Arthrobacter Psychrolactophilus Research Articles

Primer Design for Cloning of L-arabinose isomerase Gene from Arthrobacter psychrolactophillus into plasmid pET28a(+)

A novel putative L-arabinose isomerase (L-AI) called ApL-AI with the accession number WP_110486392.1 was successfully retrieved from Arthrobacter psychrolactophilus B7 genome (Accession: NZ_QJVC01000021.1) through genome mining analysis. This study aimed to obtain the L-AI gene from the Arthrobacter psychrolactophilus B7 genome and clone it into the pET28a(+) plasmid. The primers pair designed in this study successfully amplified the gene using 60 °C of PCR annealing temperature and supported the gene amplicon to insert into the pET28a(+) to form plasmid pET28a(+)-ApLAI. It was proved by the appearance of a 1557-bp amplification band on the gel electrophoresis. The sequencing analysis also revealed that the gene was inserted in the correct direction, with the gene positioned after the promoter and finished with a terminator. Therefore, the plasmid can be used to express the ApL-AI gene to produce the ApL-AI enzyme for downstream analysis and further prospecting.

Exploring an l-arabinose isomerase from cryophile bacteria Arthrobacter psychrolactophilus B7 for d-tagatose production

A novel l-arabinose isomerase (L-AI) from Arthrobacter psychrolactophilus (Ap L-AI) was successfully cloned and characterized. The enzyme catalyzes the isomerization of d-galactose into a rare sugar d-tagatose. The recombinant Ap L-AI had an approximate molecular weight of about 258 kDa, suggesting it was an aggregate of five 58 kDa monomers and became the first record as a homo-pentamer L-AI. The catalytic efficiency (kcat/Km) and Km for d-galactose were 0.32 mM−1 min−1 and 51.43 mM, respectively, while for l-arabinose, were 0.64 mM−1 min−1 and 23.41 mM, respectively. It had the highest activity at pH 7.0–7.5 and 60 °C in the presence of 0.250 mM Mn2+. Ap L-AI was discovered to be an outstanding thermostable enzyme that only lost its half-life value at 60 °C for >1000 min. These findings suggest that l-arabinose isomerase from Arthrobacter psychrolactophilus is a promising candidate for d-tagatose mass-production due to its industrially competitive temperature.

Expression and characterization of thermostable D-allulose 3-epimerase from Arthrobacter psychrolactophilus (Ap DAEase) with potential catalytic activity for bioconversion of D-allulose from d-fructose

A novel D-allulose 3-epimerase (DAEase) from Arthrobacter psychrolactophilus (Ap DAEase) was first characterized in this study. The enzyme catalyzes the epimerization of d-fructose into a functional rare sugar, D-allulose. Ap DAEase was the first record of DAEase identified as a homotrimer with the molecular weight of its subunit at approximately 34 kDa. It had an optimum activity at pH 8.5 and 70 °C in the presence of 1 mM Mg2+. Ap DAEase was found to be an excellent thermostable enzyme. The half-life value at 70 °C was 128.4 min. The kcat and catalytic efficiency of the enzyme toward d-fructose were 2920.00 s−1 and 3.953 mM−1 s−1, respectively. To the best of our knowledge, Ap DAEase possesses the highest kcat among the previously reported DAEases. The conversion ratio of 500 and 100 mg L−1d-fructose to D-allulose was approximately 27 % in 15 and 90 min, respectively. These research findings suggest that Ap DAEase is a promising candidate for the industrial production of D-allulose.

