Co-expression of Beta-Glucosidase and Laccase in Trichoderma reesei by Random Insertion with Enhanced Filter Paper Activity.

Trichoderma reesei strain Rut-C30 was modified with enhanced beta-glycosidase (BGL) activity to balance the cellulase system and generated laccase (LAC) protein for lignin degradation. Initially, the binary plasmid p1300-w1 was constructed to express T. reesei bgl2 under the control of promoter P pki and T-nos terminator. Random insertion was performed via Agrobacterium tumefaciens-mediated transformation. A total of 353 mutants were obtained, and 34PTrb2 was exceptionally stable with increased FPA and BGL activity after screening for extracellular enzyme activity. Subsequently, 34PTrb2 was used as parent strain via the same method to insert the lac gene from Fomes lignosus, with promoter P gpd , followed by cbh1 signal peptide trss and T-nos as terminator. Several mutants successfully expressed enzyme LAC with stable activity of approximately 0.13 U/mL. The mutant 15Gsslac increased activity by 40.4% FPA compared with that of the host Rut-C30.

Molecular cloning and over-expression of a fructosyltransferase from Aspergillus niger QU10.

The main commercial production of fructooligosaccharides (FOS) comes from enzymatic transformation using sucrose as substrate by microbial enzyme fructosyltransferase. A fructosyltransferase genomic DNA was isolated from Aspergillus niger QU10 by PCR. The nucleotide sequence showed a 1 941 bp size, and has been submitted to GenBank (KF699529). The cDNA of the fructosyltransferase, containing an open reading frame of 1 887 bp, was further cloned by RT-PCR. The fructosyltransferase gene from Aspergillus niger was functionally expressed both in Escherichia coli and Pichia pastoris GS 115. The highest activity value for the construction with the α-factor signal peptide reached 431 U/mL after 3 days of incubation. The recombinant enzyme is extensively glycosylated, and the active form is probably represented by a homodimer with an apparent molecular mass of 200 kDa as judged from mobility in seminative PAGE gels. The extracellular recombinant enzyme converted sucrose mostly to FOS, mainly 1-kestose and nystose, liberating glucose. FOS reached a maximal value and represented about 58% of total sugars present in the reaction mixture after 4 h reaction. The results suggest that the availability of recombinant Pichia pastoris as a new source of a FOS-producing enzyme might result of biotechnology interest for industrial application.

Molecular dynamic analysis of mutant Y195I α-cyclodextrin glycosyltransferase with switched product specificity from α-cyclodextrin to γ-cyclodextrin.

Alpha-cyclodextrin (α-CD) glycosyltransferase (α-CGTase) can convert starch into α-CD blended with various proportions of ß-cyclodextrin (ß-CD) and/or γ-cyclodextrin (γ-CD). In this study, we verified the catalytic characteristics of purified Y195I α-CGTase and elucidated the mechanism of action with molecular dynamic (MD) simulations. We found that purified Y195I α-CGTase produced less α-CD, slightly more ß-CD, and significantly more γ-CD than wild-type α-CGTase. Correspondingly, α-CD-based K m values increased, and ß-CD- and γ-CD-based K m values decreased. MD simulation studies revealed that the dynamic trajectories of the substrate oligosaccharide chain in the mutant CGTase binding site were significantly different from those in the wild-type enzyme, with reduced hydrophobic interaction, finally resulting in different product specificity and more γ-CD formation.

Novosphingobium kunmingense sp. nov., isolated from a phosphate mine.

