Showing papers on "Arthrobacter published in 2003"

Toxicity of Hexavalent Chromium and Its Reduction by Bacteria Isolated from Soil Contaminated with Tannery Waste

Abstract: An Arthrobacter sp. and a Bacillus sp., isolated from a long-term tannery waste contaminated soil, were examined for their tolerance to hexavalent chromium [Cr(VI)] and their ability to reduce Cr(VI) to Cr(III), a detoxification process in cell suspensions and cell extracts. Both bacteria tolerated Cr(VI) at 100 mg/ml on a minimal salts agar medium supplemented with 0.5% glucose, but only Arthrobacter could grow in liquid medium at this concentration. Arthrobacter sp. could reduce Cr(VI) up to 50 μg/ml, while Bacillus sp. was not able to reduce Cr(VI) beyond 20 μg/ml. Arthrobacter sp. was distinctly superior to the Bacillus sp. in terms of their Cr(VI)-reducing ability and resistance to Cr(VI). Assays with permeabilized (treated with toluene or Triton X 100) cells and crude extracts demonstrated that the Cr(VI) reduction was mainly associated with the soluble protein fraction of the cell. Arthrobacter sp. has a great potential for bioremediation of Cr(VI)-containing waste.

AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria.

Abstract: Quorum sensing is a signalling mechanism that controls diverse biological functions, including virulence, via N-acylhomoserine lactone (AHL) signal molecules in Gram-negative bacteria. With the aim of isolating strains or enzymes capable of blocking quorum sensing by inactivating AHL, bacteria were screened for AHL degradation by their ability to utilize N-3-oxohexanoyl-l-homoserine lactone (OHHL) as the sole carbon source. Among four isolates, strain IBN110, identified as Arthrobacter sp., was found to grow rapidly on OHHL, and to degrade various AHLs with different lengths and acyl side-chain substitutions. Co-culture of Arthrobacter sp. IBN110 and the plant pathogen Erwinia carotovora significantly reduced both the AHL amount and pectate lyase activity in co-culture medium, suggesting the possibility of applying Arthrobacter sp. IBN110 in the control of AHL-producing pathogenic bacteria. The ahlD gene from Arthrobacter sp. IBN110 encoding the enzyme catalysing AHL degradation was cloned, and found to encode a protein of 273 amino acids. A mass spectrometry analysis showed that AhlD probably hydrolyses the lactone ring of N-3-hexanoyl-l-homoserine lactone, indicating that AhlD is an N-acylhomoserine lactonase (AHLase). A comparison of AhlD with other known AHL-degrading enzymes, Bacillus sp. 240B1 AiiA, a Bacillus thuringiensis subsp. kyushuensis AiiA homologue and Agrobacterium tumefaciens AttM, revealed 25, 26 and 21 % overall identities, respectively, in the deduced amino acid sequences. Although these identities were relatively low, the HXDH≈H≈D motif was conserved in all the AHLases, suggesting that this motif is essential for AHLase activity. From a genome database search based on the conserved motif, putative AhlD-like lactonase genes were found in several other bacteria, and AHL-degrading activities were observed in Klebsiella pneumoniae and Bacillus stearothermophilus. Furthermore, it was verified that ahlK, an ahlD homologue, encodes an AHL-degrading enzyme in K. pneumoniae. Accordingly, the current results suggest the possibility that AhlD-like AHLases could exist in many other micro-organisms.

Respiration of 13C-labeled substrates added to soil in the field and subsequent 16S rRNA gene analysis of 13C-labeled soil DNA.

