Isolation of α-Glucuronidase Enzyme from a Rumen Metagenomic Library

α-Glucuronidase enzymes play an essential role in the full enzymatic hydrolysis of hemicellulose. Up to this point, all genes encoding α-glucuronidase enzymes have been cloned from individual, pure culture strains. Using a high-throughput screening strategy, we have isolated the first α-glucuronidase gene (rum630-AG) from a mixed population of microorganisms. The gene was subcloned into a prokaryotic vector, and the enzyme was overexpressed and biochemically characterized. The RUM630-AG enzyme had optimum activity at pH 6.5 and 40 °C. When birchwood xylan was used as substrate, the RUM630-AG functioned synergistically with an endoxylanase enzyme to hydrolyze the substrate.     

Optimization of enzymatic hydrolysis and fermentation conditions for improved bioethanol production from potato peel residues

The aim of this work was the optimization of the enzyme hydrolysis of potato peel residues (PPR) for bioethanol production. The process included a pretreatment step followed by an enzyme hydrolysis using crude enzyme system composed of cellulase, amylase and hemicellulase, produced by a mixed culture of Aspergillus niger and Trichoderma reesei. Hydrothermal, alkali and acid pretreatments were considered with regards to the enhancement of enzyme hydrolysis of potato peel residues. The obtained results showed that hydrothermal pretreatment lead to a higher enzyme hydrolysis yield compared to both acid and alkali pretreatments. Enzyme hydrolysis was also optimized for parameters such as temperature, pH, substrate loading and surfactant loading using a response surface methodology. Under optimized conditions, 77 g L^?1 of reducing sugars were obtained. Yeast fermentation of the released reducing sugars led to an ethanol titer of 30 g L^?1 after supplementation of the culture medium with ammonium sulfate. Moreover, a comparative study between acid and enzyme hydrolysis of potato peel residues was investigated. Results showed that enzyme hydrolysis offers higher yield of bioethanol production than acid hydrolysis. These results highlight the potential of second generation bioethanol production from potato peel residues treated with onsite produced hydrolytic enzymes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:397–406, 2017     

Enzymatic hydrolosis of isolated and fibre-bound galactoglucomannans from pine-wood and pine kraft pulp

Fibre-bound and isolated galactoglumanans from pine-wood and pine kraft pulp were hydrolysed with purified mannanases from Trichoderma reesei and Bacillus subtilis. The isolated galactoglucomannans from both wood and pulp could be hydrolysed fairly extensively with both enzymes. In addition to mixed oligomers, the fungal mannase produced mannobiose as the main hydrolysis product whereas the bacterial mannanase produced mannobiose, mannotriose and mannotetraose. Both enzymes hydrolysed the native galactoglucomannan in finely ground pinewood, whereas galactoglucomannan in pine kraft pulp was only hydrolysed by the T. ressei mannanase. Thus, mannanases exhibit different specificities on fibre-bound, modified substrates. In spite of the high enzyme loading, the degree of hydrolysis of fibre-bound substrates did not exceed 10% of the theoretical, probably due to poor accessibility of the substrates. Correspondence to: M. Rättö     

Effects of nucleotide analogs on ATP hydrolysis by P-type ATPases. Comparison between the canine kidney (Na++ K+) ATPase and maize root H+-ATPase

The mechanism by which chemical energy is converted into an electrochemical gradient by P-type ATPase is not completely understood. The effects of ATP analogs on the canine kidney (Na⁺+ K⁺) ATPase were compared to effects of the same analogs on the maize (Zea mays L. cv. W7551) root H⁺-ATPase in order to identify probes for the ATP binding site of the maize root enzyme and to determine potential similarities of ATP hydrolysis mechanisms in these two enzymes. Six compounds able to modify the ATP binding site covalently were compared. These compounds could be classed into three distinct groups based on activity. The first group had little or no effect on catalytic activity of either enzyme and included 7-chloro-4-nitrobenz-2-oxa-1.3-diazole. The second group, which included azido adenine analogs. fluorescein isothiocyanate and 5′-p-fluorosulfonylbenzoyladenine, were inhibitors of ATP hydrolysis by both enzymes. However, the sensitivity of the (Na⁺+ K⁺) ATPase to inhibition was much greater than that exhibited by the maize root enzyme. The third group, which included periodate treated nucleotide derivatives and 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate. inhibited both enzymes similarly. This initial screening of these covalent modifiers indicated that 2′,3′-o-(4-benzoylbenzoyl)adenosine triphosphate was the optimal covalent modifier of the ATP binding site of the maize root enzyme. Certain reagents were much more effective against the (Na⁺+ K⁺) ATPase than the maize root enzyme, possibly indicating differences in the ATP binding and hydrolysis pathway for these two enzymes. Two ATP analogs that are not covalent modifiers were also tested: the trinitrophenyl derivatives of adenine nucleotides were better than 5′-adenylylimidodiphosphate for use as an ATP binding probe.     

