The Ability of Edible Mushrooms to Act as Biocatalysts: Preparation of Chiral Alcohols Using Basidiomycete Strains

To examine the potential ability of edible mushrooms to act as biocatalysts, 19 basidiomycete strains were screened. Modified media (PG, O, and PGO medium) for liquid cultivation of these basidiomycete strains were designed and tested. Wet cells (>10 g) of 4 basidiomycete strains (Pleurotus salmoneostramineus H7, P. salmoneostramineus H13, Ganoderma lucidum NBRC31863, Flammulina velutipes NBRC31862) were harvested from PGO medium for 7 days. The stereoselective reduction of α-keto esters using the 4 strains was tested. It was found that each of these strains had a reducing activity toward 6 aliphatic α-keto esters. In the presence of L-alanine as an additive, the reduction of ethyl 2-oxobutanoate and ethyl 2-oxopentanoete by P. salmoneostramineus H7 produced the corresponding alcohol with a high conversion ratio and with excellent enantiomeric excess (>99% e.e. (R)). Furthermore, ethyl pyruvate, ethyl 2-oxobutanoate, and ethyl 2-oxopentanoate were predominantly reduced to the corresponding (R)-hydroxy ester (>99% e.e.) by G. lucidum. Thus, we found that these edible mushrooms have great potential to be used as biocatalysts for the stereoselective reduction of carbonyl compounds.

Asymmetric Syntheses Aided by Biocatalysts

This article summarizes the achievements of the authors' group in the area of biocatalyst catalyzed organic reactions in recent 10 years. A strain of Geotrichum sp. obtained by screening is capable of stereoselectively reducing a number of carbonyl compounds. In many cases, the stereochemistry is complementary with that obtained by baker's yeast. Therefore, this micro organism provides a useful pathway to the preparation of alcohol compounds with specific configurations. On the other hand, a number of plant sources have been screened for oxynitrilases and the hydrocyanation reactions of various arylcarboxaldehydes have been investigated. A “micro aqueous reaction system' was invented, by which a series of novel optically active cyanohydrins were prepared. On this basis, a high through put continuous reaction system has been designed. This paper also describes examples of the syntheses of bio active compounds by using the optically active compounds obtained from the above mentioned catalytic reactions as precursors.

Conference Paper: Green Industry Adapted to Recycling Needs of Lubumbashi City and Surrounding Areas in Democratic Republic of the Congo

The interest of this conference is agricultural, environmental, bioenergetics, and sanitary. In that context, domestic, agricultural and industrial environments produce organic waste, which needs to be collected, selected, stored and recycled properly in order to avoid environmental pollution and promote agriculture. The green Industry proposed involves the conversion of natural, non-toxic organic waste in order to efficiently produce organic fertilizers for agriculture. These types of fertilizers from biological origin are suitable because they are not toxic for human and the environment. Enzymatic reactions described in this presentation concern mainly the hydrolysis of proteins, sugars and lipids, the acidification of intermediate products from hydrolysis, the formation of acetate, and the production of methane. In other words, this review is timely as it discusses for the chemical behavior or the reactivity of different functional groups to better understand the enzymatic catalysis in the transformations of residual proteins, carbohydrates, and lipids to generate biomethane and fertilizers. In the same perspective, this review is to enrich the documentation related to organic reactions catalyzed by enzymes, which occur in the anaerobic degradation of residual organic substances, with emphasis on the structures of organic compounds and reaction mechanisms. This will allow understanding the displacement of the electrons of a reactive entity rich in electrons to another reactive entity that is poor in electrons to form new bonds in products.

Polymeric biomaterial hydrogels. I. Behavior of some ionotropic cross-linked metal-alginate hydrogels especially copper-alginate membranes in some organic solvents and buffer solutions

The change in rheological and mechanical properties for some ionotropic cross-linked metal-alginate hydrogel complexes in particularly copper-alginate membranes in the presence of some organic solvents (benzene, toluene, xylene, carbon tetrachloride, ace-tone, chloroform, dichloroethane, isobutyl alcohol and ethyl alcohol) or buffer solutions (acetates, borates and universal buffers) have been investigated. The experimental results showed a remarkable tendency of the studied hydrogels for shrinking in polar solvents, whereas no influence was observed for the hydrogels in non-polar solvents. On the other hand, the gels were found to swell or shrink in the buffer solutions depending on the pH of the buffer used. The swelling extent for hydrogel spheres was found to decrease in the order Cu > Ba ≈ Ca > Zn > Pb-alginates in universal buffers of pH = 5.33. The factors affected this behavior have been examined and discussed.

