Secretory expression of amylosucrase in Bacillus licheniformis through twin-arginine translocation pathway

Abstract   Amylosucrase (EC 2.4.1.4) is a versatile enzyme with significant potential in biotechnology and food production. To facilitate its efficient preparation, a novel expression strategy was implemented in Bacillus licheniformis for the secretory expression of Neisseria polysaccharea amylosucrase (NpAS). The host strain B. licheniformis CBBD302 underwent genetic modification through the deletion of sacB, a gene responsible for encoding levansucrase that synthesizes extracellular levan from sucrose, resulting in a levan-deficient strain, B. licheniformis CBBD302B. Neisseria polysaccharea amylosucrase was successfully expressed in B. licheniformis CBBD302B using the highly efficient Sec-type signal peptide SamyL, but its extracellular translocation was unsuccessful. Consequently, the expression of NpAS via the twin-arginine translocation (TAT) pathway was investigated using the signal peptide SglmU. The study revealed that NpAS could be effectively translocated extracellularly through the TAT pathway, with the signal peptide SglmU facilitating the process. Remarkably, 62.81% of the total expressed activity was detected in the medium. This study marks the first successful secretory expression of NpAS in Bacillus species host cells, establishing a foundation for its future efficient production. One-Sentence Summary Amylosucrase was secreted in Bacillus licheniformis via the twin-arginine translocation pathway.


Introduction
Amylosucrase (AS,EC 2.4.1.4),first discovered in Neisseria perflava (Hehre & Hamilton, 1948 ), is a glucosyltransferase enzyme from the Glycoside hydrolase (GH) family 13 (Moulis et al., 2016 ).Amylosucrase is unique for its ability to use sucrose, its natural substrate, to create amylose-type polymers.These polymers are notable for their helical structure and controlled regularity, making them functional supramolecular materials (Putseys et al., 2010 ;Seung, 2020 ).Unlike other amylopolysaccharide synthases, AS polymerizes without the need for α-D-glucosyl nucleoside diphosphate glucosyl donors, such as adenosine diphosphate (ADP)-or uridine diphosphate (UDP)-glucose (Seo et al., 2020 ).This characteristic has established AS as a valuable tool in industry for synthesizing or modifying a diverse array of polysaccharides and their derivatives.When reacting with sucrose, AS can be used to design and create amylodextrins with specific morphologies, structures, physicochemical properties, and degrees of polymerization (Potocki-Veronese et al., 2005 ).Additionally, AS can catalyze the isomerization of sucrose to produce turanose, a functional sweetener, especially at high sucrose concentrations with added fructose as a modulator (Park et al., 2016 ).In combination with maltooligosyltrehalose synthase and This study maltooligosyltrehalose trehalohydrolase, AS facilitates the onepot bioconversion of sucrose to trehalose (Jung et al., 2013 ).Furthermore, in the presence of sucrose and alternative non-natural glycosyl acceptors, AS can transfer glucose from sucrose to these acceptors, leading to the synthesis of novel compounds.Examples include carbohydrate-based dendritic nanoparticles (Putaux et al., 2006 ;Lee et al., 2022 ;) and baicalein-6-glucoside, which boasts enhanced stability, water solubility, and improved physiological properties (Kim et al., 2014 ), unusual quercetin diglucosides and isoquercitrin glucosides (Rha et al., 2020a(Rha et al., , 2020b ) ) are among the compounds whose bioavailability can be enhanced through glycosylation by AS (Moon et al., 2021 ).This enzyme can also catalyze the formation of both α and β anomers of glycosides in triterpenoids from the medicinal fungus Ganoderma lucidum (Wu et al., 2022 ), and improve the solubility of puerarin (Ding et al., 2022 ).
The exceptional and distinctive application properties of AS have spurred significant interest in the gene mining, cloning, and expression of novel AS enzymes.Since the initial biochemical identification of extracellular AS in Neisseria polysaccharea in 1983 (Riou et al., 1983 ), it has been cloned, heterologously expressed, and characterized in Escherichia coli (Büttcher et al., 1997 ).Numerous other novel ASs have since been gene cloned and/or functionally identified in a variety of organisms, including Deinococcus radiodurans (Pizzut-Serin et al., 2005 ), Deinococcus geothermalis (Emond et al., 2008 ), Alteromonas macleodii (Ha et al., 2009 ), Arthrobacter chlorophenolicus (Seo et al., 2012 ), Synechococcus sp.(Perez-Cenci & Salerno, 2014 ), Cellulomonas carboniz (Wang et al., 2017 ), and Bifidobacterium thermophilum (Choi et al., 2019 ).However, these identified AS enzymes are often fused with a glutathione-S-transferase or histidine tag and are expressed and prepared in inclusion bodies in E. coli .Despite extensive improvements in the expression efficiency of foreign proteins in E. coli (Schneider et al., 2011 ), the practicality of low-cost and large-scale enzyme preparation is hindered by low expression levels, the complexity of downstream processing, and potential endotoxin contamination.Heterologous expression in more efficient host systems, such as Bacillus subtilis , is necessary but has yet to be achieved for reasons that remain unclear (Kim et al., 2019 ).This motivates us to devise a novel routine for the highefficiency preparation of AS by employing alternative expression systems.
The current studies are designed to investigate the potential for secretory expression of NpAS in a food-grade B. licheniformis expression system.By examining peptide translocation pathways, we explored both the general transport (Sec) pathway and the twin-arginine translocation (TAT) pathway to determine if NpAS secretion is feasible through both S amyL (a Sec signal peptide) and S glmU (a TAT signal peptide).We conducted a comparative analysis of the main forms and functions of secretory recombinant NpAS and re-verified them accordingly.Establishing this new expression routine for NpAS could provide the groundwork for its future large-scale and efficient production.

