evoluted MAN47 β-mannanase displayed both higher thermal stability and activity than wide type. The evoluted enzyme M262 retained high activity after treatment at 80oC for 30 min, whereas, the wild type nearly lost activity under this condition. Meanwhile, the activity of M262 can reach to 25?U/mL, which is 4.3 times as wide type under optimum temperature. In addition, pH stability and pH range of evoluted enzyme M262 were both improved compared with wild-type enzyme. The optimum pH was estimated to be similar to that of wild-type enzyme. The sequence comparison illustrated that there were three nucleotide substitutions (T343A/C827T/T1139C) which carried corresponding amino acid changes (Ser115Thr/Thr276Met/Val380Ala). According to homologous modeling by SWISS-MODEL Repository, three mutated amino acids located at the sixth amino acid of the fourth β-sheet, the first amino acid of the sixth a-helix, the turn between the tenth and eleventh β-sheet, respectively.

Abstract:To improve the expression level and catalytic efficiency of (R)-carbonyl reductase from Candida parapsilosis in Escherichia coli, we optimized the mRNA secondary structure of (R)-carbonyl reductase gene in translational initiation region (from +1 to +78), and constructed the corresponding variant. The formation of hairpin structure was signi?cantly reduced and the Gibbs free energy was dramatically decreased from -9.5 kcal/mol to -5.0 kcal/mol after optimization. As a result, the expression level of (R)-carbonyl reductase in the variant was increased by 4-5 times and its specific activity in cell-free extracts was enhanced by 61.9% compared to the wild-type strain. When using the whole cells as catalyst and 2-hydroxyacetophenone as substrate with a high concentration of 5.0 g/L, the variant showed excellent performance to give (R)-1-phenyl-1, 2-ethanediol with optical purity of 93.1% enantiomeric excess and a yield of 81.8%, which were increased by 27.5% and 40.5% respectively than those of the wild-type. In conclusion, the optimization of mRNA secondary structure in translation initiation region can overcome the steric hindrance of translation startup, promote translation smoothly to acquire high expression of target protei1n, and favor protein folding correctly to efficiently improve the enzyme specific activity and biotransformation function.

Abstract:In the present study, through a functional strategy, a metagenome library of the marine microbes from the surface water of the South China Sea was screened for β-glucosidase and six positive clones were obtained. One of these clones, pSB47B2, was subcloned and further analysed in sequence. The result showed that there was an open reading frame for a novel β-glucosidase, which was nominated as bgl1B. Using pET22b(+) as vector and Escherichia coli BL21(DE3) as host, Bgl1B was overexpressed recombinantly with high yield obtained and substantial enzymatic activity detected. The recombinant protein (rBgl1B) was purified by Ni-NTA affinity chromatography and further biochemically characterized. The results indicated that, with pNPG as substrate, the optimum pH and temperature for the hydrolytic activity of rBgl1B were about 6.5 and 40oC respectively. Under the optimum conditions, rBgl1B hydrolyzed pNPG with an activity up to 39.7 U/mg, Km and Vmax being 0.288 mmol/L and 36.9 μmol/min respectively. In addition, rBgl1B could also hydrolyze cellobiose, with a Km of 0.173 mmol/L and a Vmax of 35 μmol/min. However, we did not detect evident hydrolytic activity of rBgl1B to lactose, maltose, sucrose, and CMC. The enzymatic activity of rBgl1B to pNPG was stimulated to certain degrees by low concentration of Ca2+ or Mn2+, whereas it exhibited significant tolerance against high Na+. Distinguished from most of the β-glucosidases derived from fungi, which display the highest activities under acidic conditions, rBgl1B exhibited relatively higher activity and stability at pH between 7.0 and 9.0.

Abstract:The aims of this research were to isolate a Aspergillus niger strain with higher β-glucosidase activity. We utilized the β-glucosidase producing strain Aspergillus niger CGMCC 3.316 as the original strain to first obtain a mutant 3-3M through ultraviolet irradiation. Then we studied the conditions of protoplast release and regeneration for strain 3-3M. We treated the protoplasts of strain 3-3M via ultraviolet irradiation and obtained another isolated mutant 60B-3D. The strain 60B-3D showed much higher beta-glucosidase production than the original strain and 3-3M strain. The β-glucosidase activity of strain 60B-3D was 23.4?IU/mL, with an improvement of 39% compared with the original strain, and 23% compared with strain 3-3M. We also studied the fermentation process of strain 60B-3D, and compared it with the original strain and strain 3-3M. We found the strain 60B-3D exhibited an improvement in xylanase production. The comparison results also showed that the strain 60B-3D secreted more protein. These results were beneficial for producing β-glucosidase through this productive mutant.

