Abstract:Enzyme engineering is a combined technology of enzymology and engineering, which is becoming the major foundation for modern biotechnology and bio-economy in near future. Recently, a breakthrough in synthetic biology demonstrates a possibility to develop artificial biosystems for biofuel and biorefinery. As one of the cores in synthetic biology, enzyme engineering has drawn more attentions all over the world. 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 discovery of new enzymes, mechanism of action modes, and production and application of enzymes.

Abstract:Engineering and redesign of enzymes are important to industrial biocatalysis. Fusion enzyme technology, based on fusion protein design, is frequently used in multifunctional enzyme construction and enzyme proximity control. Here, we reviewed the recent progress in molecular design strategy and application studies of fusion enzymes. The concept and features of fusion enzymes were introduced, followed by a systematical summary of the design strategy of fusion enzymes. In particular, the effects of different linker properties on fusion enzymes and their possible mechanisms were discussed. In addition, recent studies on fusion enzyme applications were also discussed. Finally, based on our own studies on fusion enzymes and the current research progress, the key problems in fusion enzyme technology and perspectives of this field were discussed.

Abstract:NAD(P)(H)-dependent oxidoreductase catalyzes the reduction of ketones or aldehydes to prepare a wide variety of valuable chiral alcohols or amines. However, expensive cofactors are absolutely required for the biocatalytic processes with oxidoreductases, which severely hinder their industrial applications. Consequently, the issue on reducing cofactor costs has become one of the major focuses in the field of biocatalysis. With the substantial development in recent years, a number of strategies have been proposed and implemented to solve the cofactor issues in the oxidoreductase catalyzed biocatalysis, including the establishment of cofactor regeneration system, the improvement of endogenous cofactor availability via metabolic engineering and the development of biomimetic agents to replace cofactors. In this review, recent trends and advances on these strategies are presented, and respective advantages and shortcomings are also discussed with a number of examples.

Abstract:Microorganisms contain a large number of biocatalysts, which are of great potential in industrial applications. However, the traditional cultural approaches can obtain only less than 1% of microorganisms. As a culture-independent method, metagenomics is an advanced solution by means of extracting all microbial genomic DNAs in certain environmental habitat, constructing and screening metagenomic libraries to seek novel functional genes. It serves as an effective tool for studying these uncultured microorganisms. Therefore, mining novel biocatalysts from metagenome has drawn the attention of researchers in the world. In this paper, environment sample category, genomic DNA extraction, library construction and screening strategies were reviewed. Recent examples of isolated biocatalysts from metagenomic libraries were presented. Future research directions of metagenomics were also discussed.

Abstract:Alkaline amylase is one of alkaline enzymes with optimum pH in the alkaline range, and it could keep stability and efficiently hydrolyze starch under alkaline conditions. Alkaline amylase finds wide applications in textile, detergent, pharmaceutical, food and other fields. Alkaline amylases could be produced by alkaliphilic microorganisms. In this work, the advances of alkaline amylase production and applications were reviewed.

Abstract:We cloned the lipoxygenase gene (ana-LOX) from Anabaena sp. PCC 7120 and expressed it in Escherichia coli BL21 (DE3) pLysS. We determined the active site of the recombinant ana-LOX through site-directed gene mutagenesis and obtained the shortest length of the functional gene. Meanwhile, we studied the properties of recombinant ana-LOX after purification. The C-terminal of the Aos (allene oxide synthase)-LOX fusion gene in Anabaena sp. PCC 7120 genome was found belonging to LOXs family by bioinformatics analysis. Further results of site-directed gene mutagenesis confirmed that the active sites of ana-LOX were His197, His202, His369, Asn373and Ile455. The shortest length of functional gene was identified to be 1 254 bp based on the strategy of shortening the gene length gradually. The highest activity of recombinant ana-LOX of 6 750 U/mL could be achieved when constructed to pET-32a vector and expressed at low temperature 16 °C. We purified the enzyme by Ni-NTA chelating affinity chromatography, with 60.89% yield and specific activity of 11.4×104 U/mg. The optimum reaction temperature and pH for ana-LOX were 45 oC and 6.0, respectively. Furthermore, the obtained ana-LOX was stable at room temperature. The effect of metal ions on ana-LOX was determined also. Fe2+, Mg2+, Ca2+ could markedly promote the activity of this enzyme whereas Fe3+ and Cu2+ had a strong inhibitory effect on it. Finally, the ana-LOX could improve the microscopical structure of dough. The results of this study will provide a basis for future improvements and food industrial applications of ana-LOX.

Abstract:Rare sugar is a kind of important low-energy monosaccharide that is rarely found in nature and difficult to synthesize chemically. D-allose, a six-carbon aldose, is an important rare sugar with unique physiological functions. It is radical scavenging active and can inhibit cancer cell proliferation. To obtain D-allose, the microorganisms deriving D-psicose 3-epimerase (DPE) and L-rhamnose isomerase (L-RhI) have drawn intense attention. In this paper, DPE from Clostridium cellulolyticum H10 was cloned and expressed in Bacillus subtilis, and L-RhI from Bacillus subtilis 168 was cloned and expressed in Escherichia coli BL21 (DE3). The obtained crude DPE and L-RhI were then purified through a HisTrap HP affinity chromatography column and an anion-exchange chromatography column. The purified DPE and L-RhI were employed for the production of rare sugars at last, in which DPE catalyzed D-fructose into D-psicose while L-RhI converted D-psicose into D-allose. The conversion of D-fructose into D-psicose by DPE was 27.34%, and the conversion of D-psicose into D-allose was 34.64%.