Abstract: Our goal was to develop a field soil biodegradation assay using (13)C-labeled compounds and identify the active microorganisms by analyzing 16S rRNA genes in soil-derived (13)C-labeled DNA. Our biodegradation approach sought to minimize microbiological artifacts caused by physical and/or nutritional disturbance of soil associated with sampling and laboratory incubation. The new field-based assay involved the release of (13)C-labeled compounds (glucose, phenol, caffeine, and naphthalene) to soil plots, installation of open-bottom glass chambers that covered the soil, and analysis of samples of headspace gases for (13)CO(2) respiration by gas chromatography/mass spectrometry (GC/MS). We verified that the GC/MS procedure was capable of assessing respiration of the four substrates added (50 ppm) to 5 g of soil in sealed laboratory incubations. Next, we determined background levels of (13)CO(2) emitted from naturally occurring soil organic matter to chambers inserted into our field soil test plots. We found that the conservative tracer, SF(6), that was injected into the headspace rapidly diffused out of the soil chamber and thus would be of little value for computing the efficiency of retaining respired (13)CO(2). Field respiration assays using all four compounds were completed. Background respiration from soil organic matter interfered with the documentation of in situ respiration of the slowly metabolized (caffeine) and sparingly soluble (naphthalene) compounds. Nonetheless, transient peaks of (13)CO(2) released in excess of background were found in glucose- and phenol-treated soil within 8 h. Cesium-chloride separation of (13)C-labeled soil DNA was followed by PCR amplification and sequencing of 16S rRNA genes from microbial populations involved with (13)C-substrate metabolism. A total of 29 full sequences revealed that active populations included relatives of Arthrobacter, Pseudomonas, Acinetobacter, Massilia, Flavobacterium, and Pedobacter spp. for glucose; Pseudomonas, Pantoea, Acinetobacter, Enterobacter, Stenotrophomonas, and Alcaligenes spp. for phenol; Pseudomonas, Acinetobacter, and Variovorax spp. for naphthalene; and Acinetobacter, Enterobacter, Stenotrophomonas, and Pantoea spp. for caffeine.

Arthrobacter Strain VAI-A Utilizes Acyl-Homoserine Lactone Inactivation Products and Stimulates Quorum Signal Biodegradation by Variovorax paradoxus

Abstract: Many Proteobacteria produce acyl-homoserine lactones (acyl-HSLs) and employ them as dedicated cell-to-cell signals in a process known as quorum sensing. Previously, Variovorax paradoxus VAI-C was shown to utilize diverse acyl-HSLs as sole sources of energy and nitrogen. We describe here the properties of a second isolate, Arthrobacter strain VAI-A, obtained from the same enrichment culture that yielded V. paradoxus VAI-C. Although strain VAI-A grew rapidly and exponentially on a number of substrates, it grew only slowly and aberrantly (i.e., linearly) in media amended with oxohexanoyl-HSL as the sole energy source. Increasing the culture pH markedly improved the growth rate in media containing this substrate but did not abolish the aberrant kinetics. The observed growth was remarkably similar to the known kinetics of the pH-influenced half-life of acyl-HSLs, which decay chemically to yield the corresponding acyl-homoserines. Strain VAI-A grew rapidly and exponentially when provided with an acyl-homoserine as the sole energy or nitrogen source. The isolate was also able to utilize HSL as a sole source of nitrogen but not as energy for growth. V. paradoxus, known to release HSL as a product of quorum signal degradation, was examined for the ability to support the growth of Arthrobacter strain VAI-A in defined cocultures. It did. Moreover, the acyl-HSL-dependent growth rate and yield of the coculture were dramatically superior to those of the monocultures. This suggested that the original coenrichment of these two organisms from the same soil sample was not coincidental and that consortia may play a role in quorum signal turnover and mineralization. The fact that Arthrobacter strain VAI-A utilizes the two known nitrogenous degradation products of acyl-HSLs, acyl-homoserine and HSL, begins to explain why none of the three compounds are known to accumulate in the environment.

Members of the genus Arthrobacter grow anaerobically using nitrate ammonification and fermentative processes: anaerobic adaptation of aerobic bacteria abundant in soil.

Abstract: Members of the genus Arthrobacter are usually regarded as obligate aerobic bacteria. The anaerobic growth and energy metabolism of two Arthrobacter species were investigated. Arthrobacter globiformis utilized both nitrate ammonification and lactate, acetate and ethanol producing fermentation processes for anaerobic growth. Only nitrate supported anaerobic growth of Arthrobacter nicotianae. Anaerobically induced respiratory nitrate reductase activity was detected in both strains. Neither of the tested strains used the alternative electron acceptors fumarate, dimethylsulfoxide or trimethylamine-N-oxide.