Enantioselective Conversion of Racemic Felodipine to S(-)-Felodipine by Aspergillus niger and Lipase AP6 Enzyme

The present study involves the enantioselective resolution of racemic Felodipine by using free and immobilized forms of microbial cultures as well as an enzyme (Lipase AP6). Among the microbial cultures employed in the present study, Aspergillus niger, Sphingomonas paucimobilis, Cunninghamella elegans, Escherichia coli, Pseudomonas putida and Cunninghamella blakesleeana were found to possess capability of enantioselective resolution of racemic Felodipine. The enantiomeric excess (ee%) of Felodipine after reaction catalyzed by whole-cell A. niger and S. paucimobilis was found as 81.59 and 71.67%, respectively. Immobilization enhanced the enantioselectivity (enantiomeric ratio (E)) of the biocatalysts and hence this led to enhanced enantiomeric purity of the drug. The ee% values were found to be enhanced in reactions catalyzed by A. niger and S. paucimobilis cultures after immobilization as 98.27 and 93.56%, respectively. Enantiomeric ratio (E) of the reactions catalyzed by all the biocatalysts has been improved after immobilization. E value of the reaction catalyzed by immobilized A. niger was found to be excellent (E > 100) and hence the drug showed high enantiomeric purity. In lipase AP6 catalyzed study, the enantioselectivity was enhanced after immobilization with excellent E value, which led to enhanced enantiomeric purity of the drug (99.21% ee%).

The setereoselective preparation ofβ-keto esters using baker's yeast in the presence of rice wine koji

By the addition of rice wine koji, enhancement of the reactivity was observed for the baker＇s yeast reduction of β-keto esters into （S）-β-hydroxy esters with high enantiomeric purity （73-98%）.

Hemoglobin-biocatalyzed Synthesis of Conducting Molecular Complex of Polyaniline and Lignosulfonate

A new biocatalyst route for the synthesis of a conducting polyaniline （PANI）/ lignosulfonate （LGS） complex was presented.Four different catalysts such as hemoglobin （Hb）,5,10,15,20-tetrakis （meso-hydroxyphenyl） porphyrin,iron （II） tetrasulfophthalocyanine and ferric chloride were used to polymerize aniline in the presence of a natural polyelectrolytes template LGS.The experimental results show that Hb is an effective catalyst in this case and the synthesis is simple,and the conditions are mild in that the polymerization may be carried out in lower pH （1.0-4.0） buffered solution and optimal pH of 2.0.Varying concentrations of aniline,LGS and H2O2 in feed the favorable conditions for the production of PANI were determined.UV-vis absorption,FTIR,elemental analysis,conductivity,cyclic voltammetry and thermogravimetric analyses confirm the formation of thermally stable and electroactive PANI.

Biocatalytic Synthesis of Pyruvate from DL-lactate with Enzymes in Pseudomonas sp.

A novel method of preparing pyruvate from DL-lactate catalyzed by enzymes from a bacterial strain of Pseudomonas sp. SM-6 was proposed. Catalytic processes of cell-free extract enzymes and immobilized enzymes were evaluated. The kinetic data were studied, too.

Molecular Basis for Stereospecific Hydrolysis of Ethyl Mandelate by Thermophilic Esterase

The stereospecific hydrolysis of mandelate can be effectively catalyzed by hyperthermophilic acylpeptide esterase APE 1547（Aeropyrum pernix esterase 1547）. APE 1547 used in this reaction showed a remarkable stereodi-scrimination in favour of R-mandelic acid（99% e.e.） with an enantiomeric ratio E〉200. The results of computer simulation are consistent with the experimental results. It can be inferred that the R-substrate adopted a binding mode productive of the reaction due to the formation of the hydrogen bond at the active site of APE 1547.