A yellow-pigmented, Gram-stain-negative, strictly aerobic, rod-shaped, round-ended bacterium, designated strain 18-11HK(T), was isolated from a phosphate mine situated in the suburb of Kunming in Yunnan province in south-western China. The taxonomic status of this strain was evaluated by using a polyphasic approach. On the basis of 16S rRNA gene sequence similarity, strain 18-11HK(T) was shown to belong to the genus Novosphingobium, showing the highest levels of sequence similarity with respect to 'Novosphingobium ginsenosidimutans' FW-6 (97.2%), Novosphingobium subterraneum DSM 12447(T) (96.7%), Novosphingobium aromaticivorans DSM 12444(T) (96.7%) and Novosphingobium tardaugens DSM 16702(T) (96.3%). Strain 18-11HK(T) had a genomic DNA G+C content of 65.3 mol% and Q-10 as the predominant respiratory quinone. DNA-DNA hybridizations of strain 18-11HK(T) with N. subterraneum DSM 12447(T), N. aromaticivorans DSM 12444(T) and N. tardaugens DSM 16702(T) showed low relatedness values of 29.6, 33.5 and 32.3%, respectively. The predominant fatty acids of strain 18-11HK(T) were summed feature 8 (C18 : 1ω7c and/or C18: 1ω6c), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0, and the major 2-hydroxy fatty acid was C14 : 0 2-OH. The polar lipid profile revealed the presence of sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and some unidentified lipids. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain 18-11HK(T) represents a novel species of the genus Novosphingobium, for which the name Novosphingobium kunmingense sp. nov. is proposed. The type strain is 18-11HK(T) ( = CGMCC 1.12274(T) = DSM 25975(T)).

Structural basis of a mutant Y195I α-cyclodextrin glycosyltransferase with switched product specificity from α-cyclodextrin to ß-/γ-cyclodextrin.

Cyclodextrin glycosyltransferase (EC 2.4.1.19) (CGTase) is an extracellular bacterial enzyme which has the unique capability of forming cyclodextrins from starch. Our previous investigation revealed that a mutant Y195I α-CGTase drastically altered the cyclodextrin specificity by switching toward the synthesis of both ß- and γ-CDs (Xie et al., 2013a,b). In this study, we determined one X-ray structure of the mutant Y195I α-CGTase at 2.3Å. The overall structure was similar to that of the typical ß-CGTase from Bacillus circulans 251, with minor difference in flexible domains since they showed about 70% homogeneity of amino acid sequences. The central site with isoleucine tended to be more flexible than tyrosine thus made the sugar chain, during the cyclization process, form a larger cyclodextrin like ß- and γ-CDs surrounding the central site instead of α-CD. Superposition of the structure of Y195I α-CGTase with those of ß-CGTase and γ-CGTase showed that residues Lys232, Lys89 and Arg177 at subsites +2, -3 and -7 could form smaller substrate binding cavity. In summary, the crystal structure revealed that moderate increase of mobility of the central site resulted in the switched product specificity from α-CD to ß- and γ-CDs of the mutant Y195I α-CGTase. The space differences alongside the active domain may be another factor that impacts the product specificity of the CGTase.

Mutation of tyrosine167histidine at remote substrate binding subsite -6 in α-cyclodextrin glycosyltransferase enhancing α-cyclodextrin specificity by directed evolution.

α-Cyclodextrin glycosyltransferase (α-CGTase) can convert starch into α-cyclodextrin with various proportions of ß-cyclodextrin and/or γ-cyclodextrin in the products. To improve the α-cyclodextrin-forming specificity, directed evolution on the wild-type α-CGTase was performed by constructing mutant library with error-prone PCR method. The positive mutant strains were selected in combination of starch plate screening with HPLC detection of the products. An α-CGTase from the mutant strain (assigned No. 95) was found to be able to increase the α:ß ratio in product mixture from 3.4 to 7.8 in comparison with the wild-type α-CGTase. Sequence alignment indicated that two mutations occurred in the No. 95 mutant α-CGTase, which were Y167H and A536V. Reverse mutation revealed that Y167H was responsible for this change. A series of 167 site-substituted mutants could improve the α:ß ratio to different extents as indicated by saturated mutagenesis, with Y167H as the best substitution. In conclusion, Y167 was confirmed to be one of the main subsites in the -6 domain of α-CGTase that is responsible for the α:ß ratio in the product mixture. Y167H is most preferable among all types of mutant enzymes tested at this site. The reconstructed Y167H (i.e., No. 95) α-CGTase showed better potential for α-cyclodextrin production on industrial scale.

Pseudomonas kunmingensis sp. nov., an exopolysaccharide-producing bacterium isolated from a phosphate mine.