Arthrobacter koreensis sp. nov., a novel alkalitolerant bacterium from soil

Abstract: Two Arthrobacter strains, CA15-8(T) and CA15-9, were isolated from soil, under alkali conditions, and characterized phenotypically, chemotaxonomically and genetically. These alkalitolerant organisms grew over a wide pH range (pH 7.0-12.0), with an optimum at pH 7.0-8.0. The mean G+C content of the DNA of these strains was 63+/-2 mol%. The strains contained MK-8(H(2)) and MK-9(H(2)) as the main respiratory quinones. The cell-wall peptidoglycan was Lys-Thr-Ala(2) and the whole-cell sugar was rhamnose. The major cellular fatty acids of the isolates were anteiso-C(15 : 0) and iso-C(15 : 0). A phylogenetic analysis, based on 16S rDNA sequence data, revealed that strains CA15-8(T) and CA15-9 formed an evolutionary lineage distinct from other Arthrobacter species. On the basis of morphological, physiological and chemotaxonomic characteristics, 16S rDNA sequence comparisons and DNA-DNA hybridization data, a novel species of Arthrobacter is proposed, namely Arthrobacter koreensis (type strain CA15-8(T)=KCTC 9922(T)=IFO 16787(T)).

Characterization and molecular cloning of a novel enzyme, inorganic polyphosphate/ATP-glucomannokinase, of Arthrobacter sp. strain KM

Abstract: A bacterium exhibiting activities of several inorganic polyphosphate [poly(P)]- and ATP-dependent kinases, including glucokinase, NAD kinase, mannokinase, and fructokinase, was isolated, determined to belong to the genus Arthrobacter, and designated Arthrobacter sp. strain KM. Among the kinases, a novel enzyme responsible for the poly(P)- and ATP-dependent mannokinase activities was purified 2,200-fold to homogeneity from a cell extract of the bacterium. The purified enzyme was a monomer with a molecular mass of 30 kDa. This enzyme phosphorylated glucose and mannose with a high affinity for glucose, utilizing poly(P) as well as ATP, and was designated poly(P)/ATP-glucomannokinase. The Km values of the enzyme for glucose, mannose, ATP, and hexametaphosphate were determined to be 0.50, 15, 0.20, and 0.02 mM, respectively. The catalytic sites for poly(P)-dependent phosphorylation and ATP-dependent phosphorylation of the enzyme were found to be shared, and the poly(P)-utilizing mechanism of the enzyme was shown to be nonprocessive. The gene encoding the poly(P)/ATP-glucomannokinase was cloned from Arthrobacter sp. strain KM, and its nucleotide sequence was determined. This gene contained an open reading frame consisting of 804 bp coding for a putative polypeptide with a calculated molecular mass of 29,480 Da. The deduced amino acid sequence of the polypeptide exhibited homology to the amino acid sequences of the poly(P)/ATP-glucokinase of Mycobacterium tuberculosis H37Rv (level of homology, 45%), ATP-dependent glucokinases of Corynebacterium glutamicum (45%), Renibacterium salmoninarum (45%), and Bacillus subtilis (35%), and proteins of bacteria belonging to the order Actinomyces whose functions are not known. Alignment of these homologous proteins revealed seven conserved regions. The mannose and poly(P) binding sites of poly(P)/ATP-glucomannokinase are discussed.

Naphthalene-utilizing and mercury-resistant bacteria isolated from an acidic environment.

Abstract: Soil samples were taken from areas of low pH (2.5–3.5) surrounding an outdoor coal storage pile. These samples were added to medium with naphthalene as the sole carbon source to enrich for organisms capable of degrading polycyclic aromatic hydrocarbons (PAH) at low pH. Five such bacterial strains were isolated. Sequencing of the 16S rDNA showed them to be members of the genera Clavibacter, Arthrobacter and Acidocella. These organisms were all capable of growth with naphthalene as a sole carbon source at low pH. The genes nahAc, nahAd, phnAc, nahH, xylE or GST, which are known to be associated with PAH degradation were not detected. Isolate 10, the Acidocella strain, tolerated high levels of mercury. PCR amplification and sequencing of genes from the mer operon from isolate 10 DNA suggested that mercury is transported into the bacterial cell and subsequently detoxified since the enzymes encoded by genes in this operon are involved in these processes.

