NL2030021B1

NL2030021B1 - L-ARABINOSE ISOMERASE (L-Al) DERIVED FROM LACTOCOCCUS LACTIS (L. LACTIS), AND USE THEREOF IN PREPARATION OF RARE SUGAR

Info

Publication number: NL2030021B1
Application number: NL2030021A
Authority: NL
Inventors: Zhang Susu; Liu Xinli; Wang Ting; Xu Zhenshang
Original assignee: Univ Qilu Technology
Priority date: 2021-12-03
Filing date: 2021-12-03
Publication date: 2023-06-21

Abstract

The present disclosure provides an L-arabinose isomerase (L-AI) gene derived from Laclococcus lactis (L. lactis), which corresponds to a nucleotide sequence shown in SEQ ID NO. 1 and an amino acid sequence shown in SEQ ID NO. 2. The L-AI gene and a ß- galactosidase (ß-GAL) gene derived from Streptococcus lhermophilus (S. lhermophilus) are inserted into a plasmid pETDuet-l and a resulting recombinant plasmid is transformed into Escherichia coli (E. coli) BL21 (DE3) to enable the simultaneous high- actiVity expression of the two enzymes. The recombinant E. coli strain can directly convert lactose into D-tagatose, which simpliﬁes a production process and reduces a production cost.

Description

L-ARABINOSE ISOMERASE (L-AI) DERIVED FROM LACTOCOCCUS

LACTIS (L. LACTIS), AND USE THEREOF IN PREPARATION OF RARE

SUGAR

TECHNICAL FIELD

[01] The present disclosure belongs to the field of biotechnology, and relates to novel

L-arabinose isomerase (L-Al) and use thereof, and specifically to gene cloning for L-Al of Lactococcus lactis (L. lactis), construction of recombinant Escherichia coli (E. coli) co-expressing L-Al and B-galactosidase (B-GAL), and use of the recombinant strain in the conversion of lactose to produce D-tagatose.

BACKGROUND ART

[02] As the most promising sucrose substitute, D-tagatose can be widely used in various products, including candies and soft drinks. D-tagatose is a recognized safe substance, which can present 92% of the sweetness of sucrose, but only includes 38% of the calories of sucrose. Therefore, D-tagatose is expected to serve as an additive in health foods and functional beverages to improve taste and reduce calories. D-tagatose can also serve as a substitute for sucrose or glucose to treat type 2 diabetes. As D-tagatose has low glycemic index (GI), a patient ingesting D-tagatose undergoes a relatively slow increase in the blood glucose level. In addition, many studies have shown that D-tagatose can be used as a prebiotic to help reduce the cholesterol level, increase the number of probiotics, and prevent colon cancer. Moreover, D-tagatose can also be used to prepare detergents and cosmetics with other optically active compounds and additives. In summary, D-tagatose has huge potential in creating new markets due to its diversified and unique characteristics.

[03] D-tagatose is a natural rare ketohexose present in gums and dairy products at a very low content. At present, the production of D-tagatose can be achieved through a chemical or enzymatic method. However, a chemical production process is very complicated and is accompanied by the generation of by-products, which limits the application of the chemical method. D-tagatose obtained from a biological process is more suitable for consumers, which makes the research on enzymatic production of D- tagatose get attention. With D-galactose as a substrate, D-tagatose can be obtained in vitro through the catalysis of L-Al So far, it has been reported that L-Al can be produced by many microorganisms. The thermophilic L-AI shows the highest activity at a reaction temperature of higher than 70°C. A high temperature promotes the shift of reaction equilibrium toward the formation of D-tagatose, but also exacerbates the undesirable browning reaction and the formation of by-products. In addition, the safety of genes derived from thermophilic bacteria has been questioned.

[04] Enzymatic production of D-tagatose generally uses the expensive D-galactose as a substrate. However, D-galactose can be obtained by hydrolyzing lactose or lactose- containing substances such as whey or whey permeate, which are rich and cheap by- products in the production of dairy products. From a business perspective, the one-step biosynthesis of D-tagatose with lactose as a raw material is very desirable, which requires a recombinant £. coli strain to have highly-active L-AI and B-GAL at the same time.

SUMMARY

[05] In the present disclosure, a novel L-Al gene is obtained from Z. lactis, and the gene 1s allowed to co-express with a B-GAL gene from Streptococcus thermophilus (S. thermophilus) in E. coli. The recombinant strain can directly convert lactose into D- tagatose.

[06] The present disclosure adopts the following technical solutions:

[07] A first objective of the present disclosure is to provide novel L-Al with an amino acid sequence shown in SEQ ID No. 2.

