Field of the Invention

The present invention is in the field of biotechnology and more particularly production of rare sugar disaccharides through biological route.

Background of the Invention

Despite their low natural abundance, rare disaccharides hold enormous potential for practical application.

Sucrose (table sugar) is the most widely used sweetener in food production due to its physiochemical and sensorial characteristics, however in recent decades an increased studies on alternative sweeteners is progressing rapidly, since of its high caloric value. Research on the production of oligosaccharides for foods with health benefits was started around late 1970s, and several oligosaccharides such as starch- related, sucrose-related, and lactose-related oligosaccharides have been developed.

Artificial sweeteners also called sugar substitutes were developed in recent years and are used in remarkable amounts instead of sucrose to sweeten foods and beverages but also in drugs and sanitary products. These sweeteners are not decomposed as carbohydrates and are not metabolized like sugars or only fermented slightly by the mouth microflora, which develops an artificial, metallic or licorice-like aftertaste. Therefore they often can be found blended in food to overcome this disadvantage.

Reducing disaccharides such as trehalulose (a-D-glucosylpyranosyl-l,l-D- fructofuranose) and isomaltulose (a-D-glucosylpyranosyl-l,6-Dfructofuranose) are structural isomers of sucrose (a-Dglucosylpyranosyl- l,2- -D-fructofuranoside) and are naturally present in honey as well as in sugar cane extract in very low quantities. These natural sugars display a sweetening power, bulk, organoleptic and physical properties similar to those of sucrose. Trehalulose is a non-crystalline saccharide which readily dissolves in water, does not substantially have cariogenicity, and has an about 70% sweetening power of sucrose, it is greatly expected to be used in food products, especially in foods enriched with sweeteners.

Isomaltulose is suitable sucrose replacement, since it's approximately half as sweet as sucrose, and has a similar sweetness quality and has non-carcinogenic properties. Moreover isomaltulose is also used as a raw material for production of another sweetener namely isomalt (palatint) by hydrogenation. Furthermore the reducing property of isomaltulose makes it an attractive industrial precursor for the manufacturing of biosurfactants and biocompatible polymers.

Both disaccharides: isomaltulose and trehalulose can be produced at an industrial scale through isomerization of sucrose using Sucrose Isomerases (Slase) which bio- convert sucrose into both isomaltulose and trehalulose with trace amounts of glucose and fructose.

US patent 5336617 relates to a process for preparing trehalulose and isomaltulose wherein at least the trehalulose-forming enzyme system of a trehalulose-forming microorganism is contacted with a sucrose solution to convert it into trehalulose and isomaltulose in the weight ratio of at least 4:1

Commercial isomaltulose is produced from food grade sucrose through enzymatic isomerisation with sucrose-6-glucosylmutase. In this process the biocatalyst used to convert the sucrose is obtained from non-viable, non-pathogenic P. rubrum (CBS 574.44) cells which were killed using formaldehyde before they are added to the sucrose (US 4640894 and US 5336617).

The enzymes responsible for bioconversion of sucrose in to its corresponding isomers (Sucrose to isomaltulose or trehalulose) have been reported from different microorganisms such as Protaminobacter rubrum, Serratia plymuthica, Erwinia rhaponiciti for isomaltulose production whereas trehalulose producing organisms are Pseudomonas mesoacidophila and Agrobacterium raadiobacter.

US patent 4857461 discloses the process of extraction of sucrose mutase from periplasmic membranes of Protaminobacter rubrum or Serratia plymuthica and utilization of same in a radial type bioreactor for the conversion of sucrose to isomaltulose.

In non-patent literature L. Wu et. al., 2004 and 2005 identified a sucrose isomerase from Pantoea dispersa UQ.68J having isomaltulose synthase activity and expressed in E. coli. However the recombinant isomaltulose synthase expressed in E. coli carried an additional carboxy-terminal six-His tag and moreover the expression level was low. Similarly, Nagai et al. ., 1994 showed that sucrose isomerase (also called as trehalulose synthase) activity of the P mesoacidophila MX-45. Latter Watzlawick H et. al., 2009 cloned the gene for expression in E. coli. However both authors didn't disclose their expression level of sucrose isomerase which is critical for mass production of enzymes at industrial scale.