Enzyme recovery in high-solids enzymatic hydrolysis of steam-pretreated willow: Requirements for the enzyme composition

In order to reduce the total enzyme consumption in high-solids static hydrolysis of nonwashed steam-exploded willowSalix caprea by mixed cellulase ofTrichoderma reesei + Aspergillus foetidus, two different approaches were proposed. In the first case, the enzyme activity adsorbed on residual solids after extended hydrolysis was used for hydrolysis of the newly added substrate. The initial mixing of fresh and hydrolyzed substrates was sufficient for the adsorbed enzyme redistribution and conversion of the new substrate portion, and constant mechanical stirring was not required. Feeding of two additional portions of the exploded hardwood adjusted to pH 4 with dry caustic into the reactor with simultaneous replacement of accumulated sugars with fresh buffer (pH 4.5) resulted, on average, in a 90% conversion of cellulose at the final enzyme loading of 8 IFPU per g ODM substrate, an average sugar concentration of 12%, and a glucose/xylose ratio of 5 : 1. In the second approach, weakly adsorbed cellulase fractions were used for static high-solids hydrolysis followed by their ultrafiltration recovery from the resultant sugar syrup. In contrast to the initial cellulase mixture whose residual activity in a syrup did not exceed 5–10% at the end of hydrolysis (48 h), up to 60% of weakly adsorbed enzyme fraction could be separated from sugar syrups by ultrafiltration and then reused. Weakly adsorbed enzymes displayed a hydrolysis efficiency of not less than 80% per IFPU enzyme consumed in extended hydrolysis of pretreated willow as compared to the original enzyme mixture. An electrophoretic study of the weakly adsorbed enzyme fraction identifiedT. reesei cellobiohydrolase II as the predominant component, whereas clear domination ofT. reesei cellobiohydrolase I was found by electrophoresis of proteins tightly bound to residual hydrolysis solids. Deceased     

Functional assembly and characterization of a modular xylanosome for hemicellulose hydrolysis in yeast

Five trimeric xylanosomes were successfully assembled on the cell surface of Saccharomyces cerevisiae. Three dockerin‐tagged fungal enzymes, an endoxylanase (XynAc) from Thermomyces lanuginosus, a β‐xylosidase (XlnDt) from Aspergillus niger and an acetylxylan esterase (AwAXEf) from Aspergillus awamori, were displayed for the synergistic saccharification of birchwood xylan. The surface‐expression scaffoldins were modular constructs with or without carbohydrate binding modules from Thermotoga maritima (family 22) or Clostridium thermocellum (family 3). The synergy due to enzyme–enzyme and enzyme–substrate proximity, and the effects of binding domain choice and position on xylan hydrolysis were determined. The scaffoldin‐based enzymes (with no binding domain) showed a 1.6‐fold increase in hydrolytic activity over free enzymes; this can be attributed to enzyme–enzyme proximity within the scaffoldin. The addition of a xylan binding domain from T. maritima improved hydrolysis by 2.1‐fold relative to the scaffoldin without a binding domain (signifying enzyme–substrate synergy), and 3.3‐fold over free enzymes, with a xylose productivity of 105 mg g^?1 substrate after 72 h hydrolysis. This system was also superior to the xylanosome carrying the cellulose binding module from C. thermocellum by 1.4‐fold. Furthermore, swapping the xylan binding module position within the scaffoldin resulted in 1.5‐fold more hydrolysis when the binding domain was adjacent to the endoxylanase. These results demonstrate the applicability of designer xylanosomes toward hemicellulose saccharification in yeast, and the importance of the choice and position of the carbohydrate binding module for enhanced synergy. Biotechnol. Bioeng. 2013; 110: 275–285. © 2012 Wiley Periodicals, Inc.     