Microbiome convergence following sanitizer treatment and identification of sanitizer resistant species from spinach and lettuce rinse water

Fresh produce, as a known or suspected source of multiple foodborne outbreaks, harbors large populations of diverse microorganisms, which are partially released into wash water during processing. However, the dynamics of bacterial communities in wash water during produce processing is poorly understood. In this study, we investigated the effect of chlorine (FC) and peracetic acid (PAA) on the microbiome dynamics in spinach and romaine lettuce rinse water. Treatments with increasing concentrations of sanitizers resulted in convergence of distinct microbiomes. The resultant sanitizer resistant microbiome showed dominant presence by Bacillus sp., Arthrobacter psychrolactophilus, Cupriavidus sp., and Ralstonia sp. Most of the FC and PAA resistant bacteria isolated from spinach and lettuce rinse water after sanitation were gram positive spore forming species including Bacillus, Paenibacillus, and Brevibacillus spp., while several PAA resistant Pseudomonas spp. were also isolated from lettuce rinse water. Inoculation of foodborne pathogens altered the microbiome shift in spinach rinse water under PAA treatment, but not in lettuce rinse water or FC treated samples. These inoculated foodborne pathogens were not isolated among the sanitizer resistant strains.

Impact of routine sanitation on the microbiomes in a fresh produce processing facility

Indigenous bacterial populations in fresh-cut produce processing facilities can have a profound effect on the survival and proliferation of inadvertently contaminating foodborne pathogens. In this study, environmental samples were collected from a variety of Zone 3 sites in a processing plant before and after daily routine sanitation. Viable mesophilic aerobic bacteria population was evaluated using both culturing method and quantitative real-time PCR (qPCR) after propidium monoazide treatment. Zone 3 surface microbiota were analyzed using 16S rRNA gene amplicon sequencing with the Qiime2 bioinformatic pipeline. Over 8000 bacterial species across 4 major phyla were identified in Zone 3 microbiomes in the processing facility. Overall, effective bacterial reduction was observed at the sampling sites on the production floor, while sanitation effect on peripheral surfaces was less evident. Effective sanitation resulted in both quantitative and qualitive shifts of Zone 3 microbiota. Several species were highly abundant at multiple sample sites for both winter and summer samplings. Based on the spatial and temporal distribution of the most abundant species, a Zone 3 core microbiome in the processing facility was tentatively described to included Cupriavidus sp., Pseudomonas sp., Ralstonia sp., Arthrobacter psychrolactophilus, Pseudomonas veronii, Stenotrophomonas sp., and an unknown species of the family Enterobacteriaceae.

Isolation and characterization of a psychrophilic bacterium producing cold active lactose hydrolyzing enzyme from soil of Mt. Himalaya in Nepal

Psychrophilic bacteria, which grew on lactose as a carbon source, were isolated from soil of Island Peak area at Mt. Himalaya in Nepal, and identified. 148 bacterial isolates were isolated via X-gal hydrolysis on plate. Among these, three isolates, referred to as KNOUC 401, 402 and 403, displayed good cold-adapted growth and activity for hydrolysis of o-nitrophenyl-β-D-galactopyranoside (ONPG) and lactose in cell free extracts. Strain KNOUC403 showing negative activity in β-hemolysis was selected and identified. The strain was gram-positive bacilli, non-motile, catalase-positive and aerobic. Optimum growth was done at 20°C and pH 6.5~6.8. Anteiso-C15:0 (68.14%) was predominant in the fatty acid composition of cell. The physiological and biochemical properties, cellular fatty acid composition and sequence of 16S rDNA, suggest that strain KNOUC403 indicate a taxonomic affiliation to Arthrobacter psychrolactophilus. The optimal conditions for ONPG hydrolysis of cell free extracts from the strain were 15°C and pH 6.0, and the enzyme was stable at 4°C for 42 h. Zymogram assay of cell free extract from the strain in nondenaturing polyacrylamide gel showed a distinct band of X-gal hydrolysis. Key words: Psychrophilic bacteria, Arthrobacter psychrolactophilus, cold-active lactose hydrolyzing enzyme, Mt. Himalaya.