Abstract:An enzyme-displaying yeast as a whole-cell biocatalyst seemed an alternative to immobilized enzyme, due to its low-cost preparation and simple recycle course. Here, we tried to use a recombinant Pichia pastoris displaying Candida antarctica lipase B (CALB) to catalyze the synthesis of short chain flavor esters in n-heptane. We studi some major influential factors of esterification reactions, such as carbon chain length of the substrates, alcohol structure, enzyme concentration, substrates concentration, molar ratio of the substrates. The acid conversions were determined by titration and gas chromatography analysis. About ten kinds of esters were synthesized successfully, and the acid conversions of eight esters reached as high as 90% after reaction for 6 h. The result also indicated that ethanol and hexanoic acid were the most suitable substrates for this whole-cell catalyst. Under the optimal reaction conditions (the amount of lipase 20 g/L (306.0 U/g-dry cell), hexanoic acid concentration 0.8 mol/L, the molar ratio of hexanoic acid to ethanol 1:1.1), hexanoic acid conversion reached 97.3% after reaction for 1.5 h. To our knowledge, the CALB-displaying P. pastoris whole-cell biocatalyst showed good tolerance for high substrates concentration and exhibited high reaction rate on esterification of short chain flavor esters among the present enzyme/cell reported. Thus, CALB-displaying P. pastoris whole-cell biocatalyst was promising in commercial application for flavor esters synthesis in non-aqueous phase.

Abstract:We developed a new enzymatic-catalyzing producing process of glucose laurate monoester. In the process we used Candida antarctica lipase B-displaying Pichia pastoris whole-cells as biocatalyst, glucose as the acyl acceptor and lauric acid as the acyl donor. The product glucose laurate monoester was purified by silica gel column chromatography and preparative liquid chromatography, and identified by liquid chromatography-mass spectrometry. Then we optimized the process from various aspects, such as solvent composition, ratio of dmethyl sulfoxide to 2-Methyl-2-butanol (V/V), catalyst dosage, substrate concentration, water activity and temperature. The optimal reaction conditions were: glucose 0.5 mmol/L, lauric acid 1.0 mmol/L, ratio of 2-Methyl-2-butanol to Dmethyl sulfoxide is 7:3 in 5 mL volume, temperature 60°C, the best initial water activity of whole-cells biocatalyst is 0.11. The maximum glucose conversion could be 48.7% after 72 h.

Abstract:Carnosine (β-Ala-L-His) has high antioxidant activity, and it is widely used in biology, chemical engineering, medicine and other fields. Its analogue syntheised in non-aqueous solvent and catalyzed by enzymes is high-effective but low-price, so it has great prospect. Here, we synthesized a carnosine analogue imidazole 4(5)-alanylamide-5(4)-carboxylic acid with imidazole-4,5- dicarboxylic acid and L-Alanine as substrates, α-chymotrypsin as catalyst in tetrahydrofuran (THF) solvent. Based on the orthogonal experiments, the optimized synthetic conditions are 4,5-dicarboxylic acid : L-alanine = 1:3 (m/m), α-chymotrypsin : substrates (4,5-dicarboxyl acid and L-alanine )=1:200 (m/m), pH 8 phosphate buffer:THF = 1.6:10 (V/V), reaction temperature 35°C, time 1.5 h. We separated the product with silica gel G60 thin-layer chromatography (TLC), and a new spot appeared at Rf (ratio to front) = 0.81; then the new spot was purified and characterized with UV spectra, high performance liquid chromatogram (HPLC) and 13C NMR (13C nuclear magnetic resonance). The UV spectra shows two new absorption peaks at 310 nm, and the peak in 253 nm is largely strengthened; HPLC reserve times are all 4.5 min at 253 nm, 310 nm, 330 nm; 13C NMR shows 8 carbons. Combing with the catalytic mechanism of α-chymotrypsin, structure of the analogue is confirmed, i.e. imidazole 4(5)-alanylamide-5(4)-carboxylic acid.

Abstract:The cgt gene was isolated from Paenibacillus macerans by PCR amplification and was inserted into vectors of pPIC9K and pMA5. The recombinant vectors were transformed to Pichia pastoris KM71 and Bacillus subtilis WB600, respectively. The results showed that a-CGTase activity in the culture media of recombinant P. pastoris was only 0.2 U/mL, while it was 1.9 U/mL in recombinant B. subtilis. In addition, we optimized the culture conditions of the recombinant B. subtilis strain. After cultivation at 37oC for 24 h with shake flask, the CGTase forming activity in culture media reached to 4.5 U/mL (hydrolysis activity was 3200 IU/mL), which is 9.8-fold to that of the original strain P. macerans.

