The effect of direct and counter-current flow-through delignification on enzymatic hydrolysis of wheat straw, and flow limits due to compressibility.

This article compares the processes for wheat straw lignocellulose fractionation by percolation, counter-current progressing batch percolation and batch reaction at low NaOH-loadings (3-6% of DM). The flow-through processes were found to improve delignification and subsequent enzymatic saccharification, reduce NaOH-consumption and allow reduction of thermal severity, whereas hemicellulose dissolution was unaffected. However, contrary to previous expectations, a counter-current process did not provide additional benefits to regular percolation. The compressibility and flow properties of a straw bed were determined and used for simulation of the packing density profile and dynamic pressure in an industrial scale column. After dissolution of 30% of the straw DM by delignification, a pressure drop above 100 kPa m-1 led to clogging of the flow due to compaction of straw. Accordingly, the maximum applicable feed pressure and volumetric straw throughput was determined as a function of column height, indicating that a 10 m column can be operated at a maximum feed pressure of 530 kPa, corresponding to an operation time of 50 min and a throughput of 163 kg m-3 h-1 . Biotechnol. Bioeng. 2016;113: 2605-2613. © 2016 Wiley Periodicals, Inc.

Structure modeling and functional analysis of recombinant dextransucrase from Weissella confusa Cab3 expressed in Lactococcus lactis.

The dextransucrase gene from Weissella confusa Cab3, having an open reading frame of 4.2 kb coding for 1,402 amino acids, was amplified, cloned, and expressed in Lactococcus lactis. The recombinant dextransucrase, WcCab3-rDSR was expressed as extracellular enzyme in M17 medium with a specific activity of 1.5 U/mg which after purification by PEG-400 fractionation gave 6.1 U/mg resulting in 4-fold purification. WcCab3-rDSR was expressed as soluble and homogeneous protein of molecular mass, approximately, 180 kDa as analyzed by SDS-PAGE. It displayed maximum enzyme activity at 35°C at pH 5.0 in 50 mM sodium acetate buffer. WcCab3-rDSR gave Km of 6.2 mM and Vm of 6.3 µmol/min/mg. The characterization of dextran synthesized by WcCab3-rDSR by Fourier transform infrared and nuclear magnetic resonance spectroscopic analyses revealed the structural similarities with the dextran produced by the native dextransucrase. The modeled structure of WcCab3-rDSR using the crystal structures of dextransucrase from Lactobacillus reuteri (protein data bank, PDB id: 3HZ3) and Streptococcus mutans (PDB id: 3AIB) as templates depicted the presence of different domains such as A, B, C, IV, and V. The domains A and B are circularly permuted in nature having (ß/α)8 triose phosphate isomerase-barrel fold making the catalytic core of WcCab3-rDSR. The structure superposition and multiple sequence alignment analyses of WcCab3-rDSR with available structures of enzymes from family 70 GH suggested that the amino acid residue Asp510 acts as a nucleophile, Glu548 acts as a catalytic acid/base, whereas Asp621 acts as a transition-state stabilizer and these residues are found to be conserved within the family.

Which properties of cutinases are important for applications?

Cutinases (EC 3.1.1.74) are extracellular enzymes that belong to α/ß hydrolases. They are serine esterases with the classical Ser-His-Asp triad similar to several lipases and serine proteases. In nature, cutinases catalyse the hydrolysis of the polyesters of the cuticle and the suberin layers, which protect plant surfaces. Cutinase production is typical for plant pathogenic fungi, but also, bacterial cutinases and cutinases from plant pollen have been discovered. Cutinases are promiscuous esterases catalysing reactions with a wide range of different substrates, such as short-chain soluble esters, water-insoluble medium and long-chain triacylglycerols, polyesters and waxes. In the current work, an overview is given on suggested applications of cutinases in the textile industry, in laundry detergents, in processing of biomass and food, in biocatalysis and in detoxification of environmental pollutants. The applications are discussed from the point of view of cutinase properties-which properties of cutinases are already advantageous and which would be desired. In addition, improvements that have been made on cutinase performance by protein and reaction engineering are reviewed.