The mass production of pure trehulose and/or isomaltulose is critical to meet the commercial value due to insufficient production of enzymes as biocatalysts. Thus mass production of sucrose isomerase and optimized bioconversion and downstream process is essential to make predominant product of trehulose and/or isomaltulose. Therefore heterologus expression of such enzymes is extremely desired to design a cost effective and much safe bioconversion process. Heterologus expression of gene products in different expression system is sometimes limited by the presence of codons that are infrequently used in other organisms. Expression of such genes can be enhanced by systematic substitution of the endogenous codons with codons over represented in highly expressed prokaryotic genes. Redesigning a naturally occurring gene sequence by choosing different codons without necessarily altering the encoded amino acid sequence often dramatically increased protein expression levels. One disadvantage in biocatalyst used in production of low caloric sugar such as isomaltulose or trehalulose are the production cost of the enzyme due to low expression level of enzymes in native or heterologus organisms which remains quite challenging. Although number of microorganisms such as Protaminobacter rubrum, Serratia plymuthica, Erwinia rhaponiciti, Pseudomonas mesoacidophila and Agrobacterium raadiobacter have been recognized for their ability to produce sucrose isomerase to convert sucrose in to isomaltulose and/or trehalulose at different ratios in given reaction conditions. However the mass production of pure isomaltulose and/or trehalulose is critical to meet the commercial value due to insufficient production of enzyme as biocatalysts.

Even though the enzymes are known that are capable of catalyzing the rare sugars but the gap still remain in mass production of enzymes and difficulties in their expression levels.

The inventors has identified the production constrain of rare disaccharides which is a bottleneck for industrial scaling up and identified the expression level in heterologous is low for certain nucleotide which are less preferred. In order to overcome such problem, the nucleotide sequence obtained from Pseudomonas mesoacidophila MX45 and Pantoea dispersa UQ.68J, encoding for the enzymes responsible for bio-conversion were modified to increase the expression level substantially. Such modification resulted in better expression of the enzymes sucrose isomerase of Pseudomonas mesoacidophila MX45 and isomaltulose synthase of Pantoea dispersa UQ.68J in E. coli. The E. coli host organism used in the invention is JM109 (a K-12 E. coli strain) was used for heterologus expression of recombinant Sucrose isomerase and isomaltulose synthase. It has been shown the E. coli K-12 cannot be converted into an epidemic pathogen by laboratory manipulation with r- DNA molecules and it will not colonize the human intestinal tract.

The present invention offers an alternative process for producing rare disaccharides, in which the enzymes were expressed in E. coli at a higher level by modifying the gene sequence. In other words, the present research has made and effort to genetically modify the gene responsible for the production of enzymes, namely sucrose isomerase and isomaltulose synthase to be used in bioconversion of sucrose in to trehalulose and isomaltulose, respectively. The genetic modification has resulted in increase in expression of protein in E. coli host.

Summary of the invention:

Accordingly the present invention discloses a modified gene sequence encoding for sucrose isomerase (Slase) of Pseudomonas mesoacidophila MX45 responsible for conversion of sucrose in to trehalulose and optimized expression of Slase in E. coli for mass production of biocatalyst for bioconversion of sugars in an optimum conditions.

Further, invention discloses a modified gene sequence encoding for isomaltulose synthase (ISase) of Pantoea dispersa UQ.68J responsible for conversion of sucrose in to isomaltulose and optimized expression of ISase in E. coli for mass production of biocatalyst for bioconversion of sugars in an optimum conditions.

The invention also discloses expression constructs comprising the modified genes to be expressed in E. coli.

The invention also relates to a process of producing trehalulose and isomaltulose from sucrose using the recombinant enzymes obtained from modified gene.

The inventers also found consistent conversion of sucrose in to trehalulose or isomaltulose by immobilized recombinant sucrose isomerase and isomaltulose synthases here in refered as Slase and ISase, respectively in contact with up to 60 % sucrose in the reaction mixture. Moreover the 110 units of immobilized ISase were able to achieve maximum conversion of sucrose into isomaltulose within 6 to 10 hrs. at 30°C. In the prior art for similar kind of conversion of sucrose to isomaltulose, the time consumed was 48 hours (US 4640894 and US 5336617). Detailed description of the invention:

The present invention discloses a modified gene sequence encoding for sucrose isomerase (Slase) of Pseudomonas mesoacidophila MX45, responsible for conversion of sucrose in to trehalulose and isomaltulose, and optimized expression of Slase in E. coli for mass production of biocatalyst for bioconversion of sugars in an optimum conditions.