Strains, Plasmids, and Propagation Conditions
The strains and plasmids used in this study are listed in Table 1 .Escherichia coli JM109 was used as the host for gene cloning and plasmid construction.Bacillus licheniformis CBBD302 (Niu et al., 2009 ) was employed as the parent strain for new host cell development.Temperature-sensitive replicative shuttle plasmid pUB-EX (Shen et al., 2022 ) was applied for gene deletion in B. licheniformis .pHY-WZX (Niu & Wang, 2007 ) harboring signal peptide S amyL was used for mediating the secretion and expression of NpAS or as a parent plasmid for new plasmid construction.All strains were cultivated at 37°C in Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, and 1% NaCl).Luria-Bertani plates were prepared by adding 1.5% agar powder to the LB medium.When necessary, 20 μg/ml kanamycin was supplemented.

Genetic Manipulation
The conventional laboratory DNA manipulations, including chromosomal DNA isolation, polymerase chain reaction (PCR), plasmid mini-preparation, DNA restriction endonuclease digestion, ligation, and transformation of E. coli were performed according to the established protocols (Sambrook & Russel, 2001 ).The primers used in this study are listed in Table 2 .The full nucleotide sequence of an ASase-encoding gene npas from N. polysaccharea (locus_tag: AJ011781.1)was codon-optimized and chemically synthesized by Sangon Biotech Co., Ltd., China, for expression in B. licheniformis .
The upstream and downstream fragments flanked sacB were amplified by PCR with primers LSC-up1/LSC-up2 and LSC-dn1/

For plasmid construction
LSC-dn2, respectively, using the chromosomal DNA of B. licheniformis CBBD302 as template.The amplified upstream and downstream fragments were then mixed and used as the template for the preparation of the deletion cassette, sacB ', with primer LSC-up1/LSC-dn2.It was then purified and digested by Bam HI, and cloned into Bam HI and Sma I sites of pUB-EX (Shen et al., 2022 ), resulting in pUB-sacB'.This construct was then transformed into B. licheniformis CBBD302.The sacB deletion mutant was obtained by the homologous recombination-mediated double-exchange method (Zhou et al., 2020 ) and confirmed by diagnosis PCR.The levan-deficient strain was further confirmed by cultivating it on an LB plate containing 5% sucrose at 37°C for 24 hr to observe the levan formation or inoculated at a 10% dose in 50 ml LB liquid medium (containing 5% sucrose) at 37°C and 200 rpm for 48 hr.
After fermentation, the cultures were centrifuged at 8,000 × g for 15 min.The supernatants were dealt with two volumes of chilled ethanol to precipitate polymers overnight at −20°C followed by centrifugation at 8,000 × g for 15 min, and dried by desiccation in vacuo.Polymer pellets were then re-dissolved in 50 ml of warm ddH 2 O and determined by quantifying the carbohydrate content as fructose equivalents using the phenol-sulfuric acid method (Liu et al., 2010 ).Briefly, a standard curve between the concentration of fructose and the absorbance at OD 490 was first established after incubation with sulfuric acid and phenol.Then, the content of levan could be measured according to the curve.