Preparation of Chiral Hydroxy Esters Using Actinobacteria: Biocatalyst Activity of Marine-Derived <i>Micromonospora</i>and <i>Streptomyces</i>Strains

To research the potential ability of marine-derived actinomycetes to act as biocatalysts, 8 Micromonospora strains and 5 Streptomyces strains were screened. Two recommended media (227 and 1076 media) and 2 modified media (1076-25% and P-1076-25% media) for liquid culture of these marine-derived actinomycetes were tested. As a result, 2 Micromonospora strains (Micromonospora sp. NBRC107096 and 107097) cultured with the 1076-25% medium and 2 Streptomyces strains (Streptomyces tateyamensis NBRC105048 and Streptomyces sp. NBRC105896) cultured with P-1076-25% medium showed a good growth. The stereoselective reduction of α-keto esters using these 4 actinomycetes was tested. As a result, it was found that these strains had a reducing activity toward various α-keto esters. The introduction of L-glutamate or sucrose as an additive remarkably increased the conversion ratios in the reduction of substrates by the Micromonospora strain. Furthermore, in the presence of L-alanine, Streptomyces tateyamensis NBRC105048 reduced ethyl pyruvate, ethyl 2-oxobutanoate, ethyl 2-oxopentanoate, ethyl 2-oxohexanoate, and ethyl 3-methyl-2-oxobutyrate to the corresponding α-hydroxy ester with a high conversion ratio and with excellent enantiomeric excess. Thus, we found that these marine-derived actinomycetes have great potential to be used as biocatalysts for stereoselective reduction of carbonyl compounds.

A fungal CYP from Beauveria bassiana with promiscuous steroid hydroxylation capabilities

Hydroxylation of steroid core is critical to the synthesis of steroid drugs.Direct sp^(3) C-H hydroxylation is challenging through chemical catalysis,alternatively,fungal biotransformation offers a possible solution to this problem.However,mining and metabolic engineering of cytochrome P450 monooxygenases(CYPs)is usually regarded as a more eco-friendly and efficient strategy.Herein,we report the mining and identification of a new steroid CYP(CYP68BE1)from Beauveria bassiana by transcriptomics,heterologous expression,in vivo and in vitro functional characterization.The catalytic promiscuity of CYP68BE1 was explored,and CYP68BE1 showed promiscuously and catalytically versatile,which is qualified for monohydroxylation on C11α,C1α,C6βand dihydroxylation on C1β,11αand C6β,11αof six steroids,leading to the production of key steroid intermediates required in the industrial synthesis of some indispensable steroid drugs.Molecular dynamics simulations were performed,revealing the molecular basis of different binding orientations of CYP68BE1 with different substrates.The discovery of CYP68BE1 offers a promising biocatalyst for enriching the steroid structural and functional diversity,which also can be applied to biosynthesize valuable steroid drug intermediates.

Cellulosomal hemicellulases:Indispensable players for ensuring effective lignocellulose bioconversion

The bioconversion of lignocellulose has attracted global attention,due to the significant potential of agricultural and forestry wastes as renewable zero-carbon resources and the urgent need for substituting fossil carbon.The cellulosome system is a multi-enzyme complex produced by anaerobic bacteria,which comprises cellulases,hemicellulases,and associated enzymatic and non-enzymatic components that promote biomass conversion.To enhance their efficiency in degrading recalcitrant lignocellulosic matrices,cellulosomes have been employed to construct biocatalysts for lignocellulose bioconversion,such as consolidated bioprocessing and consolidated bio-saccharification.Hemicelluloses,the second most abundant polysaccharides in plant cell walls,hold valuable application potential but can also induce inhibitory effects on cellulose hydrolysis,thus highlighting the indispensable roles of hemicellulases within the cellulosome complex.This review evaluated current research on cellulosomal hemicellulases,comparing their types,abundance,and regulation,primarily focusing on eight known cellulosome-producing species of different origins.We also reviewed their growth conditions,their hemicellulose-degrading capabilities,and the inhibitory effects of hemicellulose on cellulosome-based lignocellulose saccharification.Finally,we proposed strategies for targeted enhancement of hemicellulase in cellulosomes to improve lignocellulose bioconversion in future studies.

High-level production of𝛾γ-aminobutyric acid via efficient co-expression of the key genes of glutamate decarboxylase system in Escherichia coli

Biosynthesis of the functional factor𝛾γ-aminobutyric acid(GABA)in bacteria involves two key proteins an intra-cellular glutamate decarboxylase(GadB)and a membrane-bound antiporter(GadC).Efficient co-expression of suitable GadB and GadC candidates is crucial for improving GABA productivity.In this study,gadBΔC11 of Lacti-plantibacillus plantarum and gadCΔC41 of Escherichia coli were inserted into the designed double promoter(P T7lac and P BAD)expression system.Then,E.coli Lemo21(DE3)was chosen as the host to minimize the toxic effects of GadCΔC41 overexpression.Furthermore,a green and high-efficiency GABA synthesis system using dormant engineered Lemo21(DE3)cells as biocatalysts was developed.The total GABA yield reached 829.08 g/L with a 98.7%conversion ratio within 13 h,when engineered E.coli Lemo21(DE3)cells were concentrated to an OD 600 of 20 and reused for three cycles in a 3 M L-glutamate solution at 37℃,which represented the highest GABA productivity ever reported.Overall,expanding the active pH ranges of GadB and GadC toward physiological pH and employing a tunable expression host for membrane-bound GadC production is a promising strategy for high-level GABA biosynthesis in E.coli.