Characterization of the 4-Hydroxybenzoyl-Coenzyme A Thioesterase from Arthrobacter sp. Strain SU

Abstract: During the last century, large quantities of industrially produced 4-chlorobenzoate (4-CBA) or 4-CBA progenitors (herbicides and polychlorinated biphenyl pesticides) (8, 14, 17, 19) have been released into the environment. A wide variety of 4-CBA-degrading bacterial strains, which are capable of growth on 4-CBA as the principal source of carbon, have been isolated from soil (11, 35). In these bacteria, 4-CBA is first converted to 4-hydroxybenzoate (4-HBA), which is then further metabolized via the ortho- or meta-cleavage pathway (11). The 4-CBA dehalogenation pathway of the 4-CBA-degrading bacterium Pseudomonas sp. strain CBS3 (21) was the first to be characterized. The pathway consists of three reaction steps (28) (see Fig. ​Fig.1)1) which are catalyzed by the enzymes 4-CBA-coenzyme A (CoA) ligase, 4-CBA-CoA dehalogenase, and 4-HBA-CoA thioesterase. Each of these enzymes has been isolated, and their kinetic properties have been defined (7). The three 4-CBA dehalogenation pathway genes are organized within an operon that is under the regulatory control of 4-CBA. In some 4-CBA-degrading bacteria, the gene cluster is located within the chromosomal DNA (6, 23, 26), whereas in others it is carried on a plasmid (22, 25). FIG. 1. Illustration of the reaction steps of the bacterial 4-CBA dehalogenation pathway (28). The Arthrobacter sp. strain SU 4-CBA dehalogenation pathway operon was isolated on a 3.6-kb DNA fragment derived from a 120-kb plasmid (27). The DNA fragment was shown to contain six open reading frames, three of which (fcbA, fcbB, and fcbC) are transcribed in the same direction. Truncation mutants lacking fcbA or fcbB were unable to dehalogenate 4-CBA, whereas the truncation mutant devoid of fcbC was 10-fold less active. The fcbA gene encoded a 57-kDa protein that shares 38% sequence identity with the 57-kDa 4-CBA-CoA ligase of the Pseudomonas sp. strain CBS3 4-CBA dehalogenation pathway. The fcbB gene encoded a 30-kDa protein that shares 50% sequence identity with the 30-kDa 4-CBA-CoA dehalogenase of the Pseudomonas sp. strain CBS3 4-CBA dehalogenation pathway. The fcbA and fcbB genes were thus assumed to encode 4-CBA-CoA ligase and 4-CBA-CoA dehalogenase (27). The neighboring gene, fcbC, encoded a 16-kDa protein that did not share a significant level of sequence homology with the known 16-kDa Pseudomonas sp. strain CBS3 4-HBA-CoA thioesterase. The lack of sequence homology between the Pseudomonas thioesterase and the putative Arthrobacter thioesterase, as well as the different gene order observed in the Pseudomonas (dehalogenase-ligase-thioesterase) and Arthrobacter (ligase-dehalogenase-putative thioesterase) operons, made the assignment of the fcbC as the 4-HBA-CoA thioesterase gene uncertain. In the present study, the fcbC gene was subcloned, and its protein product was characterized.

Catabolism of arylboronic acids by Arthrobacter nicotinovorans strain PBA.

Abstract: Arthrobacter sp. strain PBA metabolized phenylboronic acid to phenol. The oxygen atom in phenol was shown to be derived from the atmosphere using (18)O(2). 1-Naphthalene-, 2-naphthalene-, 3-cyanophenyl-, 2,5-fluorophenyl-, and 3-thiophene-boronic acids were also transformed to monooxygenated products. The oxygen atom in the product was bonded to the ring carbon atom originally bearing the boronic acid substituent with all the substrates tested.

Arthrobacter gandavensis sp. nov., for strains of veterinary origin.

Abstract: Three strains of a previously undescribed, Gram-positive, coryneform bacterium, which were isolated from cattle, were subjected to polyphasic taxonomic analysis. Comparative 16S rRNA gene sequencing revealed that the unknown isolates were members of the genus Arthrobacter and were phylogenetically closely related to Arthrobacter luteolus. However, DNA–DNA hybridization indicated that the strains belonged to a new sub-lineage within the genus Arthrobacter. The unknown isolates can be distinguished from related species by biochemical tests. It is proposed that the Arthrobacter-like bacteria of veterinary origin should be classified in the genus Arthrobacter as Arthrobacter gandavensis sp. nov., with the type strain LMG 21285T (=DSM 15046T).

Influence of Al on growth, cell size and content of intracellular water of Arthrobacter sp. PI/1-95.