Strain Development
The DNA fragment encoding the signal peptide S glmU (Niu et al., 2019 ) was amplified by using B. licheniformis CBBD302 genomic DNA as template with primers Glu-F and Glu-R.The S amyL fragment in pHY-WZX was removed by reverse PCR with primers 318-F and 318-R, followed by digestion with Bam HI and ligation with Bam HI-digested S glmU fragment to create the new plasmid pTAT1.0.The whole-length gene for NpAS (named npas ) amplified pUC57-npas by PCR with primers Amy113-F and Amy113-R.The recovered npas fragment was digested by Bam HI and cloned into the Bam HI and Sma I sites of either pHY-WZX or the newly con-structed pTAT1.0.The recombinant plasmids were then transformed into B. licheniformis by electroporation and the resulting transformants were selected for subsequent studies.

Semi-quantitative Analysis of Enzyme Expression Level on Plates
Recombinant strains were cultivated to a cell density (OD 600 ) of 3.0-3.5 in 50 ml LB medium at 37°C for 12-16 hr.One μl of the culture broth was dotted on an LB plate (complemented with 5% sucrose).The plates were incubated at 37°C for 12 hr, 24 hr, or 36 hr, and the yields of AS produced by strains were evaluated using iodine vapor staining.The stained area of colonies or halo-surrounded colonies was used as a parameter for semiquantitative analysis of the enzyme production capacity (Büttcher et al., 1997 ).

Shaking Flask Fermentation of NpAS
Shake flask fermentation was performed with some modifications as described in the previous study (Niu et al., 2009 ).Recombinant colonies were inoculated into a 50 ml of LB liquid medium with 20 μg/ml kanamycin and cultured at 200 rpm and 37°C for 12-16 hr.Preparation of NpAS from recombinant strains was carried out with a 10% dose using shake flask fermentation in a 250 ml Erlenmeyer flask with a working volume of 50 ml of LB supplemented with lactose at a final concentration of 1% at 37°C and 220 rpm for 84 hr.The cell mass of recombinant NpAS expressed in the levandeficient mutant was determined by regularly measuring the optical density of the cultures at 600 nm.The dry cell weight (DCW) was measured as follows.A 50 ml of fermentation culture was collected and centrifuged at 8,000 × g at 4°C for 20 min and washed 3 times with 0.9% NaCl solution.The cell pellets were dried at 55°C to constant weight and recorded.

Sample Preparation
The culture broth was centrifuged at 8,000 × g at 4°C for 20 min, the supernatant was used for further study.The cell pellet was washed twice with ddH 2 O and resuspended in 50 mM Tris-HCl (pH 7.0) and lysed with ultrasonication (SCIENTZ-IID, Scientz, Ningbo, Zhejiang, China; output power 400 W, 20 times for 3 s, constant duty) in an ice bath.The disrupted cells were centrifuged (8,000 × g at 4°C for 20 min) and the resulting supernatant was filtered through a 0.45 μm filter used for further study.

Isolation and Purification
The broth with recombinant NpAS was precipitated using 40-70% saturated ammonium sulfate solution.The precipitants were then collected by centrifugation at 8,000 × g and 4°C for 20 min.The pellets were collected and resuspended in 50 mM Tris-HCl buffer (pH 7.0) and dialyzed against the same buffer.Subsequently, the NpAS was purified by an ÄKTA Pure system with a Sephadex G-100 (10 × 500 mm) column (Cytiva, Sweden) by eluting with 50 mM phosphate buffer (pH 7.0) at a flow rate of 0.5 ml/min.The molecular weight of NpAS was estimated based on SDS-PAGE (Zhu & Wang, 1994 ), selecting 10% (w/v) running gel and 5% (w/v) stacking gel.Protein concentration was measured using the Micro Bradford method (Bradford, 1976 ) using bovine serum albumin FV (Roche Diagnostics GmbH, Germany) as the standard.

Enzyme Activity Assay
The NpAS activity assay (total 0.4 ml) was conducted in 50 mM Tris-HCl buffer (pH 7.0) with 0.1 mol/l sucrose as a substrate at 35°C for 30 min.An inactivated enzyme sample, boiled for 10 min, served as control.After the reaction, the amount of released fructose was measured by the dinitrosalicylic acid (DNS) method (Zhu & Wang, 1994 ) with fructose as a standard.Briefly, the 0.4 ml reaction mixture was terminated by adding 0.6 ml of DNS solution, followed by heating at 100°C for 7 min and cooling on ice.Absorbance was measured at 550 nm.One unit of NpAS activity is defined as the amount of enzyme releasing 1.0 μmol of fructose per minute under the assay conditions (Kim et al., 2019 ).