Cloning and characterization of a novel acidic cutinase from Sirococcus conigenus.

A cutinase gene (ScCut1) was amplified by PCR from the genomic DNA of the ascomycetous plant pathogen Sirococcous conigenus VTT D-04989 using degenerate primers designed on the basis of conserved segments of known cutinases and cutinase-like enzymes. No introns or N- or O-glycosylation sites could be detected by analysis of the ScCut1 gene sequence. The alignment of ScCut1 with other fungal cutinases indicated that ScCut1 contained the conserved motif G-Y-S-Q-G surrounding the active site serine as well as the aspartic acid and histidine residues of the cutinase active site. The gene was expressed in Pichia pastoris, and the recombinantly produced ScCut1 enzyme was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His-tag translationally fused to the protein. The purified ScCut1 exhibited activity at acidic pH. The K(m) and V(max) values determined for pNP-butyrate esterase activity at pH 4.5 were 1.7 mM and 740 nkat mg⁻¹, respectively. Maximal activities were determined at between pH 4.7 and 5.2 and at between pH 4.1 and 4.6 with pNP-butyrate and tritiated cutin as the substrates, respectively. With both substrates, the enzyme was active over a broad pH range (between pH 3.0 and 7.5). Activity could still be detected at pH 3.0 both with tritiated cutin and with p-nitrophenyl butyrate (relative activity of 25 %) as the substrates. ScCut1 showed activity towards shorter (C2 to C3) fatty acid esters of p-nitrophenol than towards longer ones. Circular dichroism analysis suggested that the denaturation of ScCut1 by heating the protein sample to 80 °C was to a great extent reversible.

D-Tagatose production in the presence of borate by resting Lactococcus lactis cells harboring Bifidobacterium longum L-arabinose isomerase.

Bifidobacterium longum NRRL B-41409 L-arabinose isomerase (L-AI) was overexpressed in Lactococcus lactis using a phosphate depletion inducible expression system. The resting L. lactis cells harboring the B. longum L-AI were used for production of D-tagatose from D-galactose in the presence of borate buffer. Multivariable analysis suggested that high pH, temperature and borate concentration favoured the conversion of D-galactose to D-tagatose. Almost quantitative conversion (92 %) was achieved at 20 g L⁻¹ substrate and at 37.5 °C after 5 days. The D-tagatose production rate of 185 g L⁻¹ day ⁻¹ was obtained at 300 g L⁻¹ galactose, at 1.15 M borate, and at 41 °C during 10 days when the production medium was changed every 24 h. There was no significant loss in productivity during ten sequential 24 h batches. The initial D-tagatose production rate was 290 g L⁻¹ day⁻¹ under these conditions.

The role of single cell protein in cellular agriculture.

More food needs to be produced for the growing human population, but the possibilities of expanding the area of arable land are limited. Cellular Agriculture is an emerging field of biotechnology, aimed at finding alternatives to agricultural production of various commodities. As a part of Cellular Agriculture, the use of microbes and microalgae as food and feed with high protein content, so-called single cell protein (SCP), is gaining renewed scientific and commercial interest. In this review, we give an introduction to SCP production by heterotrophic microbial species, phototrophs, methanotrophs and autotrophic hydrogen oxidizers, as well as highlight some challenges and the latest developments in the growing SCP industry.

Preliminary X-ray analysis of twinned crystals of sarcosine dimethylglycine methyltransferase from Halorhodospira halochoris.