Abstract:In order to increase the production and productivity of alkaline polygalacturonate lyase (PGL), we studied the mixed carbon sources feeding strategies during the induction phase by recombinant Pichia pastoris GS115. Glycerol, sorbitol or lactic acid co-feeding with methanol all enhanced the PGL production. Among all the feeding strategies, the sorbitol co-feeding strategy was most significant. By using this strategy, the PGL activity and productivity reached 1593 U/mL and 16.7 U/(mL·h). Compared to the control, the enhancements of PGL activity and productivity were 84.6% and 45.2 % respectively, when we set the sorbitol feeding rate at 3.6 g/(h·L).

Abstract:In this study, the mature peptide sequence of a pectin lyase gene A was amplified from Aspergillus niger strain EIM-6 by using RT-PCR reverse transcription technique. The cloned gene was then inserted into a Pichia pastoris expression vector pPIC9k to produce the recombinant expression plasmid pPIC9K-pelA. By using electric shocks, we successfully transformed the recombinant pPIC9K-pelA into Pichia pastoris GS115. The activity of the engineered strain reached to 2.3 U/mL after induction with the final concentration of 1.5% methanol. SDS-PAGE analysis r evealed that the pPIC9K-pelA transformant had an additional protein band of approximately 38 kD, which was not present in the control. There were no significant differences between the recombinant and native pectin lyase with regard to their hydrolysis activities.

Abstract:We has studied the feasibility of preventing protein from denature during covalent immobilization by “conformation memory”, which was achieved by freeze-drying under enzyme active conformation and cross-linked with carrier under micro-aqueous media (MAM). Horseradish peroxidase (HRP) and chitosan beads have been used as the model enzyme and carrier. The MAM consisted of 99% dioxane and 1% water. We compared the immobilized HRP under MAM with that under traditional aqueous solvent, found that the optimum temperature of both was raised to 60°C, and the optimum pH was 6.5. However, the MAM-immobilized HRP had shown less activity loss during usage and six times higher activity than that immobilized under aqueous solvent. After 30 min incubation at 70°C, the MAM-immobilized HRP remained 75.42% activity while the aqueous-media-immobilized enzyme only 15.4%. The MAM-immobilized HRP has shown a better operation stability with 77.69% residue activity after 5 times of repeat operation while the aqueous-media-immobilized enzyme only 16.67%. In addition, the MAM-immobilized HRP had also shown more advantages when used in phenol removal. We constructed enzyme electrodes (CS-HRP-SWCNTs/Au) to further display the different properties of the two immobilized HRP. MAM-immobilized HRP-electrode has shown two times stronger response signal to H2O2 than that immobilized under aqueous media, which indicated a better enzyme activity of MAM-immobilized HRP. Our research demonstrated that the conformation memory, to some extent, did contribute to preventing protein from denaturing when use HRP as a model, and it is feasible to immobilize enzyme by covalent cross-linking method under micro-aqueous media.

Abstract:We prepared Fe3O4 magnetic nanoparticles (MNPs) with the size of 10 nm by chemical coprecipitation. The effects of six aqueous-organic solvents, including tetrahydrofuran, dioxane, acetone, N, N-Dimethylformamide, methylalcohol, and Dimethyl Sulfoxide, on peroxidase mimetic activity of Fe3O4 MNPs were studied and compared with that of horseradish peroxidase (HRP). The relative activity of Fe3O4 MNPs droped sharply as the elevation of organic solvent concentration increased from 30% to 75% (V/V). In 15% organic solutions, the optimum activity of Fe3O4 MNPs was observed around 50oC, under pH 3.6. After being treated at different temperatures and pH in 15% organic solutions, even under 75% concentration, Fe3O4 MNPs still preserved most of the activity when reacting in aqueous phase. The catalytic performances of Fe3O4 MNPs under given conditions were generally more superior to that of HRP. For it costs lower and it is easy to be prepared and segregated magnetically for recycle, to use the magnetic nanoparticles as a substitution for HRP has potential to be applied into organic catalysis.