[08] A second objective of the present disclosure is to provide a gene encoding the aforementioned novel L-Al, with a nucleotide sequence shown in SEQ ID No. 1.

[09] Further, the nucleotide sequence shown in SEQ ID No. | may be present in Z. lactis.

[10] A third objective of the present disclosure is to provide a recombinant £. coli strain. The recombinant E. coli strain is constructed as follows: A specific primer pair is used to amplify the L-AI gene described above. The primer pair includes the following two sequences:

[11] upstream primer Fl (5'-GCGCATATGTTAGAAAATACTCAAAAAG-3) (SEQ ID No. 3) and

[12] downstream primer R1 (5'-GCGCTCGAGTCATCCAAGATTAATATAAG-3") (SEQ ID No. 4).

[13] Specifically, the specific primer pair shown in SEQ ID No. 3 and SEQ ID No. 4 is used to amplify the L-AI gene shown in SEQ ID No. 1; and a PCR amplification product is subjected to double enzyme digestion with Nde 1 and Xho 1, an expression vector pETDuet-1 is linearized through the same enzyme digestion, and digestion products of the amplification product are ligated with the linearized expression vector to construct a recombinant expression vector pETDuet-ara.

[14] Further, a specific primer pair is used to amplify a B-GAL gene of S. thermophilus. The primer pair includes the following two sequences:

[15] F2 (5'-CGCGGATCCGATGAACATGACTGAAAAAATTC-3') (SEQ ID No. 5)and

[16] R2 (5'-GCGCTGCAGCTAATTTAGTGGTTCAATCATGAAG-3") (SEQ ID

No. 6).

[17] Specifically, the specific primer pair shown in SEQ ID No. 5 and SEQ ID No. 6 is used to amplify the B-GAL gene of S. thermophilus; and a PCR amplification product is subjected to double enzyme digestion with Bam HI and Pst L the recombinant expression vector pETDuet-ara is linearized through the same enzyme digestion, and products of the amplification product are ligated with the linearized recombinant expression vector to construct a recombinant expression vector pETDuet-ara-gal. The recombinant expression vector pETDuet-ara-gal is transformed into a host strain £. coli

BL21 (DE3), a transformant is cultivated, and protein expression is induced by isopropyl-B-D-thiogalactoside (IPTG) or lactose.

[18] A fourth objective of the present disclosure is to provide use of the aforementioned recombinant £. coli strain in the conversion of lactose into D-tagatose.

[19] The present disclosure has the following beneficial effects:

[20] 1. The L-AI gene provided by the present disclosure is produced by ZL. lactis, and the L. lactis is a recognized safe strain, which has been used in the food industry for a long time and shows high biological safety.

[21] 2. The recombinant £. coli strain provided by the present disclosure can directly use cheap lactose as a substrate to produce D-tagatose through one-step conversion, which greatly reduces a production cost.

[22] 3. 50°C is the optimal temperature for the conversion of the recombinant strain of the present disclosure to produce D-tagatose, which is a suitable temperature. A too- low temperature will reduce the conversion speed and conversion efficiency, and a too-

high temperature will cause the formation of by-products.

BRIEF DESCRIPTION OF THE DRAWINGS

[23] FIG. 1 shows the expression of L-Al and B-GAL in recombinant £. coli (the bands indicated by arrows represent L-Al and B-GAL, respectively; W represents whole cell components; and S represents soluble protein components).

[24] FIG. 2 shows the optimization of conditions for the conversion of galactose into

D-tagatose by recombinant E. coli (A: the activity change of recombinant E. coli under the induction conditions of 16°C (2), 20°C (0), 25°C (A), 30°C (V), and 37°C (<3); B: conversion rates of bacteria in different volumes of fermentation broth; and C: the optimal temperature for long-term reaction).

[25] FIG. 3 shows the conversion of galactose into D-tagatose by recombinant £. coli (A: lactose conversion rates (m) and D-tagatose yields (white bar) of recombinant £. coli at different lactose concentrations; and B: changes of D-galactose concentration (m) and

D-tagatose concentration (®) and yield (white bar) in a 300 g/L lactose solution with recombinant £. coli over time).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[26] To make the to-be-solved technical problems, technical solutions, and advantages of the present disclosure clearer, the present disclosure will be described in detail below with reference to the accompanying drawings and specific examples.

However, the present disclosure is definitely not limited to these examples. The following examples are merely preferred examples of the present disclosure, which are only used to explain the present disclosure, and should not be understood as a limitation to the patent scope of the present disclosure. It should be noted that any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present disclosure should be included within the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be subject to the appended claims.

