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.
Animals ; Biocatalysis ; Enzymes ; genetics ; metabolism ; Enzymes, Immobilized ; Humans ; Mutagenesis, Site-Directed ; Solvents

Enzyme engineering is a combined technology of enzymology and engineering, which is becoming one of the major fields of modem 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.
Biotechnology ; trends ; China ; Enzymes ; chemistry ; genetics ; metabolism ; Protein Engineering ; trends

To enhance recombinant protein production by CHO cells, We compared the impact of overexpression of metabolic enzymes, namely pyruvate carboxylase 2 (PYC2), malate dehydrogenase Ⅱ (MDH2), alanine aminotransferase Ⅰ (ALT1), ornithine transcarbamylase (OTC), carbamoyl phosphate synthetase Ⅰ (CPSⅠ), and metabolism related proteins, namely taurine transporter (TAUT) and Vitreoscilla hemoglobin (VHb), on transient expression of anti-hLAG3 by ExpiCHO-S. Overexpression of these 7 proteins could differentially enhance antibody production. OTC, CPSI, MDH2, and PYC2 overexpression could improve antibody titer by 29.2%, 27.6%, 24.1%, and 20.3%, respectively. Specifically, OTC and MDH2 could obviously improve early-stage antibody production rate and the culture period was shortened by 4 days compared with that of the control. In addition, OTC and MDH2 had little impact on the affinity of anti-hLAG3. In most cases, overexpression of these proteins had little impact on the cell growth of ExpiCHO-S. MDH2 and ALT1 overexpression in H293T cells could also improve antibody production. Overall, overexpression of enzymes involved in cellular metabolism is an effective tool to improve antibody production in transient expression system.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Enzymes/metabolism* ; Recombinant Proteins/genetics*

Lignocellulose is the most abundant natural biomass. Bioconversion of lignocelluloses becomes a bottleneck for biorefinery, because of its complex structures and heterogeneous composition. Besides screening or engineering approach for single free enzymes with improved properties, an alternative approach is to study synergistic pattern with hydrolysis systems or mimic natural cellulosome for better performance in cellulolytic substrate degradation. Besides, bacterial co-cultures provide another synergistic cellulolytic system. Engineered strains with modified metabolic network could facilitate consolidated bioprocess by increasing yields as well as reducing costs.
Bacteria ; genetics ; metabolism ; Biomass ; Cellulase ; genetics ; metabolism ; Cellulosomes ; genetics ; metabolism ; Enzymes ; metabolism ; Fermentation ; Lignin ; metabolism ; Metabolic Networks and Pathways

DNA Damage ; DNA Glycosylases ; genetics ; metabolism ; DNA Repair Enzymes ; genetics ; metabolism ; Humans ; Phosphoric Monoester Hydrolases ; genetics ; metabolism

Genome-scale metabolic network models have been successfully applied to guide metabolic engineering. However, the conventional flux balance analysis only considers stoichiometry and reaction direction constraints, and the simulation results cannot accurately describe certain phenomena such as overflow metabolism and diauxie growth on two substrates. Recently, researchers proposed new constraint-based methods to simulate the cellular behavior under different conditions more precisely by introducing new constraints such as limited enzyme content and thermodynamics feasibility. Here we review several enzyme-constrained models, giving a comprehensive introduction on the biological basis and mathematical representation for the enzyme constraint, the optimization function, the impact on the calculated flux distribution and their application in identification of metabolic engineering targets. The main problems in these existing methods and the perspectives on this emerging research field are also discussed. By introducing new constraints, metabolic network models can simulate and predict cellular behavior under various environmental and genetic perturbations more accurately, and thus can provide more reliable guidance to strain engineering.
Enzymes ; metabolism ; Genome ; genetics ; Metabolic Engineering ; Metabolic Networks and Pathways ; genetics ; Models, Biological ; Thermodynamics

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 studied 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.
Biocatalysis ; Candida ; enzymology ; Enzymes, Immobilized ; Esters ; metabolism ; Fungal Proteins ; Lipase ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

Industrial enzymes are the "chip" of modern bio-industries, supporting tens- and hundreds-fold of downstream industries development. Elucidating the relationships between enzyme structures and functions is fundamental for industrial applications. Recently, with the advanced developments of protein crystallization and computational simulation technologies, the structure-function relationships have been extensively studied, making the rational design and de novo design become possible. This paper reviews the progress of structure-function relationships of industrial enzymes and applications, and address future developments.
Biocatalysis ; Biotechnology ; Enzymes ; chemistry ; genetics ; metabolism ; Metabolic Engineering ; Protein Engineering ; Structure-Activity Relationship

Carcinoma, Hepatocellular ; genetics ; metabolism ; DNA Methylation ; DNA Restriction Enzymes ; metabolism ; DNA, Neoplasm ; genetics ; Glutathione S-Transferase pi ; genetics ; metabolism ; Humans ; Liver Neoplasms ; genetics ; metabolism ; Polymerase Chain Reaction ; methods

Lysins are murein hydrolases produced by bacteriophage that act on the cell wall of host bacteria to release progeny phages. Research indicated that lysins could kill bacteria effectively and specifically in vitro. To prepare recombinant bacteriophage lysin of Streptococcus (PlyC) and analyze its biological activity, we obtained two genes of PlyC named PlyCA and PlyCB by PCR amplification and inserted them into pET-32a(+), then transformed the recombinant expression vectors pET-32a(+)-PlyCA and pET-32a(+)-PlyCB into E. coli BL21(DE3) respectively. After induction with 0.7 mmol/L IPTG at 30 degrees C for 7 h, PlyCA and PlyCB were successfully expressed, SDS-PAGE analysis determined that they all constituted above 30% of the total cell proteins. After Ni(2+)-NTA affinity chromatography, the purity was more than 95%. With the denaturation and protein refolding, we gained the recombinant PlyC. To determine its biological activity, we adopted turbidimetry and plate count method. Before and after lysin treatment, the cell morphology was studied by scanning electron microscopy (SEM). The results showed that the recombinant PlyC could specifically cleavage Streptococcus pyogenes (group A beta-hemolytic streptococci). Under the incubation time of 60 min with 4 microg/mL PlyC in Streptococcus pyogenes dilution which OD600 was 0.56, the germicidal effect was up to 99.6%, while SEM observations showed that cell wall cracked and presented cell debris. This finding laid the foundation for the further study and achieving an effective treatment for streptococcal infection.
Bacteriolysis ; Enzymes ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Streptococcus pyogenes ; drug effects