Abstract:There is growing interest in biodiesel and this results in the accumulation of glycerol. The exploitation and application of glycerol has attracted more and more attention. In the current study, glycerol was biotransformed to produce 3-hydroxypropionaldehyde by genetic engineering bacteria. It is known that 3-hydroxypopionaldehyde has been widely used as an important intermediate for chemicals, effective antimicrobial agent, and fix agent for tissues. A pair of primers was designed on the basis of the sequence of both NH2-terminus and the amino acid sequence of glycerol dehydratase reported by NCBI, and a fragment about 1.6 kb was obtained by PCR amplification using the total genome DNA of Lactobacillus reuteri as template, then the fragment was cloned to the pMD18-T vector and sequenced. Two specific primers were designed according to the obtained sequence, and a fragment with length of 1674 bp was amplified using PCR with these two specific primers. Consequently, the resulting products were digested with EcoR I and Hind III and ligated using T4 DNA ligase to the pET28b vector digested with the same enzymes. The recombinant plasmid, named pET28b-dhaB, was transformed into E. coli BL21. The positive clones were induced with IPTG and the expression products were further analyzed by SDS-PAGE, indicating that protein with a molecule weight of around 65 kD was obtained. Furthermore, the glycerol dehydratase activity was evaluated and compared with the wild type strain as well.

Abstract:Lipases are important biocatalysts that are widely used in food processing and bio-diesel production. However, organic solvents could inactivate some lipases during applications. Therefore, the efficient cloning and expression of the organic solvent-tolerant lipase is important to its application. In this work, we first found out an organic solvent-tolerant lipase from Staphylococcus saprophyticus M36 and amplified the 741 bp Lipase gene lip3 (GenBank Accession No. FJ979867), by PCR, which encoded a 31.6 kD polypeptide of 247 amino acid residues. But the lipase shared 83% identity with tentative lip3 gene of Staphylococcus saprophyticus (GenBank Accession No. AP008934). We connected the gene with expression vector pET-DsbA, transformed it into Escherichia coli BL21 (DE3), and obtained the recombinant pET-DsbA-lip3. With the induction by 0.4 mmol/L of isopropyl beta-D-thiogalactopyranoside at pH 8.0, OD600 1.0, 25oC for 12 h, the lipase activity reached up to 25.8 U/mL. The lipase expressed was stable in the presence of methanol, n-hexane, and isooctane, n-heptane.

Abstract:We investigated the transesterification of crude cottonseed oil with methyl acetate to biodiesel, by using Lipozyme TL IM and Novozym 435 as catalysts. Results showed that the biodiesel yield significantly increased with the addition of methanol into the reaction system, and the highest biodiesel yield of 91.83% was achieved with the optimum conditions as follows: n-hexane as solvent, molar ratio of methyl acetate to oil 9:1, 3% methanol based on the oil mass to inhibit the creation of acetic acid, 10% Lipozyme TL IM and 5% Novozym 435 as catalyst based on the oil mass, reaction temperature 55°C and reaction time 8 h. Additionally, we explored the kinetics of lipase-catalyzed crude cottonseed oil to biodiesel, and proposed a kinetic model.

Abstract:We used adsorption method to immobilize the lipase by using nano-silica dioxide as the carrier. And we also studied different immobilization conditions effects on the catalytic activity. We got optimize immobilization conditions which were as follow the lipase quantity of 28 300 U/g, temperature of 45oC, pH of 7.5 and treated for 10 h. Under these conditions the immobilized lipase activity yield 3867 U/g carrier. The best reactive temperature for immobilized lipase was 45oC and higher than 5oC for free enzyme, and the optimal pH dropped to 5.5 compared that of free enzyme (pH 7.0). The immobilized lipase stability for thermal and pH are improved than free lipase. When temperature was below 70oC the immobilized enzyme activity was over 70% than initial activity. The free lipase activity just kept original 30% at 50°C. When pH was 5–8, the immobilized lipase activity was still more than 50% and the free lipase only remained 20%. When we used the immobilized neutrophil lipase catalyzing different oil to produce biodiesel such as soybean oil, rapeseed oil and waste oil, the esterification rate of rapeseed oil was the highest.

Abstract:In order to enhance the utilization efficiency, reduce the environmental pollution of traditional chemical treatment and the agriculture waste incineration; we studied the ligninase production by Coprinus comatus, Aspergillus niger and Trichoderma reesei through the plate screening. The results showed that C. comatus mixed culture with T. reesei have a good compatibility and higher yields of Laccase. On the basis of this pre-experiment, we studied the optimal conditions of mixed culture for enzyme production. Under the optimal conditions: the inoculation proportion of C. comatus and T. reesei (5:2), the interval of time (12?h), the temperature 26oC, the shake rotation speed 150 r/min, fermented for 3 days, the Laccase activity reached 3267.1 U/mL, increased by 106% contrasted with single culture of C. commatus.