A Gram-stain-negative, rod-shaped, exopolysaccharide-producing, strictly aerobic bacterium with a single polar flagellum, designated strain HL22-2(T), was isolated from a phosphate mine situated in a suburb of Kunmming in Yunnan province in south-western China. The taxonomic status of this strain was evaluated by using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain HL22-2(T) was related to members of the genus Pseudomonas. 16S rRNA gene sequence similarities between strain HL22-2(T) and Pseudomonas xanthomarina KMM 1447(T), Pseudomonas alcaliphila AL15-21(T) and Pseudomonas stutzeri ATCC 17588(T) were 98.9, 98.10% and 98.06%, respectively. The major cellular fatty acids were C(18 : 1)ω7c, C(16 : 0) and summed feature 3 (C(16 : 1)ω7c and/or C(16 : 1)ω6c). The DNA G+C content was 60.3 mol%. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness values, strain HL22-2(T) represents a novel species of the genus Pseudomonas, for which the name Pseudomonas kunmingensis sp. nov. is proposed. The type strain is HL22-2(T) ( = CGMCC 1.12273(T) = DSM 25974(T)).

Site-saturation mutagenesis of central tyrosine 195 leading to diverse product specificities of an α-cyclodextrin glycosyltransferase from Paenibacillus sp. 602-1.

Central tyrosine 195 plays an important role in the active site of cyclodextrin glycosyltransferase (CGTase) that is highly conservative among various CGTases. However, a detailed functional understanding of this subsite is lacking. In this study, we applied site-directed saturation mutagenesis to investigate the effect of tyrosine 195 on the hydrolytic activity and cyclization specificity of an α-CGTase. A total of 17 mutant CGTases were obtained and heterologously expressed in E. coli. The mutant Y195F α-CGTase showed similar characteristics with wild-type α-CGTase. The other mutant α-CGTases showed considerably lower activity for starch-degradation and cyclodextrin (CD) formation. Interestingly, we found that the main product of mutant Y195R α-CGTase was γ-CDs (50%), not α-CDs (35%). The mutant Y195I α-CGTase drastically altered the CD specificity of α-CGTase, which showed a switch toward the synthesis of both ß- and γ-CDs with percentages of 34% and 38%, respectively. Other mutant CGTases retained the α-CD as the main product but with lower percentages than wild-type α-CGTase. Mutant Y195F, Y195I, and Y195R CGTases showed an optimal temperature of 50°C and pH 6.5. The mutants Y195I and Y195R also showed better thermostability. These findings suggested that aromatic amino acids Tyr or Phe at the 195 position were important for the amylolytic activity and cyclization specificity of α-CGTase. The mutants Y195I CGTase and Y195R CGTase have potential applications for γ-CD production in the future.

Crystallization and preliminary X-ray diffraction studies of Tyr167His mutant α-cyclodextrin glucanotransferase from Bacillus macerans.

Improving the specificity of α-cyclodextrin glucanotransferase is a significant issue in the field of α-cyclodextrin production. In this study, a constructed Y167H mutant α-cyclodextrin glucanotransferase with enhanced α-cyclodextrin specificity was successfully expressed and purified. Single crystals were grown using PEG 4000 as a precipitating agent by the hanging-drop vapour-diffusion method at 293 K. The crystals exhibited two kinds of morphology in different crystallization conditions. The crystals diffracted to at least 2.2 Šresolution (space group P212121), with unit-cell parameters a=65.69, b=78.70, c=137.00 Å. Assuming the asymmetric cell to be occupied by a monomer of 75 kDa, the unit cell contains 43.77% solvent with a crystal volume per protein mass, VM, of 2.19 Å3 Da(-1).

[Proteomic analysis of Bacillus subtilis 168 transforming cis-propenylphosphonic acid to fosfomycin].

In this study, we investigated the mechanism of transformation by Bacillus subtilis strain 168 by proteomic analysis. B. subtilis strain 168 was able to stereoselectively transform cis-propenylphosphonic acid (cPPA) to fosfomycin. The maximal fosfomycin production was 816.6 microg/mL after two days cultivation, with a conversion rate of 36.05%. We separated the whole cellular proteins by two-dimensional gel electrophoresis (2-DE) method, and 562 protein spots were detected in the presence of cPPA in the medium, while 527 protein spots were detected in the absence of cPPA. Of them, 98 differentially expressed protein spots were found. Among them, 52 proteins were up-regulated whereas 20 were down-regulated in the presence of cPPA in the medium, and 26 induced at the presence of cPPA. The differentially expressed proteins were analyzed by combined MS and MS/MS methods. Eighty protein spots, including 45 up-regulated proteins, 17 down-regulated proteins, and 18 induced by cPPA were identified. Based on the results of proteomic analysis, we postulated two steps of transformation: in the first step, cPPA was hydrated to 2-hydroxypropylphosphonic acid; in the second step, 2-hydroxypropylphosphonic acid was transformed to fosfomycin via a dehydrogenation reaction.