[27] Unless otherwise specified, the components used in the present disclosure are all commercially available products.

[28] I. lactis used is a common commercial product.

[29] S. thermophilus used is a common commercial product.

[30] Example 1: Cloning of an L-Al gene from ZL. /actis and a B-GAL gene from SS. thermophilus

[31] LL. lactis and S. thermophilus strains were each activated overnight and then centrifuged to collect bacteria. Lysozyme and protease K were added the bacteria at an 5 appropriate amount to lyse cells and remove proteins, then 20% SDS was added, and a resulting mixture was incubated in a water bath at 65°C for 2 h. 3 mL of pre-cooled chloroform was added, and a resulting mixture was thoroughly mixed and centrifuged to obtain a supernatant. An equal volume of a pre-cooled mixed solution of phenol, chloroform, and isoamyl alcohol (25:24:1) was added to the supernatant, and a resulting mixture was thoroughly mixed by inverting up and down and then centrifuged; an equal volume of a mixture of chloroform and isoamyl alcohol (24:1) was added to a resulting upper aqueous phase, and a resulting mixture was centrifuged once again; and 0.6-fold volume of pre-cooled isopropyl alcohol (IPA) was added to a resulting aqueous phase, a resulting mixture was incubated in a water bath at room temperature to conduct alcohol precipitation for 1 h, and a resulting precipitate was collected by centrifugation. Finally, the precipitate was washed twice with 70% ethanol and then blow-dried, and DNA was dissolved in TE buffer.

[32] The L-AI gene from JL. lactis was amplified using the following primers: upstream primer F1 (S'-GCGCATATGTTAGAAAATACTCAAAAAG-3') (SEQ

ID No. 3) and downstream primer R1 (5'-

GCGCTCGAGTCATCCAAGATTAATATAAG-3") (SEQ ID No. 4). PCR reaction system (50 pL): ultrapure water (UPW): 20 ul; 2 x Taq Master Mix: 25 uL; upstream and downstream primers (10 uM): each 2 uL; and DNA template: 1 uL. PCR reaction conditions: 95°C for 5 min; 95°C for 30 s, 50°C for 30 s, and 72°C for 90 s, 30 cycles; and 72°C for 10 min. A PCR product was subjected to electrophoresis and gel extraction to obtain a purified L-AI gene PCR product.

[33] The B-GAL gene from S. thermophilus was amplified using the following primers: upstream primer F2 (5'-CGCGGATCCGATGAACATGACTGAAAAAATTC- 3) (SEQ ID No. 5) and downstream primer R2 (5'-

GCGCTGCAGCTAATTTAGTGGTTCAATCATGAAG-3') (SEQ ID No. 6). PCR reaction system (50 uL): UPW: 20 ul; 2 x Taq Master Mix: 25 pL; upstream and downstream primers (10 uM): each 2 pL; and DNA template: 1 uL. PCR reaction conditions: 95°C for 5 min; 95°C for 30 s, 50°C for 30 s, and 72°C for 3 min and 15 5,

30 cycles; and 72°C for 10 min. A PCR product was subjected to electrophoresis and gel extraction to obtain a purified B-GAL gene PCR product.

[34] Example 2: L-Al gene and B-GAL gene linking vector and protein expression

[35] The L-Al gene PCR product was subjected to double enzyme digestion with Nde land XhoI for 3 h. Digestion system (100 uL): Nae 1: 5 pL; Xho I. 5 pL; 10 x K Buffer: uL; 0.1% BSA: 10 uL; DNA: 5 pg; and sterile water. Gel extraction was conducted to obtain purified digestion fragments. A pETDuet-1 plasmid was subjected to double enzyme digestion for 3 h. Digestion system (100 pL): Nae I: 5 pL; Xho I. 5 uL; 10 x K

Buffer: 10 pL; 0.1% BSA: 10 uL; DNA: 5 ug; and sterile water. Gel extraction was 10 conducted to obtain a purified digestion plasmid. The DNA fragments and the pETDuet- 1 vector obtained after the double enzyme digestion were mixed at a molar ratio of 10:1, then T4 DNA ligase was added, and a resulting mixture was subjected to ligation overnight at 16°C. 10 pL of a ligation product was added to 100 pL of £. coli BL21(DE3) competent cells, and a resulting mixture was incubated on ice for 30 min and then subjected to heat shock for 60 s in a 42°C water bath; and then 900 pL of a LB liquid medium was added, and a resulting mixture was incubated for 1 h (37°C and 150 rpm).