Carbohydrate Profiles in NpAS-catalytic Reaction Mixture
The enzyme reaction was performed with 75 mM sucrose and 0.1 u/ml of pure NpAS at 35°C and pH 7.0 reaction for 6 hr, 24 hr, 48 hr, and 120 hr.Post-reaction, the collected samples were separated by centrifugation (8,000 × g , 4°C).The water-insoluble fractions were subsequently stained with an iodine solution to identify the generated amylose polymers according to the method described (van der Veen et al., 2006 ).Soluble products were quantified using high performance liquid chromatography (HPLC) on an Alltech Prevail Carbohydrate ES 5 u column (4.6 mm ID × 250 mm, 5 μm) with 65% (v/v) acetonitrile as mobile phase at a flow rate of 1 ml/min at 35°C.The reference standard for glucose, fructose, sucrose, and maltooligosaccharides was purchased from Jiangsu Ruiyang Biotech Co., Ltd., China.The reference standard for turanose was purchased from Sangon Biotech (Shanghai) Co., Ltd.

Construction of Levan-Deficient B. Licheniformis
Almost all isolates of B. licheniformis have been verified to produce extracellular levan polysaccharide, a naturally occurring homopolymer fructan from sucrose catalyzed by levansucrase (EC 2.4.1.10)(Liu et al., 2010 ;Nakapong et al., 2013 ).This enzyme is an extracellular enzyme encoded by gene sacB in B. licheniformis (He et al., 2018 ).It catalyzes sucrose hydrolysis and directly transferes the fructosyl moiety to another sucrose acceptor, resulting in levan formation and the release of free glucose (Klaewkla et al., 2020 ).The existence of levansucrase may complicate the purification and subsequent applications of NpAS due to they share the same substrate (sucrose) but yield different products.Consequently, it is crucial to prevent levansucrase production in the expression host by deleting the sacB gene.
The sacB gene, encoding levansucrase for the synthesis of extracellular levan (Fig. 1 a), was deleted (Fig. 1 b) from the parental B. licheniformis CBBD302 strain.The resultant sacB deletion mutant was confirmed by diagnostic PCR (Fig. 1 c) and redesignated as B. licheniformis CBBD302B.After a period of incubation on LB plates with sucrose, the colonies exhibited a rough, dry, and non-mucoid appearance, which was different from that of the parent strain (a smooth, humid, and mucoid colony) (Fig. 1 d).This changed morphology was likely due to the loss of levan synthesis in B. licheniformis CBBD302B.Subsequently, B. licheniformis CBBD302B was cultivated by shake flask fermentation.The concentration of levan in the broth was quantified and the findings are summarized in Fig. 1 e.Clearly, B. licheniformis CBBD302B revealed no detectable levan, while the parent strain could produce 10.2 g/l of levan under identical conditions.The results suggest that the production of levan in B. licheniformis CBBD302 was governed by a single sacB gene product, rendering B. licheniformis CBBD302B deficient in levan.