Sarcosine dimethylglycine methyltransferase (EC 2.1.1.157) is an enzyme from the extremely halophilic anaerobic bacterium Halorhodospira halochoris. This enzyme catalyzes the twofold methylation of sarcosine to betaine, with S-adenosylmethionine (AdoMet) as the methyl-group donor. This study presents the crystallization and preliminary X-ray analysis of recombinant sarcosine dimethylglycine methyltransferase produced in Escherichia coli. Mass spectroscopy was used to determine the purity and homogeneity of the enzyme material. Two different crystal forms, which initially appeared to be hexagonal and tetragonal, were obtained. However, on analyzing the diffraction data it was discovered that both crystal forms were pseudo-merohedrally twinned. The true crystal systems were monoclinic and orthorhombic. The monoclinic crystal diffracted to a maximum of 2.15 A resolution and the orthorhombic crystal diffracted to 1.8 A resolution.

Factors affecting the production of L-xylulose by resting cells of recombinant Escherichia coli.

Factors affecting the production of the rare sugar L-xylulose from xylitol using resting cells were investigated. An E. coli BPT228 strain that recombinantly expresses a gene for xylitol dehydrogenase was used in the experiments. The ratio of xylitol to L-xylulose was three times lower in the cytoplasm than in the medium. The effects of pH, temperature, shaking speed, and initial xylitol concentration on L-xylulose production were investigated in shaking flasks using statistical experimental design methods. The highest production rates were found at high shaking speed and at high temperature (over 44 degrees C). The optimal pH for both productivity and conversion was between 7.5 and 8.0, and the optimal xylitol concentration was in the range 250-350 g l(-1). A specific productivity of 1.09 +/- 0.10 g g(-1) h(-1) was achieved in a bioreactor. The response surface model based on the data from the shake flask experiments predicted the operation of the process in a bioreactor with reasonable accuracy.

Rate-constraining changes in surface properties, porosity and hydrolysis kinetics of lignocellulose in the course of enzymatic saccharification.

BACKGROUND: Explaining the reduction of hydrolysis rate during lignocellulose hydrolysis is a challenge for the understanding and modelling of the process. This article reports the changes of cellulose and lignin surface areas, porosity and the residual cellulase activity during the hydrolysis of autohydrolysed wheat straw and delignified wheat straw. The potential rate-constraining mechanisms are assessed with a simplified kinetic model and compared to the observed effects, residual cellulase activity and product inhibition. RESULTS: The reaction rate depended exclusively on the degree of hydrolysis, while enzyme denaturation or time-dependent changes in substrate hydrolysability were absent. Cellulose surface area decreased linearly with hydrolysis, in correlation with total cellulose content. Lignin surface area was initially decreased by the dissolution of phenolics and then remained unchanged. The dissolved phenolics did not contribute to product inhibition. The porosity of delignified straw was decreased during hydrolysis, but no difference in porosity was detected during the hydrolysis of autohydrolysed straw. CONCLUSIONS: Although a hydrolysis-dependent increase of non-productive binding capacity of lignin was not apparent, the dependence of hydrolysis maxima on the enzyme dosage was best explained by partial irreversible product inhibition. Cellulose surface area correlated with the total cellulose content, which is thus an appropriate approximation of the substrate concentration for kinetic modelling. Kinetic models of cellulose hydrolysis should be simplified enough to include reversible and irreversible product inhibition and reduction of hydrolysability, as well as their possible non-linear relations to hydrolysis degree, without overparameterization of particular factors.

Lactose- and cellobiose-derived branched trisaccharides and a sucrose-containing trisaccharide produced by acceptor reactions of Weissella confusa dextransucrase.

Dextran-producing Weissella have received significant attention. However, except for maltose, the acceptor reactions of Weissella dextransucrases with different sugars have not been investigated. The action of recombinant Weissella confusa VTT E-90392 dextransucrase was tested with several potential acceptors, particularly, analogs lactose and cellobiose. The major acceptor products of both disaccharides were identified as branched trisaccharides, with a glucosyl residue α-(1 → 2)-linked to the acceptor's reducing end. An additional product, isomelezitose (6(Fru)-α-Glcp-sucrose), was also produced when using lactose as an acceptor. This is the first report of the synthesis of isomelezitose by a dextransucrase. The NMR spectra of the three trisaccharides were fully assigned, and their structures were confirmed by selective enzymatic hydrolysis. The trisaccharides prepared from (13)C6(glc) sucrose and lactose were analyzed by ESI-MS(n), and the fragmentation patterns of these compounds were characterized.