Abstract: A distinct inhibition of the growth of a typical soil-born bacterium, Arthrobacter sp. PI/1-95, by aluminium was established. The content of intracellular water and (consequently) the cell size of the bacterium significantly increased when cells came into contact with aluminium. Both parameters were also increased when applying HCl, but Al caused the greater change, so that a distinct discrimination between the effects of aluminium on the one hand and a sole pH-effect on the other hand was possible. The presented results point to an osmo-regulative disorder connected with a malfunction of cell membrane and cell wall, which seems to be a probable mechanism for an Al-toxicity in connection with Arthrobacter sp. PI/1-95.

D-arginase of Arthrobacter sp. KUJ 8602: characterization and its identity with Zn(2+)-guanidinobutyrase.

Abstract: D-Arginase activity was found in the cells of an isolate, Arthrobacter sp. KUJ 8602, grown in the L-arginine medium, and the enzyme was purified and characterized. Its molecular weight was estimated to be about 232,000 by gel filtration, and that of the subunit was approximately 40,000 by SDS-PAGE, suggesting that the enzyme is a homohexamer. The enzyme acted on not only D-arginine but also 4-guanidinobutyrate, 3-guanidinopropionate and even L-arginine. The V(max)/K(m) values for 4-guanidinobutyrate and D-arginine were determined to be 87 and 0.81 micro mol/min/mg/mM, respectively. Accordingly, the enzyme is regarded as a kind of guanidinobutyrase [EC 3.5.3.7]. The pH optima for 4-guanidinobutyrate and D-arginine were 9.0 and 9.5, respectively. The enzyme was inhibited competitively by 5-aminovalerate, and thiol carboxylates such as mercaptoacetate served as strong mixed-type inhibitors. The enzyme contained about 1 g-atom of firmly bound Zn(2+) per mol of subunit, and removal of the metal ions by incubation with 1,10-phenanthroline resulted in loss of activity. The inactivated enzyme was reactivated markedly by incubation with either Zn(2+) or Co(2+), and slightly by incubation with Mn(2+). The nucleotide sequence of enzyme contains an open reading frame that encodes a polypeptide of 353 amino acid residues (M(r): 37,933). The predicted amino acid sequence contains sequences involved in the binding of metal ions and the guanidino group of the substrate, which show a high homology with corresponding sequences of Mn(2+)-dependent amidinohydrolases such as agmatinase from Escherichia coli and L-arginase from rat liver, though the homology of their entire sequences is relatively low (24-43%).

Purification and properties of a heat-stable inulin fructotransferase from Arthrobacter ureafaciens.

Abstract: An inulin fructotransferase producing difructose dianhydride I (EC 2.4.1.200) was purified from Arthrobacter ureafaciens A51-1. It had maximum activity at pH 5.5 and 45 °C, and was stable up to 80 °C. This is the highest thermal stability for this enzyme reported to date. The molecular mass was estimated to be 38 000 by SDS-PAGE, and 61 000 by gel filtration. It was therefore estimated to be a dimer.

Proteolytic activity of a yeast cell wall lytic Arthrobacter species

Abstract: B . F . A D A M I T S C H , F . K A R N E R A N D W . H A M P E L . 2003. Aims: To investigate properties of the proteolytic activity of a yeast cell wall lytic soil bacterium identified as an Arthrobacter species. Methods and Results: The organism was grown at pH 7AE5 and 30� C in shake flasks on media with different complex subtrates. Highest proteolytic activity assayed with azocaseine was detected in media with wheat gluten. In addition, L-leucine, L L-alanine exopeptidase activity and esterase activity were found. The proteolytic activity showed stability up to pH 12, with a maximum at pH 11. The temperature optimum was at 55� C, but there was a loss in enzyme activity of 50% within 2 h. The proteolytic activity was inhibited by 3,4-dichloroisocumarin, whereas there was little or no effect with EDTA, pepstatin A or E64. Conclusions: The proteolytic activity is highly alkaline stable. The formation of the enzyme can be induced by media with high protein content.