Abstract:3-ketosteroid-9α-hydroxylase (KSH), a key enzyme in the microbial steroid degradation, is highly valuable for the production of some stroid drugs. Degenerate primers were designed by comparing the ksh from Rhodococcus erythropolis SQ1 and its homologous sequences in the reported genome of Mycobacteria. Subsequently, a gene fragment of KSH was cloned from Mycobacterium sp. NwIB-01, a sterol-transforming bacterium isolated from soil in our lab. According to the conservative sequence, the full-length 1188 bp gene encoding ksh (designated as M.S.-ksh) was obtained by chromosome walking, which showed 85% identity with the ksh of M. smegmatis mc2155. The heterologous expression of KSH was achieved in Escherichia coli BL21(DE3) using the pET-32a-c(+) vector system. The expressed KSH protein was mostly in soluble form after IPTG induction at 30oC and accounted for more than 30% of total bacterial proteins according to SDS-PAGE electrophoresis. The molecular mass of KSH was about 45 kD, which was exactly the size predicted. After Ni2+ affinity chromatography, the purity of the target protein was more than 90%. Our work will definitely contribute to the industrial production?of some steroid drugs by developing KSH genetically engineered bacteria.

Abstract:We optimized the medium for recombinant dextransucrase expression in engineering strain Escherichia coli BL21 (DE3)/pET28-dexYG by an Orthogonal experiment. After the medium had been decided, we studied the effect of temperature, sucrose concentration and pH value on the yield. The results indicated that optimal conditions were adding IPTG of 0.25 mmol/L when OD600 reached 2.0 and cultivation lasted for 4 h at 25°C. Under the selected medium and these conditions, the dextransucrase activity expressed by the engineering strain was high activity. Maximal activity reached 110.16 U/mL sucrose concentration effects the dextran yield grately. The results for dextransucrase expression would provide foundation for industrial application of dextransucrase.

Abstract:A sensitive electrochemical biosensor based on Aflatoxin-Oxidase (AFO) was developed for detection of sterigmatocystin (ST). The enzyme was immobilized on chitosan-single-walled carbon nanotubes (CS-SWCNTs) hybrid film, which attached to the poly-o-phenylenediamine (POPD)-modified Au electrode. The fabricated procedures of the biosensor were characterized with atomic force microscopy (AFM), fourier transform-infrared spectroscopy (FT-IR), and electrochemical impedance spectroscopy (EIS). The cyclic voltammetric results of the biosensor indicated that AFO exhibited a surface-controlled and quasi-reversible electrochemical redox behavior with a formal potential of -0.436 V (vs. Ag/AgCl) in 0.1 mol/L PBS (pH 7.0), which resulted from the direct electron transfer between entrapped AFO and the underlying electrode. The enzymatic electrode exhibited an excellent electrocatalytic response to ST. The linear range of ST determination was from 10 ng/mL to 310 ng/mL with correlation coefficient of 0.997, the detection limit was 3 ng/mL (S/N=3), and the response time was less than 10 seconds. The apparent Michaelis-Menten constant () was estimated to be 7.13 mmol/L. The biosensor had the advantages of good repeatability and stability, remaining 85.6% of its original current value after storage at 4oC for a month, and the RSD for 11 replicate determination of 20 ng/mL ST was 3.9%. This AFO/CS-SWCNTs/POPD/Au modified electrode showed high selectivity and sensitivity in real sample analysis, giving values of recovery in the range of 87.6%-105.5%. The proposed method can be applied to the determination of ST in real samples with satisfactory results.

Abstract:We immobilized Candida sp. lipase onto seven kinds of industrial adsorption and ion exchange resins. By determining the activity of each immobilized enzyme, the weakly basic anionic exchange resin of D301 showed the best results for the immobilization of Candida sp. lipase. Comparing the scanning electron micrographs of D301 with Novozym 435 (immobilized Candida antarctica lipase B from Novo Nordisk Corp.), we selected D301 as a carrier for the immobilization of Candida sp. lipase. And we pretreated the resin D301 with the bifunctional agent glutaraldehyde and crosslinked it with Candida sp. lipase. The optimal conditions for the immobilization of Candida sp. lipase were as follows: 8 mL of the amount of 5% glutaraldehyde solution, five hours of the time pretreated D301 with glutaraldehyde, 1.0 g/L the concentration of Candida sp. lipase used, pH of the phosphate buffered, 6.0 and 10 hours of time for immobilization, respectively. The activity of immobilized enzyme was over 35 U/mg and the efficiency of immobilization was around 3.5 U/(mg·h).