A resulting bacterial solution was centrifuged, and 800 uL of a resulting supernatant was removed; and the bacteria were resuspended in the remaining medium, and a resulting bacterial suspension was spread on an LB plate with 100 pg/mL ampicillin. A transformant was sequenced, and a sequencing result was consistent with the nucleotide sequence of the L-Al gene (as shown in SEQ ID NO. 1). The plasmid pETDuet-1 integrated with the L-AI gene was named pETDuet-ara.

[36] The B-GAL gene PCR product was subjected to double enzyme digestion with

Bam HI and Pst 1 for 3 h. Digestion system (100 pL): Bam HI: 5 uL; Pst 1: 5 uL; 10 x K

Buffer: 10 pL; 0.1% BSA: 10 uL; DNA: 5 ug; and sterile water. Gel extraction was conducted to obtain purified digestion fragments. The plasmid pETDuet-ara was subjected to double enzyme digestion for 3 h. Digestion system (100 uL): Bam HT: 5 ul,

PstT: 5 uL; 10 = K Buffer: 10 pL; 0.1% BSA: 10 uL; DNA: 5 ug; and sterile water. Gel extraction was conducted to obtain a purified digestion plasmid. The DNA fragments and the pETDuet-ara vector obtained after the double enzyme digestion were mixed at a molar ratio of 10:1, then T: DNA ligase was added, and a resulting mixture was subjected to ligation overnight at 16°C. 10 uL of a ligation product was added to 100 uL of £. coli BL21 (DE3) competent cells, and a resulting mixture was incubated on ice for

30 min and then subjected to heat shock for 60 s in a 42°C water bath; and then 900 pL of a LB liquid medium was added, and a resulting mixture was incubated for 1 h (37°C and 150 rpm). A resulting bacterial solution was centrifuged, and 800 uL of a resulting supernatant was removed; and the bacteria were resuspended in the remaining medium, and a resulting bacterial suspension was spread on an LB plate with 100 pg/mL ampicillin. A transformant was sequenced, and a sequencing result was consistent with the nucleotide sequence of the B-GAL gene. The plasmid pETDuet-ara integrated with the B-GAL gene was named pETDuet-ara-gal.

[37] A recombinant expression strain was inoculated into an LB liquid medium and cultivated overnight at 37°C. A resulting bacterial solution was inoculated into 100 mL of an LB liquid medium at an inoculation volume of 1%, and cultivated at 37°C; and after the bacteria grew to an OD of 0.6, IPTG or 2% lactose was added at a final concentration of 0.5 mM to induce protein expression. Polyacrylamide gel electrophoresis (PAGE) was used to detect the protein expression. As shown in FIG. 1, both L-AI and B-GAL underwent soluble expression in large amounts.

[38] Example 3: Determination of the optimal conversion conditions for recombinant £. coli

[39] In atemperature range of 30°C to 60°C, the optimal temperature for conversion of lactose into D-tagatose by recombinant £. coli was studied. In a pH range of 4.0 to 9.0, the optimal pH for conversion of lactose into D-tagatose by recombinant £. coli was studied. Results showed that the optimal temperature was 50°C and the optimal pH was 8.0.

[40] The effects of induction temperature, cell dosage, and long-term reaction temperature on the reaction were further studied. The £. coli was inoculated into a fermentation broth and subjected to induction cultivation at 16°C, 20°C, 25°C, 30°C, and 37°C, separately. As shown in FIG. 2A, the maximum activity was detected after 24 h of incubation at 20°C. Cells were collected at regular intervals to determine the conversion efficiency. Bacteria were collected from 0.5 ml, 1 ml, 3 ml, 5 ml, 7 ml, 9 ml, 11 ml, and 13 mL of fermentation broth, separately, and the effects of cell dosages on the production of D-tagatose were compared. Results showed that the optimal dosage was 9 mL of fermentation broth (FIG. 2 B). A reaction mixture was incubated at 30°C to 60°C for 24 h to determine the optimal long-term reaction temperature. As shown in

FIG. 2C, the maximum yield of D-tagatose was detected at 50°C.

[41] Example 4: Conversion of lactose into D-tagatose by the recombinant strain

[42] Lactose solutions with different concentrations were used as substrates to compare the conversion efficiencies of the recombinant strain to produce D-tagatose.

Bacterial cells (about 1.7 g of wet cells) in 90 mL of fermentation broth were collected, and added to 100 mM phosphate buffers (pH 8.0) with final lactose concentrations of 50 g/L, 100 g/L, 150 g/L, 200 g/L, 250 g/L, and 300 g/L, and bioconversion was conducted at 50°C. The bioconversion was conducted for 24 h, during which samples were collected every 4 h to quantitatively analyze the concentrations of lactose, D-galactose, and D-tagatose through HPLC.