Production of xylitol from D-xylose by recombinant Lactococcus lactis.

The D-xylose reductase from Pichia stipitis CBS 5773 and the xylose transporter from Lactobacillus brevis ATCC 8287 were expressed in active form in Lactococcus lactis NZ9800. Xylitol production was investigated using non-growing recombinant cells in high cell-density under microaerobic conditions in the presence of xylose and glucose. Besides xylose, the recombinant strain with xylose reductase activity reduced l-arabinose and D-ribose in significant extent to the corresponding pentitols. The ratio of xylitol produced per glucose consumed was almost 10-fold higher under glucose limitation than the ratio in the presence of excess initial glucose. The co-expression of the xylose transporter with the xylose reductase did not increase the efficiency of xylitol production appreciably when compared to the strain in which only the xylose reductase gene was expressed. A fed-batch experiment with high initial xylose concentration (160 gl(-1)) under glucose limitation was carried out using the strain co-expressing xylose reductase and xylose transporter genes. The xylitol yield from xylose was 1.0 mol mol(-1) and the ratio of xylitol produced per glucose consumed was 2.5 mol mol(-1). The volumetric productivity was 2.72 gl(-1)h(-1) at 20 h. Of the xylose initially present, 34% was consumed. Analysis of the fermentation metabolites revealed a shift from homolactic to mixed acid fermentation at early stages of the experiment.

Cloning and characterization of a Weissella confusa dextransucrase and its application in high fibre baking.

Wheat bran offers health benefits as a baking ingredient, but is detrimental to bread textural quality. Dextran production by microbial fermentation improves sourdough bread volume and freshness, but extensive acid production during fermentation may negate this effect. Enzymatic production of dextran in wheat bran was tested to determine if dextran-containing bran could be used in baking without disrupting bread texture. The Weissella confusa VTT E-90392 dextransucrase gene was sequenced and His-tagged dextransucrase Wc392-rDSR was produced in Lactococcus lactis. Purified enzyme was characterized using (14)C-sucrose radioisotope and reducing value-based assays, the former yielding K(m) and V(max) values of 14.7 mM and 8.2 µmol/(mg ∙ min), respectively, at the pH optimum of 5.4. The structure and size of in vitro dextran product was similar to dextran produced in vivo. Dextran (8.1% dry weight) was produced in wheat bran in 6 h using Wc392-rDSR. Bran with and without dextran was used in wheat baking at 20% supplementation level. Dextran presence improved bread softness and neutralized bran-induced volume loss, clearly demonstrating the potential of using dextransucrases in bran bioprocessing for use in baking.

A Cutinase from Trichoderma reesei with a lid-covered active site and kinetic properties of true lipases.

Cutinases belong to the α/ß-hydrolase fold family of enzymes and degrade cutin and various esters, including triglycerides, phospholipids and galactolipids. Cutinases are able to degrade aggregated and soluble substrates because, in contrast with true lipases, they do not have a lid covering their catalytic machinery. We report here the structure of a cutinase from the fungus Trichoderma reesei (Tr) in native and inhibitor-bound conformations, along with its enzymatic characterization. A rare characteristic of Tr cutinase is its optimal activity at acidic pH. Furthermore, Tr cutinase, in contrast with classical cutinases, possesses a lid covering its active site and requires the presence of detergents for activity. In addition to the presence of the lid, the core of the Tr enzyme is very similar to other cutinase cores, with a central five-stranded ß-sheet covered by helices on either side. The catalytic residues form a catalytic triad involving Ser164, His229 and Asp216 that is covered by the two N-terminal helices, which form the lid. This lid opens in the presence of surfactants, such as ß-octylglucoside, and uncovers the catalytic crevice, allowing a C11Y4 phosphonate inhibitor to bind to the catalytic serine. Taken together, these results reveal Tr cutinase to be a member of a new group of lipolytic enzymes resembling cutinases but with kinetic and structural features of true lipases and a heightened specificity for long-chain triglycerides.