Abstract:Exploring excellent new pullulanase genes, and enriching pullulanase theory are of great importance to realize the industrialization of pullulanase. Three genes, pulA, pulB and pulC, encoding pullulanases, were cloned from Bacillus cereus GXBC-3 by bioinformatics analyzing the open reading frame in Bacillus cereus, annotated as putative I and II pullulanases in the GenBank database. Characteristics of these recombinant enzymes were inducible intracellular expressed in Escherichia coli, the results showed PulA was typical II pullulanase. Recombinant PulA could hydrolyze α-l,4- and α-l,6-glycosidic bonds. Its specific activity was 32.89 U/mg with an optimum temperature of 40 °C and optimum pH 6.5 using pullulan as substrate. And for soluble starch substrate, its specific activity was 25.71 U/mg with an optimum temperature of 50 °C and optimum pH 7.0. PulB and PulC were I pullulanases and only hydrolyzed α-l,6-glycosidic bond. The specific activities, optimum temperature and optimum pH of PulB and PulC for pullulan substrate were 228.54 U/mg, 45 °C, 7.0 and 229.65 U/mg, 45 °C, 6.5, respectively.

Abstract:Activity losing during the covalent immobilization of enzyme is a serious problem. Here we studied organic phase immobilization by using glucose oxidase (GOD) as a model. After lyophilized at optimum pH, GOD is covalently immobilized onto glutaraldhyde-activated chitosan microsphere carrier under the condition of water, 1, 4-dioxane, ether and ethanol separately. The special activities, enzyme characterization and kinetic parameters are determined. Results show that all of the organic phase immobilized GODs have higher special activities and larger Kcat than that of aqueous phase. Under the conditions of 0.1% of glutaraldehyde,1.6% moisture content with 80 mg of GOD added to per gram of carrier, 2.9-fold of the special activity and 3-fold of the effective activity recovery ratio were obtained, and 3-fold of the residue activity was demonstrated after 7 runs when compares1, 4-dioxane phase immobilized GOD with water phase immobilized one. In addition, kinetic study shows that 1,4-dioxane immobilized GOD (Kmapp=5.63 mmol/L, Vmax=1.70 μmol/(min·mg GOD), Kcat=0.304 s?1) was superior to water immobilized GOD (Kmapp=7.33 mmol/L, Vmax=1.02 μmol/(min·mg GOD), Kcat=0.221 s?1). All above indicated GOD immobilized in proper organic media presented a better activity with improved catalytic performance. Organic phase immobilization might be one of the ways to overcome the conformational denature of enzyme protein during covalent modification.

Abstract:Cold-adapted lipases are attractive biocatalysts that can be used at low temperatures as additives in food products, laundry detergents, and the organic synthesis of chiral intermediates. Cold-adapted lipases are normally found in microorganisms that survive at low temperatures. A fungi strain XMZ-9 exhibiting lipolytic activity was isolated from the soil of glaciers in Xinjiang by the screening plates using 1% tributyrin as the substrate and Victoria blue as an indicator. Based on morphological characteristics and phylogenetic comparisons of its 18S rDNA, the strain was identified as Penicillium sp. The partial nucleotide sequences of these two lipase related genes, LipA and LipB, were obtained by touchdown PCR using the degenerate primers designed according to the conservative domains of lipase. The full-length sequences of two genes were obtained by genome walking. The gene lipA contained 1 014 nucleotides, without any intron, comprising one open reading frame encoding a polypeptide of 337 amino acids. The gene lipB comprised two introns (61 bp and 49 bp) and a coding region sequence of 1 122 bp encoding a polypeptide of 373 amino acids. cDNA sequences of both lipA and lipB were cloned and expressed in Escherichia coli BL21 (DE3). The recombinant LipA was mostly expressed as inclusion bodies, and recovered lipase activity at low temperature after in vitro refolded by dilution. Differently, the recombinant LipB was expressed in the soluble form and then purified by Ni-NTA affinity chromatography Column. It showed high lipase activity at low temperature. These results indicated that they were cold-adapted enzymes. This study paves the way for the further research of these cold-adapted lipases for application in the industry.

Abstract:3-Hydroxypropionic acid is an important building block to synthesize lots of industrially valuable chemicals. In this study, we firstly investigated the effects of cell, substrate and product concentrations on biosynthesis of 3-hydroxypropionic acid from 1,3-propanediol by Gluconobacter oxydans ZJB09112 in 50-mL shake flask containing 10 mL transformation liquid. To avoid the inhibition of substrate and product, we adopted fed-batch biotransformation and fed-batch biotransformation coupled with in situ product removal in 2-L bubble column reactor containing 1 L transformation liquid. The results show that high concentrations of substrate and product could inhibit the biotransformation by decreasing the initial reaction rate, and the optimal reaction conditions were as follows: cell concentration 6 g/L, pH 5.5. Fed-batch biotransformation in which the substrate concentration was maintained at 15–20 g/L could obtain product concentration of 60.8 g/L after 60 h, which gave a productivity of 1.0 g/(L·h) and a yield of 84.3%. Furthermore, fed-batch biotransformation coupled with in situ product removal could achieve the total product concentration of 76.3 g/L after 50 h, which gave a productivity of 1.5 g/(L·h) and a yield of 83.7%. The results obtained here may be useful for the application of G. oxydans in biocatalysis industry by using its characteristic of incomplete oxidation of alcohols.

Abstract:Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and I410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced Km value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.