[43] Results showed that, with the increase of the substrate lactose concentration, the

D-tagatose yield continued to increase, with a maximum value of 2.65 g/L/h. The conversion rate of D-galactose increased first and then remained unchanged, and the conversion rate was 42.4% at a lactose concentration of 300 g/L. (FIG. 3A). The production of D-tagatose at a lactose concentration of 300 g/L was tracked. At the initial 3 h, the substrate lactose was completely hydrolyzed, and the conversion rate reached 20.5%. The maximum conversion rate of 42.4% was achieved at 15 h (FIG. 3B).

Sequence Listing <110> Xu Zhenshang

Wang Ting

Liu Xinli

Zhang Susu <120> L-ARABINOSE ISOMERASE (L-Al) DERIVED FROM LACTOCOCCUS

LACTIS (L. LACTIS), AND USE THEREOF IN PREPARATION OF RARE SUGAR <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1425 <212> DNA <213> L. lactis <400> 1 atgttagaaa atactcaaaa agaattttgg tttgttacag ggtctcaatt tttatatggt 60 ccagaagctt tggcgactgt tgaaaaaaat gctcgaaaag tagttgacga aatgaataat 120 tctggtagcc tcccttatcc aattattttt aaaatagttg caacaactgc tgaaaatatt 180 actcaaatta tgaaggaagc aaattatcag gatgaagttg cgggcgtgat tacatggatg 240 cacacctttt ctccagccaa aaactggatt cgtgggacac aacttttaaa taaaccctta 300 ctacacttag ctactcaatt tttgaaccat attccttaca aaacgattga ttttgattat 360 atgaatgtaa atcaatcagc tcatggtgac cgagaatatg cttttatcaa tgcacgtttg 420 aaaaaaaata ataagattat atttggccat tgggaagatg atgaaattag acaacaaatc 480 gctaaatgga tgaatgttge ggtggcttac aatgaatcct ataaaataaa aatcgttaca 540 tttgctgata aaatgcgaga tgtcgctgtt acggacggag ataaagttga agcgcaaatt 600 aaatttggct ggacggttga ttattgggga gtaggtgatt tagtagaata tgttaatget 660 gtagcagaga gtgatattaa tcagctttat aaagatttac aaagtaaata tgattatatt 720 gaaggaaaca atagtccgga aaaattcgaa aaaaatgtta aatatcaatt acgtgaatat 780 ttaggaatta agaaatttat ggacgataaa ggctatagtg cctttactac taactttgaa 840 gacttgatag gcttagaaca attacctgga cttgcagcac aattactttt ggctgatggt 900 tatggattcg ctggagaagg agattggaaa acagcagcac ttgtccgtct gttcaaaatt 960 atggcgcata ataaagaaac agtctttatg gaagattata ctttggattt gcgtcaagga 1020 cacgaagcaa ttcttgggtc acacatgctt gaagttgatc cttcaatagc atcagacaag 1080 ccacgagttg aggttcatcc gttagatatt ggtggtaagt cagatccagc acgettagta 1140 ttcacaggta tgattggtga agctgttgat attactatgg cagattatgg aaatgaattt 1200 aagttaattt cttatgaggt tactggaaat aaaccagaag aagaaactcc gttcttacca 1260 gttgctaagc agctctggac gcctaaacaa ggattaaaag ttggggctga acaatggtta 1320

IO aaagcaggag ggggacatca tactgtttta tcttttgtgg ttgattcaga acaaattggt 1380 gatttgtgtg caatgtttgg actaacttat attaatcttg gatga 1425 <210> 2 <211> 474 <212> PRT <213> L. lactis <400> 2