Screening of microbes for novel acidic cutinases and cloning and expression of an acidic cutinase from Aspergillus niger CBS 513.88.

Isolates from gardening waste compost and 38 culture collection microbes were grown on agar plates at pH 4.0 with the cutinase model substrate polycaprolactone as a carbon source. The strains showing polycaprolactone hydrolysis were cultivated in liquid at acidic pH and the cultivations were monitored by assaying the p-nitrophenyl butyrate esterase activities. Culture supernatants of four strains were analyzed for the hydrolysis of tritiated apple cutin at different pHs. Highest amounts of radioactive hydrolysis products were detected at pHs below 5. The hydrolysis of apple cutin by the culture supernatants at acidic pH was further confirmed by GC-MS analysis of the hydrolysis products. On the basis of screening, the acidic cutinase from Aspergillus niger CBS 513.88 was chosen for heterogeneous production in Pichia pastoris and for analysis of the effects of pH on activity and stability. The recombinant enzyme showed activity over a broad range of pHs with maximal activity between pH 5.0 and 6.5. Activity could be detected still at pH 3.5.

Bifidobacterium longum L-arabinose isomerase--overexpression in Lactococcus lactis, purification, and characterization.

Bifidobacterium longum NRRL B-41409 L-arabinose isomerase (L-AI) was cloned and overexpressed in Lactococcus lactis using a phosphate-depletion-inducible expression system. The purified B. longum L-AI was characterized using D-galactose and L-arabinose as the substrates. The enzyme was active and stable at acidic pH with an optimum at pH 6.0-6.5. The enzyme showed the highest activity at 55 °C during a 20-min incubation at pH 6.5. The K(m) value was 120 mM for L-arabinose and 590 mM for D-galactose. The V(max) was 42 U mg(-1) with L-arabinose and 7.7 U mg(-1) with D-galactose as the substrates. The enzyme had very low requirement for metal ions for catalytic activity, but it was stabilized by divalent metal ions (Mg(2+), Mn(2+)). The enzyme bound the metal ions so tightly that they could not be fully removed from the active site by EDTA treatment. Using purified B. longum L-AI as the catalyst at 35 °C, equilibrium yields of 36 % D-tagatose and 11 % L-ribulose with 1.67 M D-galactose and L-arabinose, respectively, as the substrates were reached.

Production of L-xylose from L-xylulose using Escherichia coli L-fucose isomerase.

L-Xylulose was used as a raw material for the production of L-xylose with a recombinantly produced Escherichia coli L-fucose isomerase as the catalyst. The enzyme had a very alkaline pH optimum (over 10.5) and displayed Michaelis-Menten kinetics for L-xylulose with a K(m) of 41 mM and a V(max) of 0.23 µmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6h 50 min and 1h 31 min, respectively. The reaction equilibrium between L-xylulose and L-xylose was 15:85 at 35 °C and thus favored the formation of L-xylose. Contrary to the L-rhamnose isomerase catalyzed reaction described previously [14]L-lyxose was not detected in the reaction mixture with L-fucose isomerase. Although xylitol acted as an inhibitor of the reaction, even at a high ratio of xylitol to L-xylulose the inhibition did not reach 50%.

Methods for identifying lipoxygenase producing microorganisms on agar plates.