Sec Translation Pathway in B. Licheniformis Was Unable to Mediate the Secretion of NpAS
Many mechanisms and biochemical types are involved in protein translocation with different selections or affinities (Anne et al., 2017 ).Of which, the Sec pathway is ubiquitous and essential to directly export a majority number of proteins under their unfolded forms through a well-organized cargo across the cell membrane and cell wall in a process of either co-translational or post-translational (Tsirigotaki et al., 2017 ).The Sec pathway can overexpress and efficiently prepare many kinds of industrially important enzymes (Niu et al., 2009(Niu et al., , 2022 ; ;Shen et al., 2022 ).Therefore, we tried to examine the possibility of the Sec pathway in B. licheniformis for secretory expression of NpAS.
The npas gene encoding the matured peptide of NpAS (Büttcher et al., 1997 ) was codon-optimized and chemically synthesized.It was then cloned in-frame after the S amyL in the expression plasmid pHY-WZX (Niu & Wang, 2007 ).The resulting recombinant plasmid pHY-AS1 was transformed into B. licheniformis CBBD302B, yielding a recombinant strain BL-AS1 ( B. licheniformis CBBD302B harboring pHY-AS1).Clear and amylose-specific materials formed in these cells after cultivating on sucrose-containing plates and an altered iodine staining pattern changed from red (12 hr) to blue (36 hr) (Fig. 2 a), which indicated that the recombinant strain BL-AS1 functionally expressed NpAS.Consequently, the NpAS yields were further examined in shake-flask fermentation experiments.Contrary to expectations, no detectable NpAS activity in the broth was found, although there existed an obvious NpAS activity inside the cells (Fig. 2 b).These results strongly indicated that NpAS molecules were unable to be translocated through the Sec pathway in B. licheniformis .The possible reason is that the NpAS peptide may fold to form well-folded NpAS too fast to be recognized and handled by Sec translocase in B. licheniformis (Denks et al., 2014 ).The results illustrated that NpAS was actively expressed in B. licheniformis , however, it could not be translocated extracellularly through the Sec pathway.
Alternatively, the TAT pathway, another well-characterized secretion pathway, provides a fascinating alternative for protein secretion when proper folding occurred within the cytoplasm prior to secretion (Berks, 2015 ).Proteins such as GFP (Albiniak et al., 2013 ), protein glutaminase (Niu et al., 2019 ), disulfide bondcontaining protein (Arauzo-Aguilera et al., 2023 ), and laccases (Valimets et al., 2023 ) have been successfully secreted using the TAT pathway.Next, we will consider the possibility of NpAS being targeted for export via the TAT pathway.

The TAT Translation Pathway in B. Licheniformis Can Mediate the Secretion of NpAS
A typical TAT translocation pathway was found by mining the genome of B. licheniformis .A novel expression plasmid (designated as pTAT1.0)was constructed by replacing the nucleotide sequence coding for AmyL signal peptide in the plasmid pHY-WZX (Niu & Wang, 2007 ) with the nucleotide sequences encoding for GlmU twin-arginine signal peptide (S glmU ) (Niu et al., 2019 ).Subsequently, the npas gene was cloned in-frame after S glmU , yielding a recombination plasmid pTAT1.0-AS1.A recombinant strain BL-1.0AS1 was developed by transforming pTAT1.0-AS1into B. licheniformis CBBD302B and cultivated on LB plates (containing 5% sucrose) for 36 hr.Post-incubation, a clear and amylose-specific halo was formed in the periphery of the colonies (Fig. 3 a), which indicated that NpAS was functionally expressed and may be extracellularly translocated.Consequently, the NpAS yields were conducted to evaluate using shake-flask fermentation experiments, and the intracellular and extracellular activities of NpAS were assayed and summarized in Fig. 3 b.After cultivation for 84 hr, the extracellular activity of NpAS reached 428.21 u/g (DCW), which was about Studies have shown that altering the permeability of the cell wall can significantly enhance the secretion efficiency of recombinant proteins (Zhao et al., 2017 ;Yang et al., 2022 ).Consequently, we investigated the impact of various cell wall/membraneweakening chemicals on the extracellular secretion of NpAS.Our findings indicated a considerable increase in extracellular NpAS when glycine was added, with the activity reaching approximately 6.13 times that of the control when 2.5% glycine was included (Fig. 4 a).The enhanced secretion of NpAS may be attributed to increased cell permeability as glycine replaces L-alanine and Dalanine in the peptidoglycan of the cell wall during ongoing cell growth, resulting in a significantly weakened cell wall (Hammes et al., 1973 ).Other chemicals, such as sodium penicillin G, SDS, and Triton X-100, had little or negative effects on enzyme production (Fig. 4 b-D).Specifically, sodium penicillin G was found to negatively impact NpAS yield (Fig. 4 b).The addition of SDS and Triton X-100 significantly affected the dry cell weight, with extracellular activity increasing to about 3.82-fold (Fig. 4 c) and 3.13-fold (Fig. 4 d) more than the control when 0.5% Triton X-100 and 0.01% SDS were added, respectively.These results suggest that an optimal amount of glycine in the culture medium is more conducive to the secretion level of NpAS.
To further check if the recombinant NpAS retained the same unique functions as that of the wild-type NpAS, extracellular NpAS from strain BL-1.0AS was prepared and purified.The formation of turanose and α-1,4-glucans (amylose) from sucrose by purified NpAS were examined.The resulting products were determined by using HPLC and iodine staining methods.Turanose, a specific product from sucrose through the isomerization activity of NpAS, was identified (Fig. 5 a).α-1,4-Glucans, another specific product from sucrose through its transglucosylase activity were detectable at 6 hr and gradually increased over the course of the reaction (Fig. 5 b).The results clearly indicated that recombinant NpAS expressed in B. licheniformis via the TAT pathway preserves the unique characters of the wild NpAS in synthesizing amylose type polymers and turanose (Wang et al., 2012 ;De Montalk et al., 2000 ).
The research results conclusively showed that NpAS could be expressed and secreted extracellularly in B. licheniformis via the TAT pathway.It was also demonstrated that the fetal peptide of the expressed NpAS in B. licheniformis might fold concurrently.It would be a plausible explanation that only the TAT pathway but not the Sec pathway in B. licheniformis can effectively transport the expressed NpAS (Berks, 2015 ), although a relatively high percentage of NpAS activity remained intracellularly.Enhancing the yield and translocation efficiency of NpAS in B. licheniformis through TAT pathway engineering is feasible, as will be detailed in subsequent studies.