Arthrobacter psychrochitiniphilus sp. nov., a psychrotrophic bacterium isolated from Antarctica

A psychrotrophic Arthrobacter strain, GP3(T), was isolated from Adélie penguin guano from Antarctica and characterized. The 16S rRNA gene sequence of the novel strain showed the highest similarity (97.8 %) with that of Arthrobacter psychrolactophilus B7(T). The novel strain showed a morphological change from rod to coccus. The growth temperature range of strain GP3(T) was 0-25 degrees C, with optimal growth at 20 degrees C. The strain grew over a salinity range of between 0 and 3 % (w/v) NaCl and the optimal pH for growth was pH 6-8. Strain GP3(T) contained anteiso-C(15 : 0) as the major fatty acid. The major menaquinone was MK-9(H(2)). The cell-wall peptidoglycan type was of the A3alpha variant. The DNA G+C content of strain GP3(T) was 58.5 mol%. Strain GP3(T) was able to hydrolyse chitin, Tween 80, starch, cellulose and lactose, but not gelatin, lecithin or urea. In addition to these physiological characteristics, DNA-DNA hybridization studies clearly differentiated strain GP3(T) from A. psychrolactophilus. The combined results of phylogenetic, physiological and chemotaxonomic studies indicated that strain GP3(T) represents a novel species of the genus Arthrobacter. The name Arthrobacter psychrochitiniphilus sp. nov. (type strain GP3(T)=JCM 13874(T)=CGMCC 1.6355(T)) is proposed in recognition of the strain's strong chitin-utilizing ability.

Selection of a cold-adapted bacterium for bioremediation of wastewater at low temperatures

Amongst more than 1000 isolates collected in various cold environments, the strain Arthrobacter psychrolactophilus Sp 31.3 has been selected for its ability to grow and to produce exoenzymes at low temperatures, its inability to grow at 37 degrees C, its non-halophilic character and its growth versatility on various media. This non-pathogenic strain displays a strong resistance to desiccation and storage at room temperature and is suitable for the production of freeze-dried bacterial starters. When grown in a synthetic wastewater at 10 degrees C, the strain induces a complete clarification of the turbid medium and efficiently hydrolyses proteins, starch and lipids in the broth. Furthermore, this strain has a remarkable capacity to improve the biodegradability of organic compounds in wastewater as indicated by a BOD(5)/COD ratio of 0.7.

Use of 16S rRNA gene sequencing analysis to characterize culturable intestinal bacteria in Atlantic salmon (Salmo salar) fed diets with cellulose or non-starch polysaccharides from soy

Duplicate groups of Atlantic salmon (Salmo salar L.), kept in seawater, were fed fish meal-based cold-pelleted diets. Diets with non-starch polysaccharides (NSP), either cellulose, purified soybean NSP or extruded purified soybean NSP at a dietary level of 100 g kg−1, were compared with a diet without supplemental NSP and a diet with soybean meal in a 28-day feeding trial. Isolation and characterizations were limited to culturable bacteria and population levels of aerobic and facultative aerobic heterotrophic autochthonous (adherent) and allochthonous (transient) bacteria present in the mid and distal intestines of Atlantic salmon fed the five different diets estimated using traditional bacteriological techniques. The presence of an autochthonous microbiota was demonstrated using transmission electron microscopy. No significant effects of diet composition were observed on total population levels of culturable bacteria present in the digestive tract, but the study showed that the composition of the gut microbiota (autochthonous or allochthonous) was sensitive to dietary changes. A total of 752 culturable isolates from the intestines were characterized by biochemical and physiological properties. Of these, 188 isolates were further characterized by partial sequencing the 16S rRNA genes. Among these, 146 isolates belonged to 31 phylotypes that were >94% identical to previously described species. However, 42 isolates showed similarity <94% to species available at the National Center of Biotechnology Information. Several of the phylotypes identified in the present study have not been reported previously in the gastrointestinal (GI) tract of fish, including the Gram-negative bacteria Gelidibacter salicanalis, Pseudoalteromonas elyakovii, Psychrobacter aquimaris, Psychrobacter cibarius, Psychrobacter fozii, Psychrobacter maritimus, Psychrobacter okhotskensis and Psychrobacter psychrophilus. Among the Gram-positive bacteria identified were Arthrobacter bergeri, Arthrobacter psychrolactophilus, Arthrobacter rhombi, Bacillus pumilus, Bacillus subtilis, Exiguobacterium spp., Microbacterium oxydans, Planococcus maritimus, Sporosarcina ginsengisoli and several bacteria that have been described as unculturable previously. In addition, we identified Carnobacterium inhibens, a lactic acid bacterium that is not frequently isolated from the GI tract of fish. Psychrobacter cibarius was the dominant bacterial species and was isolated from the digestive tract of all fish investigated.