Met Leu Glu Asn Thr Gln Lys Glu Phe Trp Phe Val Thr Gly Ser Gln 1 5 10 15

Phe Leu Tyr Gly Pro Glu Ala Leu Ala Thr Val Glu Lys Asn Ala Arg 20 25 30

Lys Val Val Asp Glu Met Asn Asn Ser Gly Ser Leu Pro Tyr Pro Ile 35 40 45

Ile Phe Lys Ile Val Ala Thr Thr Ala Glu Asn Ile Thr Gln Ile Met 50 55 60

Lys Glu Ala Asn Tyr Gln Asp Glu Val Ala Gly Val Ile Thr Trp Met 65 70 75 80

His Thr Phe Ser Pro Ala Lys Asn Trp Ile Arg Gly Thr Gln Leu Leu 85 90 95

Asn Lys Pro Leu Leu His Leu Ala Thr Gln Phe Leu Asn His Ile Pro 100 105 110

Tyr Lys Thr Ile Asp Phe Asp Tyr Met Asn Val Asn Gln Ser Ala His

115 120 125

Gly Asp Arg Glu Tyr Ala Phe Ile Asn Ala Arg Leu Lys Lys Asn Asn 130 135 140

Lys Ile Ile Phe Gly His Trp Glu Asp Asp Glu Ile Arg Gln Gln Ile 145 150 155 160

Ala Lys Trp Met Asn Val Ala Val Ala Tyr Asn Glu Ser Tyr Lys Ile 165 170 175

Lys Ile Val Thr Phe Ala Asp Lys Met Arg Asp Val Ala Val Thr Asp 180 185 190

Gly Asp Lys Val Glu Ala Gln Ile Lys Phe Gly Trp Thr Val Asp Tyr 195 200 205

Trp Gly Val Gly Asp Leu Val Glu Tyr Val Asn Ala Val Ala Glu Ser 210 215 220

Asp Ile Asn Gln Leu Tyr Lys Asp Leu Gln Ser Lys Tyr Asp Tyr lle 225 230 235 240

Glu Gly Asn Asn Ser Pro Glu Lys Phe Glu Lys Asn Val Lys Tyr Gln 245 250 255

Leu Arg Glu Tyr Leu Gly Ile Lys Lys Phe Met Asp Asp Lys Gly Tyr 260 265 270

Ser Ala Phe Thr Thr Asn Phe Glu Asp Leu Ile Gly Leu Glu Gln Leu 275 280 285

Pro Gly Leu Ala Ala Gln Leu Leu Leu Ala Asp Gly Tyr Gly Phe Ala 290 295 300

Gly Glu Gly Asp Trp Lys Thr Ala Ala Leu Val Arg Leu Phe Lys Ile 305 310 315 320

Met Ala His Asn Lys Glu Thr Val Phe Met Glu Asp Tyr Thr Leu Asp 325 330 335

Leu Arg Gln Gly His Glu Ala Ile Leu Gly Ser His Met Leu Glu Val 340 345 350

Asp Pro Ser Ile Ala Ser Asp Lys Pro Arg Val Glu Val His Pro Leu 355 360 365

Asp Ile Gly Gly Lys Ser Asp Pro Ala Arg Leu Val Phe Thr Gly Met 370 375 380

Ile Gly Glu Ala Val Asp Ile Thr Met Ala Asp Tyr Gly Asn Glu Phe 385 390 395 400

Lys Leu Ile Ser Tyr Glu Val Thr Gly Asn Lys Pro Glu Glu Glu Thr 405 410 415

Pro Phe Leu Pro Val Ala Lys Gln Leu Trp Thr Pro Lys Gln Gly Leu 420 425 430

Lys Val Gly Ala Glu Gln Trp Leu Lys Ala Gly Gly Gly His His Thr 435 440 445

Val Leu Ser Phe Val Val Asp Ser Glu Gln Ile Gly Asp Leu Cys Ala 450 455 460

Met Phe Gly Leu Thr Tyr Ile Asn Leu Gly 465 470 <210> 3 <211> 28 <212> DNA <213> L. lactis <400> 3 gcgcatatgt tagaaaatac tcaaaaag 28 <210> 4 <211> 29 <212> DNA <213> L. lactis <400> 4 gcgctcgagt catccaagat taatataag 29 <210> 5 <211> 32 <212> DNA <213> L. lactis

<400> 5 cgcggatccg atgaacatga ctgaaaaaat tc 32 <210> 6 <211> 34 <212> DNA <213> L. lactis <400> 6 gcgctgcagc taatttagtg gttcaatcat gaag 34

SEQLTXT

SEQUENCE LISTING

<110> Zhenshang, Xu

Ting, Wang

Xinli, Liu

Susu, Zhang <120> L-ARABINOSE ISOMERASE (L-AI) DERIVED FROM LACTOCOCCUS LACTIS (L.