[Increasing of product specificity of gamma-cyclodextrin by mutating the active domain of alpha-cyclodextrin glucanotransferase from Paenibacillus macerans sp. 602-1].

We studied the mutation effect of subsites -3(Lys47), -7(146-152), and cyclization center (Tyr195) in active domain on product specificity of alpha-cyclodextrin glucanotransferase (alpha-CGTase) from Paenibacillus macerans sp. 602-1. The Lys47 was replaced by Thr47 and Tyr195 by Ile195, and the amino acids from 146 to 152 were replaced by Ile (named as delta6). All these mutant alpha-CGTases were actively expressed in E. coli BL21. Compared with the wild-type alpha-CGTase, the starch-degrading activities of all the mutant enzymes were declined. For mutant Y195I, the percentage of alpha-CD was decreased from 68% to 30%, and beta-CD was raised from 22.2% to 33.3%. Interestingly, gamma-CD was increased from 8.9% to 36.7% and became the main product, while the actual yield was increased from 0.4 g/L to 1.1 g/L. Mutant K47T and delta6 still produced alpha-CD as main product though the percentage of beta- and gamma-CD increased. Purified Y195I CGTase showed similar optimum temperature with the wild-type alpha-CGTase, but its optimum pH shifted from 5.0 to 6.0 with better pH stability. In summary, mutant Y195I CGTase has the potential to produce gamma-CD as the main product.

Overexpression of a recombinant amidase in a complex auto-inducing culture: purification, biochemical characterization, and regio- and stereoselectivity.

Rhodococcus erythropolis AJ270 metabolizes a wide range of nitriles via the two-step nitrile hydratase/amidase pathway. In this study, an amidase gene from R. erythropolis AJ270 was cloned and expressed in Escherichia coli BL21 (DE3). The activity reached the highest level of 22.04 U/ml in a complex auto-inducing medium using a simplified process of fermentation operation. The recombinant amidase was purified to more than 95% from the crude lysate using Ni-NTA affinity chromatography and Superose S10-300 gel filtration. The V(max) and K(m) values of the purified enzyme with acetamide (50 mM) were 6.89 µmol/min/mg protein and 4.12 mM, respectively, which are similar to those of the enzyme from the wild-type cell. The enzyme converted racemic α-substituted amides, O-benzylated ß-hydroxy amides, and N-benzylated ß-amino amides to the corresponding (S)-acids with remarkably high enantioselectivity. The ionic liquid [BMIm][PF6] (1-butyl-3-methylimidazolium hexafluorophosphate) enhanced the activity by 1.5-fold compared with water. The adequate expression of the enzyme and excellent enantioselectivity of the recombinant amidase to a broad spectrum of amides suggest that the enzyme has prospective industrial-scale practical applications in pharmaceutical chemistry.

Purification and characterization of four keratinases produced by Streptomyces sp. strain 16 in native human foot skin medium.

Four extracellular keratinases (designated KI, KII, KIII, and KIV) were produced during submerged aerobic cultivation in a medium containing native human foot skin (NHFS) for enzyme synthesis. The molecular weights, determined by SDS-PAGE, were 25, 50, 34, and 19 kDa, respectively. Gel filtration of the four purified enzymes in native conditions indicated that active keratinase KI is a novel homo-octamer, KII a homo-dimer, and KIII and KIV monomers. All four keratinases exhibited high activities at pH 8.0-10.0 with an optimal pH of 9.0. The optimal temperature for keratinolytic activity of KI, KII, and KIII was approximately 50, and 60 degrees C for KIV. One millimolar of PMSF completely inhibited the keratinolytic activities of the four enzymes. The N-terminal sequences of KI, KII, and KIII showed that they were different from previously described enzymes, whereas KIV shared an identical N-terminal sequence with two other peptidases from Streptomyces.