Visible light assisted enzyme-photocatalytic cascade degradation of organophosphorus pesticides

The worldwide application of organophosphorus pesticides(OPs)has promoted agricultural development,but their gradual accumulation in soil and water can seriously affect the central nervous system of humans and other mammals.Organophosphorus hydrolase(OPH)is an effective enzyme that can catalyze the degradation of the residual OPs.However,the degradation products such as p-nitrophenol(p-NP)is still toxic.Thus,it is of great significance to develop a multi-functional support that can be simultaneously used for the immobilization of OPH and the further degradation of p-NP.Herein,a visible light assisted enzyme-photocatalytic integrated catalyst was constructed by immobilizing OPH on hollow structured Au-TiO_(2)(named OPH@H-Au-TiO_(2))for the degradation of OPs.The obtained OPH@H-Au-TiO_(2)can degrade methyl parathion to p-NP by OPH and then degrade p-NP to hydroquinone with low toxicity by using H-Au-TiO_(2)under visible light.OPH molecules were immobilized on HAu-TiO_(2)through adsorption method to prepare OPH@H-Au-TiO_(2).After 2.5 h of reaction,methyl parathion is completely degraded,and about 82.64%of the generated p-NP is further degraded into hydroquinone.After reused for 4 times,the OPH@H-Au-TiO_(2)retains more than 80%of the initial degradation activity.This research presents a new insight in designing and constructing multi-functional biocatalyst,which greatly expands the application scenarios and industrial value of enzyme catalysis.

High-fructose corn syrup production and its new applications for 5-hydroxymethylfurfural and value-added furan derivatives:Promises and challenges

High fructose corn syrup has been industrially produced by converting glucose to fructose by glucose isomerases,tetrameric metalloenzymes widely used in industrial biocatalysis.Advances in enzyme engineering and commercial production of glucose isomerase have paved the way to explore more efficient variants of these enzymes.The 5-hydroxymethylfurfural can be produced from high fructose corn syrup catalytic dehydration,and it can be further converted into various furanic compounds chemically or biologically for various industrial applications as a promising platform chemical.Although the chemical conversion of 5-hydroxymethylfurfural into furanic compounds has been extensively investigated in recent years,bioconversion has shown promise for its mild conditions due to the harsh chemical reaction conditions.This review discusses pro-tein engineering potential for improving glucose isomerase production and recent advancements in bioconversion of 5-hydroxymethylfurfural into value-added furanic derivatives.It suggests bi-ological strategies for the industrial transformation of 5-hydroxymethylfurfural.

Enantioselective Resolution of (±)-l-Phenylethyl Acetate by Using the Whole Cells of Deep-sea Bacterium Bacillus sp. DL-2

Bacillus sp. DL-2 was isolated from the deep sea of the Western Pacific and further utilized as novel biocatalysts to efficiently asymmetrically hydrolyze (±)-1-phenylethyl acetate. After the optimization of hydrolytic reactions, chiral chemicals (R)1-phenylethanol and (S)-l-phenylethyl acetate were obtained with high optical purities (96% and 99.8%, respectively). Our research is about the asymmeric hydrolysis of (±)-1-phenylethyl acetate using whole-cell biocatalysts. In addition, the optical purity of (S)-l-phenylethyl acetate generated through the kinetic resolution of (±)-1-phenylethyl acetate using the whole-cells of Bacillus sp. DL-2 was the highest report so far. Using the whole cells of deep sea bacterium Bacillus sp. DL-2 as the biocatalysts is an enviromnentally friendly method and will play critical roles in industrial asymmetric synthesis.