Synergism of cellulase enzymes in mixed culture solid substrate fermentation

Sugar cane bagasse was subjected to a mixed culture, solid substrate fermentation with Trichoderma reesei QM9414 and Aspergillus terreus SUK-1 to produce cellulase and reducing sugars. The highest cellulase activity and reducing sugar amount were obtained in mixed culture. The percentage of substrate degradation achieved employing mixed culture was 26% compared to 50% using separate cultures of the two molds. This suggests that the synergism of enzymes in mixed culture solid substrate fermentation have lower synergism than in pure culture.     

On the Modes of Action of Three Xylanases Produced by a Strain of Aspergillus niger van Tieghem

The modes of action of three xylanases (I, II and III) produced by Aspergillus niger van Tieghem on several substrates were investigated. Xylanase I possesed the strongest activity against xylooligosaccharides among the three enzymes and converted them into xylose and xylobiose. Xylanase II and III catalyzed a glycosylating reaction and produced higher polymerized xylooligosaccharides from xylotetraose or xylopentaose. Among three enzymes, xylanase II could split α1,3-arabinofuranosidic bond of arabinose-xylose mixed oligosaccharides.

In the case of hydrolysis by three xylanases on xylan and arabinoxylan, the maximum hydrolysis degree and the reaction products were compared with each other. From the results, some speculation were made concerning the modes of action of the enzymes.     

Adsorption and recovery of cellulases during hydrolysis of newspaper

The adsorption of cellulases from Trichoderma viride was studied during the hydrolysis of newspaper. By measuring individual enzyme activities it was found that in the early stage of hydrolysis enzyme components showing C_xA; were adsorbed preferentially to those showing C₁A; afterwards ths situation was inverted. Electrophoretic resolution of proteins in hydrolysates showed a continuous decrease of enzyme proteins in solution, and furthermore suggested that the enzymes once adsorbed remained immobilized on the substrate (even after extensive digestion). Experiments to recover the enzyme that had remained in solution after typical hydrolysis showed a potential saving of up to 40%.     

Functional analyses and application of microbial lactonohydrolases

Microbial lactonohydrolases (intramolecular ester bond-hydrolyzing enzymes) with unique properties were found. The lactonohydrolase fromFusarium oxysporum catalyzes enantioselective hydrolysis of aldonate lactones andd-pantoyl lactone (d-PL). This enzyme is useful for the large-scale optical resolution of racemic PL. TheAgrobacterium tumefaciens enzyme catalyzes asymmetric hydrolysis of PL, but the stereospecificity is opposite to that of theFusarium enzyme. Dihydrocoumarin hydrolase (DHase) fromAcinetobacter calcoaceticus is a bifunctional enzyme, which catalyzes not only hydrolysis of aromatic lactones but also bromination of monochlorodimedon in the presence of H₂O₂ and dihydrocoumarin. DHase also hydrolyzes several linear esters, and is useful for enantioselective hydrolysis of methyldl-β-acetylthioisobutyrate and regioselective hydrolysis of methyl cetraxate.     

Isolation and purification of d-mannose from palm kernel

An economically viable procedure for the isolation and purification of d-mannose from palm kernel was developed in this research. The palm kernel was catalytically hydrolyzed with sulfuric acid at 100 °C and then fermented by mannan-degrading enzymes. The solution after fermentation underwent filtration in a silica gel column, desalination by ion-exchange resin, and crystallization in ethanol to produce pure d-mannose in a total yield of 48.4% (based on the weight of the palm kernel). Different enzymes were investigated, and the results indicated that endo-β-mannanase was the best enzyme to promote the hydrolysis of the oligosaccharides isolated from the palm kernel. The pure d-mannose sample was characterized by FTIR, ¹H NMR, and ¹³C NMR spectra.     

Production of parathion hydrolase activity

Summary A mixed bacterial culture which was obtained in a previous enrichment grew on parathion, an organophosphate insecticide, as a sole carbon and energy source. A cell-free enzyme preparation from this culture detoxified by hydrolysis eight commercially used organophosphate insecticides.Fermentation procedures for the production of this parathion hydrolase activity were examined to determine if this enzyme activity could be produced economically. The mixed culture was grown using sterile or non-sterile procedures in 4 or 11 continuous and batch culture fermentations. A pure Pseudomonas sp isolated from the mixed culture expressed parathion hydrolase activity when grown under axenic fermentation conditions on industrially used media such as meat extract, soya bean meal, and corn extract. The optimal conditions for production of parathion hydrolase activity were determined for both pure and mixed cultures. The yield of parathion hydrolase activity/ of fermentation broth per hour was improved 22 fold by growing the pure culture on an industrial meat extract medium instead of the mixed culture on parathion.     