Overexpression and functional analysis of cold-active β-galactosidase from Arthrobacter psychrolactophilus strain F2

Cold-active β-galactosidase from Arthrobacter psychrolactophilus strain F2 was overexpressed in Escherichia coli using the Cold expression system and the recombinant enzyme, rBglAp, was characterized. The purified rBglAp exhibited similar enzymatic properties to the native enzyme, e.g., (i) it had high activity at 0 °C, (ii) its optimum temperature and pH were 10 °C and 8.0, respectively, and (iii) it was possible to rapidly inactivate the rBglAp at 50 °C in 5 min. Moreover, rBglAp was able to hydrolyze both ONPG and lactose with K m values of 2.7 and 42.1 mM, respectively, at 10 °C. One U of rBglAp could hydrolyze about 70% of the lactose in 1 ml of milk in 24 h, and the enzyme produced trisaccharide from lactose. We conclude that rBglAp is a cold-active enzyme that is extremely heat labile and has significant potential application to the food industry.

Purification and molecular characterization of cold-active β-galactosidase from Arthrobacter psychrolactophilus strain F2

In this study, we purified and molecularly characterized a cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2. The purified beta-galactosidase from strain F2 exhibited high activity at 0 degrees C, and its optimum temperature and pH were 10 degrees C and 8.0, respectively. It was possible to inactivate the beta-galactosidase rapidly at 45 degrees C in 5 min. The enzyme was able to hydrolyze lactose as a substrate, as well as o-nitrophenyl-beta-D-galactopyranoside (ONPG), the Km values with ONPG and lactose being calculated to be 2.8 mM and 50 mM, respectively, at 10 degrees C. Moreover, the bglA gene encoding the beta-galactosidase of strain F2 was cloned and analyzed. The bglA gene consists of a 3,084-bp open reading frame corresponding to a protein of 1,028 amino acid residues. BglAp, the gene product derived from bglA, had several conserved regions for glycosyl hydrolase family 2, e.g., the glycosyl hydrolase 2 (GH2) sugar binding domain, GH2 acid-base catalyst, GH2 triosephosphate isomerase barrel domain, GH2 signature 1, and several other GH2 conserved regions. From these facts, we conclude that the beta-galactosidase from A. psychrolactophilus strain F2, which is a new member of glycosyl hydrolase family 2, is a cold-active enzyme that is extremely heat labile and could have advantageous applications in the food industry.

Characteristics of the amylase of Arthrobacter psychrolactophilus

Properties of the extracellular amylase produced by the psychrotrophic bacterium, Arthrobacter psychrolactophilus, were determined for crude preparations and purified enzyme. The hydrolysis of soluble starch by concentrated crude preparations was found to be a nonlinear function of time at 30 and 40 degrees C. Concentrates of supernatant fractions incubated without substrate exhibited poor stability at 30, 40, or 50 degrees C, with 87% inactivation after 21 h at 30 degrees C, 45% inactivation after 40 min at 40 degrees C and 90% inactivation after 10 min at 50 degrees C. Proteases known to be present in crude preparations had a temperature optimum of 50 degrees C, but accounted for a small fraction of thermal instability. Inactivation at 30, 40, or 50 degrees C was not slowed by adding 20 mg/ml bovine serum albumin or protease inhibitor cocktail to the preparations or the assays to protect against proteases. Purified amylase preparations were almost as thermally sensitive in the absence of substrate as crude preparations. The temperature optimum of the amylase in short incubations with Sigma Infinity Amylase Reagent was about 50 degrees C, and the amylase required Ca(+2) for activity. The optimal pH for activity was 5.0-9.0 on soluble starch (30 degrees C), and the amylase exhibited a K (m) with 4-nitrophenyl-alpha-D-maltoheptaoside-4,6-O-ethylidene of 120 microM at 22 degrees C. The amylase in crude concentrates initially hydrolyzed raw starch at 30 degrees C at about the same rate as an equal number of units of barley alpha-amylase, but lost most of its activity after only a few hours.