The nucleotide sequence is also modified to increase the expression of isomaltulose synthase (ISase) of Pantoea dispersa UQ.68J which is responsible for conversion of sucrose in to isomaltulose predominantly.

The invention also discloses expression constructs comprising the modified genes to be expressed in E. coli. The invention also relates to a process of producing trehalulose and isomaltulose from sucrose using the enzyme obtained from modified genes.

Upon comparing with the expression level of the native sucrose isomerase and isomaltulose synthase, it was found that the modification carried out in the native gene resulted in an increase in expression level in the range of 14% to 19% of the total cellular protein.

The present invention also discloses a novel and inventive protocol to assess the protein degradation and leaching during immobilization and bioconversion process using protein specific antibodies. Till date, no process has been shown to distinguish the whole length protein from degraded one. All available processes involve epitope tag to identify the whole length protein and therefore the degraded protein is not taken into consideration. For this process the recombinant proteins were expressed with 6xHIS epitope tag by using the pET23-SI and pET15-IS (Fig. IB and 2B) constructs and the recombinant proteins were purified by one step purification using an appropriate affinity matrix. The pure proteins were used as immunogen to generate polyclonal antibody in New Zealand white rabbits. The purified protein showed strong immunogenic response and anti sera were purified by affinity chromatography using Protein A-Sepharose 4B. Affinity purified antibody used in analytical process mentioned in the embodiment.

Gene encoding for sucrose isomerase was modified for enhanced expression in Escherichia coli was synthesized using gene modification. The modified gene sequence is represented as SEQ ID NO 1. Similar modification was done to increase the expression of isomaltulose synthase in E. coli is represented as SEQ ID NO 2. Both sequence ID NOs 1 and 2 were cloned in to pETll using Ndel and BamHI restriction enzyme site to generate pETll-SI and pETll-IS constructs. Cloned gene sequences were confirmed by sequence analysis.

In a further aspect of the invention, a recombinant plasmid DNA (pETll-SI and pETl- IS) were transformed into E. coli expression host JM109 by electro transformation method to express sucrose isomerase and isomaltulose synthase, respectively. A stable transformants were selected and deposited in international depository, namely MTCC, Chandigarh bearing accession number MTCC5785 and MTCC5784 for sucrose isomerase and isomaltulose synthase, respectively.

In another embodiment of the invention, the large scale production of the above enzymes is disclosed. Importantly, the medium used for this purpose comprises no components of animal origin. The components of the medium were di-ammonium hydrogen phosphate, potassium dihydrogen phosphate and citric acid, which were sterilized in situ in the fermenter. Post sterilization a solution containing glucose, metal ions, trace elements and EDTA were added to the basal salt medium. Liquor ammonia was used as an alkali and nitrogen source. The temperature of the fermentation was maintained at 30 to 37 °C at a pH 5 to 8 and oxygen level was maintained not less than 40%, throughout the fermentation. The fermentation process at 2L scale yields about 30-40 g/l biomass.

The organism containing the synthesized gene is able to produce more enzyme as a result of genetic modification of the native nucleic acid sequence of Pseudomonas mesoacidophila MX45 and Pantoea dispersa UQ68J. Besides the production of soluble enzyme, the inclusion bodies formed in the process is solubilized and refolded in vitro into active form using standard refolding conditions. In addition production of more soluble proteins in vivo were also achieved by co-expression of modified gene constructs (pETll-SI and pETll-IS) together with chaperone plasmids such as: pG-KJE8 or pGro7 or pKJE7 or pG-Tf2 or pTfl6 (Takara).

Another aspect of the present invention is the immobilization of purified or partially purified enzymes in a suitable matrix known in the art for continuous operation. In one more aspect of the invention relates to immobilization of the enzymes, namely, Slase and ISase using a suitable matrix. Protein retention was found to be about 70-80 % (w/v).

The optimization of process parameters for the production of trehalulose and isomaltulose were carried out with varying pH and temperature, which were used for the production.