Chemical modification of proteases for wool cuticle scale removal

Over the last few decades several enzymatic processes to improve properties of wool fabrics like felting tendency, shrink resistance, dyeing ability and handling characteristics have been described. Previous investigations into the use of proteases to hydrolyse the cuticles at the surface of wool fibres, resulted in high strength and weight losses. Therefore restriction of the enzyme activity to the wool surface or control of enzyme diffusion to the cortex cells is required.

To change the diffusion behaviour of proteases in wool fibres, the soluble polymer PEG was covalently attached to a protease from Bacillus lentus. Modified enzymes with different molecular weights were compared. These modified enzymes retained up to 80% of their activity in the standard assay while hydrolysis of wool fibres was successfully restricted to cuticles, resulting in a 90% decrease in weight losses compared to non-modified enzymes.     

The sole cysteine residue (Cys301) of tetrathionate hydrolase from Acidithiobacillus ferrooxidans does not play a role in enzyme activity

Cysteine residues are absolutely indispensable for the reactions of almost all enzymes involved in the dissimilatory oxidation pathways of reduced inorganic sulfur compounds. Tetrathionate hydrolase from the acidophilic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans (Af-Tth) catalyzes tetrathionate hydrolysis to generate elemental sulfur, thiosulfate, and sulfate. Af-Tth is a key enzyme in the dissimilatory sulfur oxidation pathway in this bacterium. Only one cysteine residue (Cys301) has been identified in the deduced amino acid sequence of the Af-Tth gene. In order to clarify the role of the sole cysteine residue, a site-specific mutant enzyme (C301A) was generated. No difference was observed in the retention volumes of the wild-type and mutant Af-Tth enzymes by gel-filtration column chromatography, and surprisingly the enzyme activities measured in the cysteine-deficient and wild-type enzymes were the same. These results suggest that the sole cysteine residue (Cys301) in Af-Tth is involved in neither the tetrathionate hydrolysis reaction nor the subunit assembly. Af-Tth may thus have a novel cysteine-independent reaction mechanism.     

Engineering Saccharomyces cerevisiae for direct conversion of raw,uncooked or granular starch to ethanol

The production of raw starch-degrading amylases by recombinant Saccharomyces cerevisiae provides opportunities for the direct hydrolysis and fermentation of raw starch to ethanol without cooking or exogenous enzyme addition. Such a consolidated bioprocess (CBP) for raw starch fermentation will substantially reduce costs associated with energy usage and commercial granular starch hydrolyzing (GSH) enzymes. The core purpose of this review is to provide comprehensive insight into the physiological impact of recombinant amylase production on the ethanol-producing yeast. Key production parameters, based on outcomes from modifications to the yeast genome and levels of amylase production, were compared to key benchmark data. In turn, these outcomes are of significance from a process point of view to highlight shortcomings in the current state of the art of raw starch fermentation yeast compared to a set of industrial standards. Therefore, this study provides an integrated critical assessment of physiology, genetics and process aspects of recombinant raw starch fermenting yeast in relation to presently used technology. Various approaches to strain development were compared on a common basis of quantitative performance measures, including the extent of hydrolysis, fermentation-hydrolysis yield and productivity. Key findings showed that levels of α-amylase required for raw starch hydrolysis far exceeded enzyme levels for soluble starch hydrolysis, pointing to a pre-requisite for excess α-amylase compared to glucoamylase for efficient raw starch hydrolysis. However, the physiological limitations of amylase production by yeast, requiring high biomass concentrations and long cultivation periods for sufficient enzyme accumulation under anaerobic conditions, remained a substantial challenge. Accordingly, the fermentation performance of the recombinant S. cerevisiae strains reviewed in this study could not match the performance of conventional starch fermentation processes, based either on starch cooking and/or exogenous amylase enzyme addition. As an alternative strategy, the addition of exogenous GSH enzymes during early stages of raw starch fermentation may prove to be a viable approach for industrial application of recombinant S. cerevisiae, with the process still benefitting from amylase production by CBP yeast during later stages of cultivation.     