LACTIS), AND USE THEREOF IN PREPARATION OF RARE SUGAR <130> HKJP202110327 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 1425 <212> DNA <213> Lactococcus lactis <400> 1 atgttagaaa atactcaaaa agaattttgg tttgttacag ggtctcaatt tttatatggt 60 ccagaagctt tggcgactgt tgaaaaaaat gctcgaaaag tagttgacga aatgaataat 120 tctggtagcc tcccttatcc aattattttt aaaatagttg caacaactgc tgaaaatatt 180 actcaaatta tgaaggaagc aaattatcag gatgaagttg cgggcgtgat tacatggatg 240 cacacctttt ctccagccaa aaactggatt cgtgggacac aacttttaaa taaaccctta 300 ctacacttag ctactcaatt tttgaaccat attccttaca aaacgattga ttttgattat 360 atgaatgtaa atcaatcagc tcatggtgac cgagaatatg cttttatcaa tgcacgtttg 420 aaaaaaaata ataagattat atttggccat tgggaagatg atgaaattag acaacaaatc 480 gctaaatgga tgaatgttgc ggtggcttac aatgaatcct ataaaataaa aatcgttaca 540 tttgctgata aaatgcgaga tgtcgctgtt acggacggag ataaagttga agcgcaaatt 600 aaatttggct ggacggttga ttattgggga gtaggtgatt tagtagaata tgttaatgct 660 gtagcagaga gtgatattaa tcagctttat aaagatttac aaagtaaata tgattatatt 720 gaaggaaaca atagtccgga aaaattcgaa aaaaatgtta aatatcaatt acgtgaatat 780 ttaggaatta agaaatttat ggacgataaa ggctatagtg cctttactac taactttgaa 840 gacttgatag gcttagaaca attacctgga cttgcagcac aattactttt ggctgatggt 900

Pagina 1

SEQLTXT tatggattcg ctggagaagg agattggaaa acagcagcac ttgtccgtct gttcaaaatt 960 atggcgcata ataaagaaac agtctttatg gaagattata ctttggattt gcgtcaagga 1020 cacgaagcaa ttcttgggtc acacatgctt gaagttgatc cttcaatagc atcagacaag 1080 ccacgagttg aggttcatcc gttagatatt ggtggtaagt cagatccagc acgcttagta 1140 ttcacaggta tgattggtga agctgttgat attactatgg cagattatgg aaatgaattt 1200 aagttaattt cttatgaggt tactggaaat aaaccagaag aagaaactcc gttcttacca 1260 gttgctaagc agctctggac gcctaaacaa ggattaaaag ttggggctga acaatggtta 1320 aaagcaggag ggggacatca tactgtttta tcttttgtgg ttgattcaga acaaattggt 1380 gatttgtgtg caatgtttgg actaacttat attaatcttg gatga 1425 <210> 2 <211> 474 <212> PRT <213> Lactococcus lactis <400> 2