In one more feature of the invention is that the production of trehalulose from sucrose was carried out by using 25 to 100 units of immobilized Slase enzymes with varying amount of sucrose as a substrate. The conversion of sucrose to trehalulose reached saturation at higher substrate concentration of more than 50% (w/w) at enzyme concentration of 100 to 200 Units preferably 120 to 150 units of enzyme with a reaction time of about 8 hrs. The sugar solution produced in the process passed through cation and anion exchange resins to remove salt and ions present in buffer solutions and concentrated by any known means, preferably using rotary vacuum evaporator to obtain 90% purity.

In one more feature of the invention is that the production of isomaltulose form sucrose was carried out by using 25 to 100 units of immobilized ISase enzymes with varying amount of sucrose as a substrate. The reaction was carried out with substrate concentration ranging from 10% to 60% at a temperature in the range of 10° to 50°C and the pH in the range of 5 to 9. The conversion of sucrose to trehalulose reached saturation at higher substrate concentration of more than 50% (w/w) at enzyme concentration of 100 to 200 Units preferably 120 to 150 units of enzyme with a reaction time of about 8 hrs (Tables 3 and 4).

The isomaltulose sugar solution passed through cation and anion exchange resins to remove salt and ions present in buffer solutions till the time the traces removed. The sugar solution was concentrated using rotary vacuum evaporator system and subsequently passed through a column packed with activated charcoal, in order to remove the color. The purity of the product was analyzed by HPLC and was found to be about 83%. Brief Description of the Drawings

Figure 1: Schematic view of a gene construct generated for expression of sucrose isomerase in E. coli

A: Sucrose isomerase (SI) was cloned into pETlla using Ndel and BamHI sites. Sucrose isomerase (SI) gene is flanked by Bglll, Xbal and Ndel at 5'end, and BamHI at 3'end. During cloning procedure Nhel site was removed. The properties of plasmid are: T7 promoter, T7 terminator and Ampicillin resistance marker.

B: Sucrose isomerase encoding sequence (SI) was cloned into pET23a using BamHI and Hindi 11 sites. Sucrose isomerase (SI) gene is flanked by Bglll, Xbal, Ndel, Nhel and BamHI at 5'end, and Hindi 11, Notl and Xhol at 3'end. During cloning procedure EcoRI, Sacl and Sail sites were removed. The properties of plasmid are: T7 promoter, T7 terminator, Epitope tag: 6x HIS and Ampicillin resistance marker. Figure 2: Schematic view of a gene construct generated for expression of isomaltulose synthase in E. coli

A: Isomaltulose synthase encoding sequence (IS) was cloned into pETlla using Ndel and BamHI sites. Isomaltulose synthase (IS) gene is flanked by Bglll, Xbal and Ndel at 5'end, and BamHI at 3'end. During cloning procedure Nhel site was removed. The properties of plasmid are: T7 promoter, T7 terminator and Ampicillin resistance marker.

B: Isomaltulose synthase encoding sequence (IS) was cloned into pET15b using Ndel and BamHI sites. Isomaltulose synthase (IS) gene is flanked by Ncol and Ndel at 5'end and Hindi 11 at 3'end. During cloning procedure Xhol site was removed. The properties of plasmid are: T7 promoter, T7 terminator, Epitope tag: 6x HIS and Ampicillin resistance marker.

Figure 3: Expression analysis of recombinant sucrose isomerase in E. coli.

A. Control and recombinant E. coli cells [JM109 carrying pETll-SI] were induced for protein expression by addition of 0.5 mM IPTG into media. Cells were lysed and supernatant and pellet fractions were subjected to 10 % SDS-PAGE. Control strain: Lane 1 and 2 are uninduced and induced total cell lysate. Recombinant strain: Lane 3 and 4 are uninduced and induced total cell lysate. Cell fractions of recombinant strains: Lane 6 and 7 are uninduced cell supernatant and pellet, Lane 8 and 9 are two hrs induced supernatant and pellet, Lane 10 and 11 are four hrs induced supernatant and pellet. Abbreviations are: M : Protein molecular weight marker and kDa = Kilo Dalton.

B. Identity analysis of recombinant protein by Western blot analysis. Lanel and 2: Host cell lysate un-induced and induced. Lane 3 and 4: Recombinant strain uninduced and induced. Immuno-detection was carried our using protein specific antibodies. Figure 4: Expression analysis of recombinant isomaltulose synthase in E. coli.