Steroids hydroxylation catalyzed by the monooxygenase mutant 139-3 from Bacillus megaterium BM3

The search of new substrates with pharmaceutical and industrial potential for biocatalysts including cytochrome P450 enzymes is always challenging. Cytochrome P450 BM3 mutant 139-3, a versatile biocatalyst, exhibited hydroxylation activities towards fatty acids and alkanes. However, there were limited reports about its hydroxylation activity towards steroids. Herein, an Escherichia coli–based whole-cell extract containing the recombinant 139-3 protein was used as the biocatalyst to screen 13 steroids. Results revealed that 139-3 was able to specifically hydroxylate androstenedione(1) at 1α-position, generating a hydroxylated steroid 1α-OH-androstenedione(1a). To investigate whether C-1αhydroxylation catalyzed by BM3 mutant 139-3 could be industrially used, an optimization of catalyzing conditions was performed. Accordingly, the BM3 mutant 139-3 enzyme was observed to display maximum activity at 37 1C, under p H 7.0 for 4 h, with 37% transformation rate. Moreover, four 139-3variants were generated by random mutagenesis with the aim of improving its activity and expanding substrate scope. Surprisingly, these mutants, sharing a common mutated site R379 S, lost their activities towards androstenedione(1). These data clearly indicated that arginine residue located at site 379 played key role in the hydroxylation activities of 139-3. Overall, these new findings broadened the substrate scope of 139-3 enzyme, thereby expanding its potential applications as a biocatalyst on steroids hydroxylation in pharmaceutical industry.

Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity

Surface display is effectively utilized to construct a whole-cell biocatalyst.Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast.Here,the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae,and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor,recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed.Compared with the wild-type ROL-displaying yeast,the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate.To our knowledge,this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction.Consequently,the yeast whole-cell ROL biocatalyst was constructed with high activity.The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C.Furthermore,this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.

Improved L -phenylglycine synthesis by introducing an engineered cofactor self-sufficient system

L-phenylglycine(L-phg)is a valuable non-proteinogenic amino acid used as a precursor to β-lactam antibiotics,antitumor agent taxol and many other pharmaceuticals.L-phg synthesis through microbial bioconversion allows for high enantioselectivity and sustainable production,which will be of great commercial and environmental value compared with organic synthesis methods.In this work,an L-phg synthesis pathway was built in Escher-ichia coli resulting in 0.23 mM L-phg production from 10 mM L-phenylalanine.Then,new hydroxymandelate synthases and hydroxymandelate oxidases were applied in the L-phg synthesis leading to a 5-fold increase in L-phg production.To address 2-oxoglutarate,NH_(4)^(+),and NADH shortage,a cofactor self-sufficient system was introduced,which converted by-product L-glutamate and NAD^(+)to these three cofactors simultaneously.In this way,L-phg increased 2.5-fold to 2.82 mM.Additionally,in order to reduce the loss of these three cofactors,a protein scaffold between synthesis pathway and cofactor regeneration modular was built,which further improved the L-phg production to 3.72 mM with a yield of 0.34 g/g L-phe.This work illustrated a strategy applying for whole-cell biocatalyst converting amino acid to its value-added chiral amine in a cofactor self-sufficient manner.

Green synthesis,characterization,and catalytic application of a supported and magnetically isolable copper-iron oxide-sodium alginate

Herein,we report a green and facile preparation and characterization procedure of copper nanoparticles stabilized on magnetically separable sodium alginate support(Cu NPs-Fe_(3)O_(4)-SAlg)using Silybum marianum seeds extract and investigate their catalytic reductive chemistry for reduction of environmental pollutants.Indeed,the phytochemical content of the plant extract are responsible to biosynthesis of copper(Cu)nanoparticles and their stabilization by functionalizing the surface of magnetic alginate.Characterization of the biocatalyst was undertaken with FT-IR,XPS,XRD and STEM equipped with EDS.A standard suite of model compounds on which the reductive chemistry was tested were 4-nitrophenol(4-NP),potassium hexacyanoferrate(K3[Fe(CN)6]),and a variety of organic dyes including Congo Red(CR).The as-synthesized Cu NPs-Fe_(3)O_(4)-SAlg catalyst was highly effective in reducing 4-NP to 4-aminophenol in water and in the presence of sodium borohydride as a reducing agent.Also,a high reduction performance was observed with CR and K3[Fe(CN)6]in presence of the Cu NPs-Fe_(3)O_(4)-SAlg catalyst.The catalyst acted within 190 and 35 s respectively,at room temperature in the presence of NaBH4.Thus,the Cu NPs-Fe_(3)O_(4)-SAlg catalyst is a highly efficient catalyst which is also reusable due to its magnetic nature.This work reports a green synthesis of a high-performance Cu NPs-Fe_(3)O_(4)-SAlg catalyst using an environmental friendly method for reduction of 4-NP,CR,and K3[Fe(CN)6]over a short time.