Production of amylase by Arthrobacter psychrolactophilus

Arthrobacter psychrolactophilus ATCC 700733 grew with a doubling time of 1.5-2.3 h (22 degrees C) and produced up to 0.2 units/mL (soluble starch assay) of extracellular amylase in tryptic soy broth without dextrose (TSBWD) containing 0.5% or 1.0% (w/v) soluble starch or maltose as the fermentable substrate. Time-course experiments in media containing soluble starch as substrate showed that amylolytic activity appeared in cultures at 24 h (after exponential growth had ceased), reached peak levels in 72-96 h, and declined rapidly after reaching peak levels. Peak levels were highest in TSBWD containing 1.0% soluble starch. Proteolytic activity appeared at about the same time as amylolytic activity and increased during the period of amylase production. Significant amylase production was not observed in cultures in TSBWD with 0.5% glucose or in cultures grown at 28 degrees C, but low levels of amylase were observed in TSBWD cultures grown at 19-23 degrees C which contained no added carbohydrate. A single band of activity was observed after electrophoresis of supernatant fractions in non-denaturing gels, followed by in situ staining for amylolytic activity. The amylase possessed a raw starch-binding domain and bound to uncooked corn, wheat or potato starch granules. It was active in the Phadebas assay for alpha-amylase. Activity was maximum on soluble starch at a temperature between 40 degrees C and 50 degrees C. The amylase after purification by affinity chromatography on raw starch granules exhibited two starch-binding protein bands on SDS gels of 105 kDa and 26 kDa.

Polyphasic taxonomic study of strain CCM 2783 resulting in the description of Arthrobacter stackebrandtii sp. nov.

Strain CCM 2783, previously classified as representing Arthrobacter aurescens, was subjected to a polyphasic taxonomic study. 16S rRNA gene sequence analysis and chemotaxonomic characteristics such as peptidoglycan type A3alpha Lys-Ala(2), major menaquinone MK-9(H(2)) and fatty acid composition confirmed assignment of the strain to the genus Arthrobacter. The results of phylogenetic analysis, DNA-DNA relatedness experiments and physiological and chemotaxonomic characteristics indicate that CCM 2783 differs from its nearest phylogenetic relative Arthrobacter psychrolactophilus and from other recognized Arthrobacter species. Therefore, a novel species, Arthrobacter stackebrandtii sp. nov., is proposed with the type strain CCM 2783(T) (=DSM 16005(T)).

Isolation and characterization of psychrophiles producing cold-active beta-galactosidase.

The present study was conducted to screen for psychrophilic micro-organisms that are able to hydrolyse lactose at low temperature, and to examine the cold-active beta-galactosidase produced by the isolated psychrophilic micro-organisms. Psychrophilic bacteria, which grow on lactose as a sole carbon source, were isolated from soil from Hokkaido, Japan. The phenotype and sequence of 16S rDNA of the isolated strains indicated a taxonomic affiliation to Arthrobacter psychrolactophilus. The isolated A. psychrolactophilus strains were able to grow on lactose at below 5 degrees C, and showed cold-active beta-galactosidase activity, which was highly specific at even 0 degrees C. Facts in this study may indicate the possibility that the isolated strains produce novel beta-galactosidases that are able to hydrolyse lactose at low temperature, although some strains have isozymes. It may be possible that the cold active beta-galactosidases from the isolated strains can be applied to the food industry, e.g. processing of milk and whey below 5 degrees C.