Control and recombinant E. coli cells [JM109 carrying pETll-IS] were induced for protein expression by addition of 0.5 mM IPTG into media. Cells were lysed and supernatant and pellet fractions were subjected to 10 % SDS-PAGE. Control strain: Lane 1 and 2 are uninduced and induced total cell lysate. Recombinant strain: Lane 3 and 4 are uninduced and induced total cell lysate. Cell fractions of recombinant strains: Lane 6 and 7 are uninduced cell supernatant and pellet, Lane 8 and 9 are two hrs induced supernatant and pellet, Lane 10 and 11 are four hrs induced supernatant and pellet. Abbreviations are: M : Protein molecular weight marker and kDa = Kilo Dalton.

B. Identity analysis of recombinant protein by Western blot analysis. Lanel and 2: Host cell lysate un-induced and induced. Lane 3 and 4: Recombinant strain uninduced and induced. Immuno-detection was carried our using protein specific antibodies.

Figure 5: HPLC analysis of recombinant sucrose isomerase activity for substrate to product conversion.

The reaction mixtures were subjected to HPLC analysis to confirm the residual substrate and product formation. The product peaks were confirmed with commercially available sucrose and isomaltulose, trehalulose as substrate and product standards, respectively.

Figure 6: HPLC analysis of recombinant isomaltulsoe synthase activity for substrate to product conversion.

The reaction mixtures were subject to HPLC analysis to confirm the residual substrate and product formation. The product peaks were confirmed with commercially available Sucrose and isomaltulose as substrate and product standards, respectively. Figure 7: Analysis of purified Slase

A. Different fractions and purified protein were separated on 12 % SDS-PAGE and stained by coomassie brilliant blue R250. Loading pattern are Lane 1 : Marker; Lane 2: Total cell Lysate; Lane 3: Cell lyste before loading in columnl; Lane 4: Column 1 purified Slase; Lane 5: Column 2 purified Slase.

B. Identity analysis of purified recombinant protein by Western blot analysis. Lane 1: Marker; Lane 2: Total cell Lysate; Lane 3: Cell lyste before loading in columnl; Lane 4: Column 1 purified Slase; Lane 5: Column 2 purified Slase. Immuno-detection was carried our using protein specific antibodies.

Figure 8: Analysis of purified ISase

A. Different fractions and purified protein were separated on 12 % SDS-PAGE and stained by coomassie brilliant blue R250. Lane 1: Molecular weight marker, Lane 2 to 6: Fractions 1 to 7, Lane 9: Crude cell lysate.

B. Identity analysis of purified recombinant protein by Western blot analysis. Lane 1: Total cell Lysate, Lane 2 and 3: Supernatant and Cell lyste of recombinant cell lysate, Lane 4: Purified ISase. Immuno-detection was carried our using protein specific antibodies.

Figure 9: Activity of a sucrose isomerase against reaction pH and reaction temperature. The reaction mixture containing sucrose and purified Slase were incubated at different pH and temperature as indicated. Bioconversion reaction stopped by boiling the reaction mixture at 95 °C. HPLC analysis of the reaction mixture confirmed the residual substrate and product formation. The product peaks were confirmed with commercially available sucrose and isomaltulose, trehalulose as substrate and product standards

Figure 10: Activity of an isomaltulose synthase against reaction pH and reaction temperature. The reaction mixture containing sucrose and purified ISase were incubated at different pH and temperature(s) as indicated. The bioconversion the reaction stopped by boiling the reaction mixture at 95 °C. HPLC analysis confirmed the residual substrate and product formation. The product peaks were confirmed with commercially available sucrose and isomaltulose as substrate and product standards

Figure 11: Sequence alignment analysis of modified gene sequence with native gene sequence encoding for sucrose isomerase.

Modified gene sequence (represented as "modified") was subjected to sequence alignment with native gene sequence (represented as "native") of Pseudomonas mesoacidophila MX45 using multiple sequence alignment tool (ClustalW2). The nucleotides of modified gene sequence were marked as (.) and homology shared to native sequence was marked as (*). In the modified gene 22% of nucleotides were changed and compared to native gene sequence, in addition 66 nucleotides (22 codons) were removed after ATG start codon which codes for 22 amino acid predicted signal sequence in P. mesoacidophila MX-45.

Figure 12: Sequence alignment analysis of modified gene sequence with native gene sequence encoding for isomaltulose synthase.