Phospholipase A2 activity on mixed lipid monolayers: Inhibition and activation of phospholipases A2 from porcine pancreas and Crotalus adamanteus by anionic and neutral amphiphiles

The effects of anionic and neutral amphiphiles on porcine pancreatic and Crotalus adamanteus phospholipases A2 were studied in a monolayer system as a function of surface pressure. The insoluble amphiphile, dicetyl phosphate (DCP), inhibited the hydrolysis of didecanoylphosphatidylcholine (DDPC) by both enzymes below their normal cutoff pressures with pure DDPC. DCP, however, enhanced enzyme penetration and thus activated the pancreatic enzyme above its normal cutoff pressure. The soluble surfactants, 3,5-dibromo- and 3,5-diiodosalicyclate, acetyl salicylate, and salicylic acid, had similar effects. 1,2-Didecanoin inhibited the hydrolysis of DDPC below the normal cutoff pressures and increased the cutoff pressures for both enzymes. Zwitterionic detergents, N-dodecyl- and N-tetradecyl-N,N-dimethyl-3-aminopropanesulfonate, were found to be potent inhibitors of the pancreatic enzyme on DDPC monolayers. Relative substrate specificities for both enzymes were determined as a function of surface pressure with phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid. Pancreatic phospholipase A2 was more active and penetrated to higher pressures with the anionic phospholipids, while the venom enzyme was more active with phosphatidylcholine.     

二氧化碳固定酶固碳效率及其对地衣芽孢杆菌代谢的影响

【背景】随着代谢工程与合成生物学的快速发展,通过对异养微生物进行代谢改造,利用生物法进行二氧化碳固定成为一个新的趋势。生物代谢途径中存在着大量固碳酶,这些酶尚待挖掘与应用,不同的酶固碳效率之间也缺少比较。【目的】在体外和体内对固碳功能和效率进行评价。【方法】选取3种固碳酶,即核酮糖1,5-二磷酸羧化加氧酶(ribose 1,5-diphosphate carboxylation oxygenase, RuBisCo)、磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate carboxykinase, PCK)和乙酰辅酶A羧化酶(acetyl coenzyme A carboxylase, ACC)在大肠杆菌中异源表达并纯化。测定纯酶的酶活,并建立无细胞催化实验-液质联用评价酶固碳能力的方法。在厌氧发酵条件下检测代谢指标,比较过表达固碳酶的地衣芽孢杆菌相较于原始菌的代谢差异。【结果】3种酶均实现可溶性表达,纯酶的比酶活分别为66.43、1.16和12.52 U/mg。通过体外无细胞催化实验,ACC在3种酶中表现出最高的固碳效率。分别过表达了PCK、ACC的重组地衣芽孢杆菌,厌氧发酵主产物乳酸的转化率从48.6%分别提升至58.1%和59.7%。【结论】可以通过体外、体内结合的方式对固碳酶的效率进行评价,该研究可为固碳酶在微生物遗传改造中理性、精准地应用提供参考。     

Phytase from Wheat Bran

myo-Inositol mono-, di-, tri-, tetra-, and pentaphosphate were prepared by enzymic hydrolysis of myo-inositol hexaphosphate with a 1,500-fold purified phytase preparation from wheat bran and the subsequent Dowex 1 column chromatography. Relative initial rates of hydrolysis of these inositol phosphates by phytase were nearly the same each other and the activation energy of hydrolysis was about 11,000 cal. per mole for all these substrates. Km values did not vary widely with the substrates. The hydrolysis of inositol phosphates proceeded in a complicated way, except inositol monophosphate, where the reaction was of the first order. The enzyme hydrolyzed the substrates in the manner that removed phosphate group of them one by one. When mixed substrate was used the enzyme showed a preferential attack on the highest member of the phosphates present. From the mixed substrate test, it was concluded that wheat bran phytase is a single enzyme.     

Thermostable Aminoacylase from Bacillus thermoglucosidius: Purification and Characterization

We studied the distribution of aminoacylase, an enzyme catalyzing the hydrolysis of N-acylamino acids, in thermophilic bacteria, and found Bacillus thermoglucosidius DSM 2542 to be the best producer of the enzyme. The enzyme, purified 13,400-fold to homogeneity in an overall yield of 34%, has a molecular weight of about 175,000, and is composed of four subunits identical in molecular weight (43,000). The enzyme contains 4g atoms of zinc per mol of enzyme protein. The enzyme catalyzes hydrolysis of various kinds of N-acyl-l-amino acids with very high molecular activity compared to those of fungal and mammalian enzymes: V_max and K_m for TV-acetyl-l-methionine are 3410 units/mg protein and 7.9 mm, respectively. Great stability at high temperatures and with organic solvents and protein denaturants is a characteristic of the enzyme.