Stereoselective epoxidation of cis-propenylphosphonic acid to fosfomycin by a newly isolated bacterium Bacillus simplex strain S101.

In industry, fosfomycin is mainly prepared via chemical epoxidation of cis-propenylphosphonic acid (cPPA). The conversion yield of fosfomycin is less than 50% in the whole process and a large quantity of waste is produced. Biotransformation by microorganisms is an alternative method of preparation. This kind of conversion is more delicate, environmentally friendly, and the conversion yield of fosfomycin would be higher. In this work, an aerobic bacterium capable of transforming cPPA to fosfomycin was isolated. The organism, designated as strain S101, was identified as Bacillus simplex by morphological and physiological characteristics as well as by analysis of the gene encoding the 16S rRNA. Fosfomycin was assayed by two means, bioassay and gas chromatography (GC). Glycerol was a good carbon source for growth and cPPA conversion of strain S101. When cPPA was used as the sole carbon source, neither growth nor conversion to fosfomycin occurred. The optimum cPPA concentration in the conversion medium was 2,000 microg ml(-1). After 6 days of incubation, the concentration of fosfomycin reached its maximum level (1,838.2 microg ml(-1)), with a conversion ratio of 81.3%. Air was indispensable for the growth but not for the conversion to fosfomycin. Furthermore, vanadium ions were found to be essential for the conversion. High concentrations of cPPA had fewer inhibitory effects on the growth of strain S101.

Laccase-mediator system in the decolorization of different types of recalcitrant dyes.

Phloroglucinol, thymol, and violuric acid (VIO) were selected as laccase mediators after screening 14 different compounds with indigo carmine (indigoid dye) as a substrate. With the presence of these three mediators, a nearly complete decolorization (90-100%) was attained in 1 h. Thus, these three compounds were used as mediators for the decolorization of other four dyes. The results indicated that VIO was effective mediator in decolorization of Remazol brilliant blue R (RBBR, anthraquinoid dye) and Coomassie brilliant blue G-250 (CBB, triphenylmethane dyes), and Acid red (diazo dye). In presence of VIO, the four dyes described above attained 70% decolorization. Thymol was able to mediate decolorization of RBBR and Azure A (heterocyclic dye). Phloroglucinol has no mediating capability in decolorization of the four dyes analyzed. Mediator concentration, pH, and copper ion have an effect on the decolorization of the RBBR. Our data suggested that the decolorization capabilities of laccase/mediator system were related to the types of mediator, the dye structure and decolorization condition.

Purification, characterization, and substrate specificity of two 2,3-dihydroxybiphenyl 1,2-dioxygenase from Rhodococcus sp. R04, showing their distinct stability at various temperature.

The genes of two 2,3-dihydroxybiphenyl 1,2-dioxygenases (BphC1 and BphC2) were obtained from the gene library of Rhodococcus sp. R04. The enzymes have been purified to apparent electrophoretic homogeneity from the cell extracts of the recombinant harboring bphC1 and bphC2. Both BphC1 and BphC2 were hexamers, consisting of six subunits of 35 and 33 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The enzymes had similar optimal pH (pH 9.0), but different temperatures for their maximum activity (30 degrees C for BphC1, 80 degrees C for BphC2). In addition, they exhibited distinct stability at various temperatures. The enzymes could cleave a wide range of catechols, with 2,3-dihydroxybiphenyl being the optimum substrate for BphC1 and BphC2. BphC1 was inhibited by 2,3-dihydroxybiphenyl, catechol and 3-chlorocatechol, whereas BphC2 showed strong substrate inhibition for all the given substrates. BphC2 exhibited a half-life of 15 min at 80 degrees C and 50 min at 70 degrees C, making it the most thermostable extradiol dioxygenase studied in mesophilic bacteria. After disruption of bphC1 and bphC2 genes, R04DeltaC1 (bphC1 mutant) delayed the time of their completely eliminating biphenyl another 15 h compared with its parent strain R04, but R04DeltaC2 (bphC2 mutant) lost the ability to grow on biphenyl, suggesting that BphC1 plays an assistant role in the degrading of biphenyl by strain R04, while BphC2 is essential for the growth of strain R04 on biphenyl.