Modified gene sequence (represented as "modified") was subjected to sequence alignment with native gene sequence (represented as "native") of Pantoea dispersa UQ.68J using multiple sequence alignment tool (ClustalW2). The nucleotides of modified gene sequence were marked as (.) and homology shared to native sequence was marked as (*). In the modified gene 20% of nucleotides were changed and compared to native gene sequence, in addition 96 nucleotides (32 codons) were removed after ATG start codon which codes for 22 amino acid predicted signal sequence in Pantoea dispersa UQ.68J.

Examples:

The following examples are given by way of illustration, which should not be construed to limit the scope of the invention. Example 1:

Gene construction

Gene encoding for sucrose isomerase (SI) was modified for enhanced expression in Escherichia coli was synthesized using gene synthesis approach. The modified gene sequence is represented as SEQ I D NO 1. Similar modification was done to increase the expression of isomaltulose synthase in E. coli as represented in SEQ. I D NO 2. Both sequence I D NOs 1 a nd 2 were cloned in to pUC57 using EcoRV restriction enzyme site to generate pUC57-SI and pUC57-IS constructs. Cloned gene sequence was confirmed by sequence a nalysis.

The DNA fragment encoding for sucrose isomerase was PCR amplified using gene specific primers, and sub cloned into pETlla using Ndel and BamHI restriction enzyme sites to generate pETll-SI (Fig. 1A). I n addition the coding region was PCR amplified without stop codon using gene specific primers and sub cloned into E. coli expression vector pET23a (Fig. IB) using BamHI and Hindi 11 restriction enzymes to generate pET23-SI-HIS construct expressing sucrose isomerase with C-terminal 6x Histidine tag. The recom bina nt plasmid ca rrying sucrose isomerase gene (pETll-SI and pET23-SI) was confirmed by restriction digestion ana lysis and followed by DNA sequencing.

The DNA fragment encoding for isoma ltulose synthase was PCR amplified using gene specific primers, and sub cloned into pETlla using Ndel and BamHI restriction enzyme sites to generate pETll-IS (Fig. 2A). I n addition the coding region was PCR amplified without stop codon using gene specific primers and sub cloned into E. coli expression vector pET15b (Fig. 2B) using Ndel and Hindi 11 restriction enzymes to generate pET15-IS-HIS construct expressing isomaltulose synthase with C-terminal 6x Histidine tag. The recombinant plasmid ca rrying isomaltulose synthase gene (pETll- IS and pET15-IS) was confirmed by restriction digestion analysis and followed by DNA sequencing. Example 2:

Development of recombinant E. coli with gene constructs

For sucrose isomerase

Recombina nt plasmid DNA (pETll-SI) was transformed into E. coli expression host JM 109 by electro transformation method a nd grown on Luria-Bertani (LB) agar plates containing ampicillin (50 g/ml). I ndividual colonies were picked and grown on LB liquid or defined media containing ampicillin (75 g/m l) for overnight at 37 °C. Overnight culture was re-inoculated into 0.1 OD ₆ oo in LB liquid or defined media without ampicillin and grown up to 0.6 OD ₆ oo and the cells were induced for protein expression by addition of 0.5mM of I PTG (Isopropyl β-D-l-thiogalactopyra noside) and incubated at 37 °C. An aliquot of E. coli culture was collected at different time points. The cell lysate was subjected to SDS-PAGE and Western blot ana lysis to verify the protein expression (Fig.3). For isomaltulose synthase

Recombina nt plasmid DNA (pETll-IS) was transformed into E. coli expression host JM 109 by electro transformation method a nd grown on Luria-Bertani (LB) agar plates containing ampicillin (50 g/ml). I ndividual colonies were picked and grown on LB liquid or defined media containing ampicillin (75 g/m l) for overnight at 37 °C. Overnight culture was re-inoculated into 0.1 OD ₆ oo in LB liquid or defined media without ampicillin and grown up to 0.6 OD ₆ oo and the cells were induced for protein expression by addition of 0.5mM of I PTG (Isopropyl β-D-l-thiogalactopyra noside) and incubated at 37 °C. An aliquot of E. coli culture was collected at different time points. The cell lysate was subjected to SDS-PAGE and Western blot ana lysis to verify the protein expression (Fig.4).