Isolation of novel phenol-degrading bacteria from activated sludge using feed water medium (fwm)

Abstract: 100 isolates were obtained from feed water solid media using spread-plate method with diluted activated sludge ( 10 -5 dilution rate). All the isolates were capable of growing on a synthetic medium with phenol (500 ~ 800 mg L -1 ) as a sole carbon source and classified into 4 operational taxoriomic units (OTUs) by amplified ribosomal DNA restriction analysis (ARDRA). The full-length 16S rDNA gene of one representative strain for each OTU was amplified, cloned, sequenced and blasted against the GenBank database. The results showed that 97 isolates were closely related to Alcaligenes faecalis with 99% homology; two isolates showed 99% identity to the sequence of Arthrobacter nicotianae and Klebsiella sp. respectively; and the last one exhibited 96% homology with Ochrobactrum sp. The specific amplification of the gene encoding the large subunit of the multi-component phenol hydroxylase (LmPHs) revealed that three out of four OTUs employed the metabolic pathway catalyzed by multicomponent phenol hydroxylase to degrade phenol, while the specific amplification from the Arthrobacter nicotianae-like strain did not show any products, indicating a possibly different phenol-degrading pathway. Fig 2, Tab 2, Ref 11

Hsp70 from arthrobacter

Abstract: The hsp70 gene from an Arthrobacter species has been isolated and sequenced. The encoded protein is believed to be highly immunogenic, especially in fish, and also has utility as a non-specific adjuvant, and as an adjuvanting carrier for heterologous antigens.

Investigation of Pyrimidine Salvage Pathways to Categorize Indigenous Soil Bacteria of Agricultural and Medical Importance and Analysis of the Pyrimidine Biosynthetic Pathway's Enzyme Properties for Correlating Cell Morphology to Function in All Phases of Growth

Abstract: This dissertation comprises three parts and is presented in two chapters. Chapter 1 concerns Arthrobacter, a bacterium with an intriguing growth cycle. Whereas most bacteria exist as either a rod or coccus, this bacterium shares the rod/coccus lifestyle. It therefore seemed important to examine the growth regulatory pathways from the rod and coccus. The committed step, that catalyzed by aspartate transcarbamoylase (ATCase), in the pyrimidine biosynthetic pathway was chosen. The ATCase in Arthrobacter is like the well known Pseudomonas enzyme except that it has an active dihydroorotase (DHOase) associated. Included in Chapter 1 is the description of a microorganism, Burkholderia cepacia, whose ATCase has characteristics that are at once reminiscent of bacteria, mammals, and fungi. It differs in size or aggregation based on environmental conditions. In addition, it has an active DHOase associated with the ATCase, like Arthrobacter. B. cepacia is important both medically and for bioremediation. Since B. cepacia is resistant to most antibiotics, its unique ATCase is a prime target for inhibition. Whereas the first chapter deals with the de novo pathway to making pyrimidines, which is found mainly in the lag and log phase, Chapter 2 addresses the salvage pathway, which comes more into play during the stationary phase. This section focuses on the isolation, identification, and grouping of a number of natural soil bacteria from various soil locations. These organisms are important agriculturally, medically, and industrially. Addition of these soil isolates to poor soils has been found to improve the soil. In a previous study by D.A. Beck, the salvage schemes for a number of laboratory strains of microorganisms were determined. Nine separate classes of salvage were designated by determining the salvage enzymes present. In this study emphasis has been placed on soil bacteria, which had not previously been analyzed. A number of species of soil bacteria were identified using the MIDI. The salvage enzymes were then determined for these organisms and a comparison of these isolates to the previous study was performed in order to group the new organisms into 19 salvage schemes, that is 10 more than in the previous study.

Some Properties of the Xylose/Glucose Isomerase of Immobilized Arthrobacter sp. Cells

Abstract: The study of the xylose/glucose isomerase-containing Arthrobacter sp B-5 cells immobilized in cobalt hydroxide gel showed that immobilization increases the substrate affinity of the enzyme, its thermo- and pH-optima of action and stability, and makes the addition of stabilizing cobalt ions to the isomerization medium unnecessary

Optimization of conditions for shaking fermentation of #beta#-fructofuranosidase by Arthrobacter

Abstract: Through single factor experiment and response surface analysis (RSA), the optimum composition of culture medium were sucrose 4.45%, yeast extract 1.40%, peptone 0.93%, (NH_(4))_(2)HPO_(4) 0.4%, MgSO_(4)·7H_(2)O 0.13%. Under this condition the output of Lactosucrose was 16.38%.