Plate assays for lipoxygenase producing microorganisms on agar plates have been developed. Both potassium iodide-starch and indamine dye formation methods were effective for detecting soybean lipoxygenase activity on agar plates. A positive result was also achieved using the ß-carotene bleaching method, but the sensitivity of this method was lower than the other two methods. The potassium iodide-starch and indamine dye formation methods were also applied for detecting lipoxygenase production by Trichoderma reesei and Pichia pastoris transformants expressing the lipoxygenase gene of the fungus Gaeumannomyces graminis. In both cases lipoxygenase production in the transformants could be identified. For detection of the G. graminis lipoxygenase produced by Aspergillus nidulans the potassium iodide-starch method was successful. When Escherichia coli was grown on agar and soybean lipoxygenase was applied on the culture lipoxygenase activity could clearly be detected by the indamine dye formation method. This suggests that the method has potential for screening of metagenomic libraries in E. coli for lipoxygenase activity.

Suberin of potato (Solanum tuberosum var. Nikola): comparison of the effect of cutinase CcCut1 with chemical depolymerization.

Chemical and enzymatic depolymerizations of suberin isolated from potato peel ( Solanum tuberosum var. Nikola) were performed under various conditions. Enzymatic hydrolysis with cutinase CcCut1 and chemical methanolysis with NaOMe of suberin yielded monomeric fragments, which were identified as TMS derivatives with GC-MS and GC-FID. The solid, hydrolysis-resistant residues were analyzed with solid state (13)C CPMAS NMR, FT-IR, and microscopic methods. Methanolysis released more CHCl(3)-soluble material than the cutinase treatment when determined gravimetrically. Interestingly, cutinase-catalyzed hydrolysis produced higher proportions of aliphatic monomers than hydrolysis with the NaOMe procedure when analyzed by GC in the form of TMS derivatives. Monomers released by the two methods were mainly alpha,omega-dioic acids and omega-hydroxy acids, but the ratios of the detected monomers were different, at 40.0 and 32.7% for methanolysis and 64.6 and 8.2% for cutinase, respectively. Thus, cutinase CcCut1 showed higher activity toward ester bonds of alpha,omega-dioic acids than toward the bonds of omega-hydroxy acids. The most abundant monomeric compounds were octadec-9-ene-1,18-dioic acid and 18-hydroxyoctadec-9-enoic acid, which accounted for ca. 37 and 28% of all monomers, respectively. The results of the analyses of the chemical and enzymatic hydrolysis products were supported by the spectroscopic analyses with FT-IR and CPMAS (13)C NMR together with the analysis of the microstructures of the hydrolysis residues by light and confocal microscopy.

Contributions of biotechnology to the production of mannitol.

Mannitol is a 6-carbon sugar alcohol that has been produced traditionally by chemical catalysis. Biotechnology has brought advances to the production in terms of substrate purity, process equipment requirements and safety. Enzymatic methods have improved the yields and the use of microbes has brought versatility to the range of substrates that can be used in the processes. Some of the microbial processes are already industrially feasible and could be taken to further use. The most promising production strategy reported so far has been the utilization of non-growing cells of heterofermentative lactic acid bacteria for converting fructose to mannitol. The latest developments in the field have dealt with the use of recombinant strains in mannitol production.

A new and efficient phosphate starvation inducible expression system for Lactococcus lactis.

A new expression system for Lactococcus lactis was developed. The system is based on a phosphate starvation inducible pstF promoter of L. lactis MG1363. Intracellular beta-galactosidase and secreted alpha-amylase were produced using this tightly regulated system. No evidence of regulatory sites in regions of the 5'-end of the pstF coding sequence was found. High expression levels of the beta-galactosidase gene were obtained using the original pstF RBS in a phosphate-depleted medium. The results suggested that with the phosphate starvation inducible system, it is possible to achieve expression levels comparable to the ones obtained with the widely used nisin-controlled gene expression system (NICE). A specific beta-galactosidase activity of 670 microkat g(-1) using a phosphate-depleted medium and an alpha-amylase activity of 3.6 microkat l(-1) in a bioreactor cultivation were produced. The advantages of the current expression system include that no prior removal of phosphate from the medium in bioreactor scale is required, and no additions of inducing agents are needed. Furthermore, the system can be operated in L. lactis without introduction of regulatory genes into the host.