Met Leu Glu Asn Thr Gln Lys Glu Phe Trp Phe Val Thr Gly Ser Gln 1 5 10 15

Phe Leu Tyr Gly Pro Glu Ala Leu Ala Thr Val Glu Lys Asn Ala Arg

Lys Val Val Asp Glu Met Asn Asn Ser Gly Ser Leu Pro Tyr Pro Ile

Ile Phe Lys Ile Val Ala Thr Thr Ala Glu Asn Ile Thr Gln Ile Met 60

Lys Glu Ala Asn Tyr Gln Asp Glu Val Ala Gly Val Ile Thr Trp Met 65 70 75 80

His Thr Phe Ser Pro Ala Lys Asn Trp Ile Arg Gly Thr Gln Leu Leu 85 90 95

Asn Lys Pro Leu Leu His Leu Ala Thr Gln Phe Leu Asn His Ile Pro 100 105 110

Pagina 2

SEQLTXT

Tyr Lys Thr Ile Asp Phe Asp Tyr Met Asn Val Asn Gln Ser Ala His 115 120 125

Gly Asp Arg Glu Tyr Ala Phe Ile Asn Ala Arg Leu Lys Lys Asn Asn 130 135 140

Lys Ile Ile Phe Gly His Trp Glu Asp Asp Glu Ile Arg Gln Gln Ile 145 150 155 160

Ala Lys Trp Met Asn Val Ala Val Ala Tyr Asn Glu Ser Tyr Lys Ile 165 170 175

Lys Ile Val Thr Phe Ala Asp Lys Met Arg Asp Val Ala Val Thr Asp 180 185 190

Gly Asp Lys Val Glu Ala Gln Ile Lys Phe Gly Trp Thr Val Asp Tyr 195 200 205

Trp Gly Val Gly Asp Leu Val Glu Tyr Val Asn Ala Val Ala Glu Ser 210 215 220

Asp Ile Asn Gln Leu Tyr Lys Asp Leu Gln Ser Lys Tyr Asp Tyr Ile 225 230 235 240

Glu Gly Asn Asn Ser Pro Glu Lys Phe Glu Lys Asn Val Lys Tyr Gln 245 250 255

Leu Arg Glu Tyr Leu Gly Ile Lys Lys Phe Met Asp Asp Lys Gly Tyr 260 265 270

Ser Ala Phe Thr Thr Asn Phe Glu Asp Leu Ile Gly Leu Glu Gln Leu 275 280 285

Pro Gly Leu Ala Ala Gln Leu Leu Leu Ala Asp Gly Tyr Gly Phe Ala 290 295 300

Gly Glu Gly Asp Trp Lys Thr Ala Ala Leu Val Arg Leu Phe Lys Ile 305 310 315 320

Pagina 3

SEQLTXT

Met Ala His Asn Lys Glu Thr Val Phe Met Glu Asp Tyr Thr Leu Asp 325 330 335

Leu Arg Gln Gly His Glu Ala Ile Leu Gly Ser His Met Leu Glu Val 340 345 350

Asp Pro Ser Ile Ala Ser Asp Lys Pro Arg Val Glu Val His Pro Leu 355 360 365

Asp Ile Gly Gly Lys Ser Asp Pro Ala Arg Leu Val Phe Thr Gly Met 370 375 380

Ile Gly Glu Ala Val Asp Ile Thr Met Ala Asp Tyr Gly Asn Glu Phe 385 390 395 400

Lys Leu Ile Ser Tyr Glu Val Thr Gly Asn Lys Pro Glu Glu Glu Thr 405 410 415

Pro Phe Leu Pro Val Ala Lys Gln Leu Trp Thr Pro Lys Gln Gly Leu 420 425 430

Lys Val Gly Ala Glu Gln Trp Leu Lys Ala Gly Gly Gly His His Thr 435 440 445

Val Leu Ser Phe Val Val Asp Ser Glu Gln Ile Gly Asp Leu Cys Ala 450 455 460

Met Phe Gly Leu Thr Tyr Ile Asn Leu Gly 465 470 <2105 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> upstream primer F1 for amplifying the L-AI gene <400> 3 gcgcatatgt tagaaaatac tcaaaaag 28

Pagina 4

SEQLTXT

<2105 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> downstream primer R1 for amplifying the L-AI gene <400> 4 gcgctcgagt catccaagat taatataag 29 <210>5 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer F2 for amplifying a |A-GAL gene of S. thermophilus <400> 5 cgcggatccg atgaacatga ctgaaaaaat tc 32 <210> 6 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer R2 for amplifying a |A-GAL gene of S. thermophilus <400> 6 gcgctgcagc taatttagtg gttcaatcat gaag 34

Pagina 5

Claims

Conclusions l. Novel gene encoding L-arabinose isomerase (L-Al), wherein the novel gene encoding L-AI has a nucleotide sequence shown in SEQ ID NO. 1.

2. Novel L-arabinose isomerase (L-AD), wherein the novel L-AI has an amino acid sequence shown in SEQ ID NO. 2.

The novel gene encoding L-arabinose isomerase (L-A1) according to claim 1, wherein the sequence is present in Lactococcus lactis (L. lactis).

A method of preparation for a recombinant strain of Escherichia coli (E. coli), the method comprising: using specific primers F1 (5'-GCGCATATGTTAGAAAATACTCAAAAAG-3') (SEQ ID No. 3) and RI (5- GCGCTCGAGTCATCCAAGATTAATATAAG-3") (SEQ ID No. 4) to amplify the novel gene encoding L-arabinose isomerase (L-AT) according to claim 1; subjecting PCR amplification product to double enzyme digestion with Neel and Xhol, linearizing an expression vector pETDuet-1 by the same enzyme digestion, and ligating digestion products of the amplification product with the linearized expression vector to construct a recombinant expression vector pETDuet-ara; using specific primers F2 (5-CGCGGATCCGATGAACATGACTGAAAAAATTC-3") ( SEQ ID No. 5) and R2 (5'-GCCGCTGCAGCTAATTTAGTGGTTCAATCATGAAG-3') (SEQ ID No. 6) to amplify a β-galactosidase (β-GAL) gene from Streptococcus thermophilus (S. thermophilus); subjecting a PCR amplification product to double digestion with Bam HI and PST, linearizing the recombinant expression vector pETDuet-ara by the same enzyme digestion, and ligating digestion products of the amplification product with the linearized recombinant expression vector to form a recombinant expression vector pETDuet- ara-gal, and transforming the recombinant expression vector pETDuet-ara-gal into a host strain E. coli BL21

(DE3), culturing a transformant, and inducing protein expression by isopropyl-β-D-thiogalactoside (IPTG) or lactose.

The preparation method of a recombinant strain of Escherichia coli (E. coli) according to claim 4, wherein the strain can directly convert lactose into D-tagatose.

The preparation method of a recombinant strain of Escherichia coli (E. coli) according to claim 5, wherein the optimal conditions include: temperature: 50°C, pH: 8.0 and substrate lactose concentration: 300 g/L.