Conclusion
In this study, the new expression strategy was developed successfully for the secretory expression of NpAS in B. licheniformis .Neisseria polysaccharea amylosucrase, when expressed in B. licheniformis , is likely to fold rapidly and be translocated extracellularly via the TAT pathway rather than the Sec pathway.This innovative expression system for NpAS lays the groundwork for its large-scale and efficient preparation.

Fig. 1 .
Fig. 1.Verification and characterizations of sacB deletion in B. licheniformis .(a) Schematic illustration representing the results of sacB deletion in B. licheniformis CBBD302.(b) Design of the primers used in the verification of the double-crossover mutant when screening the integrons of sacB cassette.LSC-F and LSC-R were primer pairs used for PCR validation and pUB-sacB' was used for the deletion of sacB .(c) Verification of the sacB deleting strains by colony PCR.(M) DNA molecular weight marker; lane 1: PCR product of parent strain with an amplified fragment of 2.8 kb; lane 2: PCR product of mutant B. licheniformis CBBD302 with an amplified fragment of 1.3 kb.(d) Levan forming assay on LB plate.The levan-deficient strain and parent strain were cultured on an LB plate (containing 5% sucrose) at 37°C for 24 hr to observe the formation of levan.(e) Levan yield by B. licheniformis CBBD302 and B. licheniformis CBBD302B in 250 ml Erlenmeyer flask with a working volume of 50 ml.The incubation was carried out at 37°C and 200 rpm for 48 hr.The samples were collected and the DCW and levan contents were determined.Values are means of three replications ± standard deviation.

Fig. 2 .
Fig. 2. The expression of NpAS by Sec translation pathway in B. licheniformis .(a) After cultured for 12 hr, 24 hr, and 36 hr, amylose forming assay on an LB plate (containing 5% sucrose) by BL-AS1 and B. licheniformis CBBD302B stained with iodine vapor.(b) NpAS yield by BL-AS1 in 250 ml Erlenmeyer flask with a working volume of 50 ml.The incubation was carried out at 37°C and 220 rpm for 84 hr.The samples were collected and their NpAS activity and DCW were determined.The error bar indicates the standard deviation from three parallel experiments.

Fig. 3 .
Fig. 3.The expression of NpAS by TAT translation pathway in B. licheniformis .(a) After cultured for 12 hr, 24 hr, and 36 hr, amylose forming assay on LB plate (containing 5% sucrose) by BL-1.0AS1 stained with iodine vapor.(b) NpAS yield by BL-1.0AS1 in 250 ml Erlenmeyer flask with a working volume of 50 ml.The incubation was carried out at 37°C and 220 rpm for 84 hr.The samples were collected, and their NpAS activity and DCW were determined.The error bar indicates the standard deviation from three parallel experiments.

Fig. 5 .
Fig. 5. Characteristic of the products synthesized by NpAS.(a) HPLC profile of the reaction medium supernatant after 120 hr reaction in the presence of 75 mM sucrose (sample S).Standard is turanose.(b) The water-insoluble solution was stained with a dilute iodine solution.A total of 400 μl of each reaction mixture contained 75 mM sucrose and 0.1 u/ml of the enzymes in 50 mM Tris-HCl buffer (pH 7.0) and incubated at 35°C for 6 hr, 24 hr, 48 hr, and 120 hr.

Table 2 .
Nucleotide sequence of primers