Efficient expression in E. coli of an enantioselective nitrile hydratase from Rhodococcus erythropolis.

The genes encoding an enantioselective nitrile hydratase (NHase) from Rhodococcus erythropolis AJ270 have been cloned and an active NHase has been produced in Escherichia coli. Maximal activity was found when the genes encoding the alpha- and beta-subunits were transcribed as one unit and the gene encoding the P44k activator protein as a separate ORF on a single replicon. Addition of n-butyric acid and FeSO(4 )could improve NHase activity. Coexpression of the GroEL-GroES chaperone proteins increased activity in the absence of P44k protein but had no effect in the presence of P44k. The recombinant enzyme was highly enantioselective in the synthesis of S-(+)-3-benzoyloxy- 4-cyanobutyramide from the prochiral substrate 3-benzoyloxyglutaronitrile.

Screening for a new Streptomyces strain capable of efficient keratin degradation.

Keratinous wastes could be degraded by some microorganisms in nature. Native human foot skin (NHFS) was used as sole nitrogen source to screen microorganisms with keratin-degrading capability. From approximately 200 strains, a strain of Streptomyces sp. strain No.16 was found to possess the strongest keratinolytic activity, and the total activity in the culture was 110 KU/ml with specific activity of 2870 KU/mg protein (KU: keratinase unit). Substrate specificity test indicated that the crude keratinase could degrade keratin azure, human hair, cock feathers and collagen. The optimal pH of the crude keratinase ranged from 7.5 to 10 and the temperature ranged from 40 degrees C to 55 degrees C. Metal chelating agent ethylenediamine tetraacetic acid obviously stimulated the keratinolytic activity but suppressed the proteolytic activity. To our knowledge, this is the first report on specific induction of keratinases by NHFS from an actinomycete. Moreover, excellent characteristics of its crude keratinase may lead to the potential application in waste treatment and recovery, poultry and leather industry, medicine, and cosmetic development.

Molecular evolution of Fome lignosus laccase by ethyl methane sulfonate-based random mutagenesis in vitro.

In order to improve the laccase activity, mutant libraries are constructed through ethyl methane sulfonate-based (EMS) random mutagenesis. Mutagenesis improved expression 3.7-fold to 144 mgl(-1) laccase in yeast, together with a 1.4-fold increase in K(cat). Thus, the total activity is enhanced 5-fold for 2,2'-azino-bis 3-ethylbenzothiaoline-6-sulfonic acid (ABTS). In the presence of 0.6mM copper, the highest activity value reached 30 Uml(-1) after a 3-day cultivation at a temperature of 30 degrees C(.) In comparison with the wild type, the best mutant enzymatic properties (K(m) for ABTS and guaiacol, thermo- and pH stability, optimal pH) are not changed. Moreover, amino acid sequence analysis indicates that there are four substitutions in the best mutant laccase (Gly160Asp, Ala167Thr, Gly174Asp, and Glu234Gly). The best mutant laccase model showed that the Gly160 and Ala167 are to be found near the water channel; especially the distance of Ala167 to the Cu3a is 14.46 A. This implies that it is likely involved in the formation of water channel and that it helps facilitate the easy incoming and outgoing of water.

High resolution X-ray molecular structure of the nitrile hydratase from Rhodococcus erythropolis AJ270 reveals posttranslational oxidation of two cysteines into sulfinic acids and a novel biocatalytic nitrile hydration mechanism.

The crystal structure of Fe-type nitrile hydratase from Rhodococcus erythropolis AJ270 was determined at 1.3A resolution. The two cysteine residues (alphaCys(112) and alphaCys(114)) equatorially coordinated to the ferric ion were post-translationally modified to cysteine sulfinic acids. A glutamine residue (alphaGln(90)) in the active center gave double conformations. Based on the interactions among the enzyme, substrate and water molecules, a new mechanism of biocatalysis of nitrile hydratase was proposed, in which the water molecule activated by the glutamine residue performed as the nucleophile to attack on the nitrile which was simultaneously interacted by another water molecule coordinated to the ferric ion.