Example 3:

Production of enzymes, namely, sucrose isomerase and isomaltulose synthase

For la rge scale production of the above enzymes same protocols were followed. The medium used comprises no components of animal origin. The components of the medium were 4.0 g/L di-ammonium hydrogen phosphate, 13.3 g/L potassium dihydrogen phosphate and 1.7 g/L citric acid, 28 g/L glucose, 1.2 g/L MgSo4.7H20, 45mg/L Thiamine HCL, lg/L CoCI2.6H20, 6g/L MnCI2.4H20, 0.9 g/L CuSo4.5H20, 1.2g/L H3B03, 0.9 g/L NaMo04, 13.52 g/L Zn (CH3COO-), 40 g/L Fe-Citrate and 14.1 g/L EDTA. Liquor ammonia was used as an alkali and nitrogen source. The temperature of the fermentation was maintained at 37 °C at a pH 6.9 and oxygen level was maintained not less than 40%, throughout the fermentation. The fermentation process at 2L scale yields 30 -40g/l biomass.

Example 4:

Purification of enzymes

After completion of the fermentation the cells were centrifuged at 5000 g for 10 min and resuspend in 20 mM Tris-EDTA (TE) buffer, pH 8.0. The cells were lysed using the cell disruptor at 25 KPsi twice and the resulted cell lysate was clarified by centrifugation. The crude cell-free extract obtained from the supernatant following centrifugation at 27 OOOg for 30 min at 4^C was used for the purification. Clarified crude cell lysate was applied onto a Q.-Sepharose column (GE, Healthcare) pre- equilibrated with 20 mM Tris-HCI buffer pH 8.0 and washed with five column volume of same buffer containing 100 mM NaCI. The bound proteins were eluted with NaCI gradient (0.1-0.4 M) in the same buffer, followed by step elution with 0.5 M and 1M NaCI wash in the same buffer. Fractions were collected and tested for sucrose isomerase and isomaltulose synthase activity and purity by SDS-PAGE (Fig. 7and 8). The purification yield, activity recovery and fold purification for sucrose isomerase and isomaltulose synthase were shown in Table 1 and Table 2, respectively. Fractions containing the purified protein were dialyzed against 20 mM Tris pH 8.0 for 16 hours at 4 °C and concentrated by ultrafiltration using Centricon YM-10 devices (Millipore) prior to immobilization or stored with 20% glycerol at -20^C.

Example 5:

Immobilization of enzymes:

The same protocol was followed for Slase and ISase. Partially purified or purified Slase and ISase wre dialyzed against 20 mM Tris buffer (pH 8.0) for 16 hours at 4 °C followed by mixing with equal volume of 4% sodium alginate (final concentration of sodium alginate was 2 % w/v). The Slase or ISase containing sodium alginate solution was dropped by a surgical needle into chilled 0.2 M CaCI ₂ solution with constant stirring. Immobilized beads were kept in CaCI ₂ overnight at 4 °C, followed by water wash and kept on a blotting paper for drying at 4 ^c. Protein retention was found to be 85 % w/v with 2 % w/v of sodium alginate.

Example 6:

Production of rare disaccharides

Production of trehalulose by recombinant Slase

The optimization of process parameters for the production of trehalulose was carried out with varying pH and temperature, which were used for the production of trehalulose. Results are shown in Fig. 9. Production of trehalulose form sucrose was carried out by using 110 units of immobilized SI enzymes with 10%, 20% , 30% and 40% sucrose solution in 20 mM Sodium Acetate, 10 mM CaCI ₂ buffer pH 6.5 at 14°C.

The sugar solution was subjected to cation and anion exchange resins to remove salt and ions present in buffer solutions.

The sugar solution was concentrated using rotary vacuum evaporator system and subsequently passed through a column packed with activated charcoal, in order to remove the color. The purity of the product was analyzed by HPLC (Fig. 5) and ions contaminations were analyzed in ion chromatography (Dionex). Physico-chemical properties and purity of the product were carried out using standard techniques to confirm the safety aspects of produced trehalulose in this process. Bioconversion of trehalulose from sucrose was observed to be ~92 %. Production of isomaltulose by recombinant ISase

The optimization of process parameters for the production of isomaltulose was carried out with varying pH and temperature, which were used for the production isomaltulose. Results are shown in Fig. 10.

Production of isomaltulose from sucrose was carried out by using 110 units of immobilized ISase with 100 g/l, 200 g/l and 400 g/l sucrose solution was used in 20 mM Tris buffer, 5 mM MnCI ₂ (pH 8.0) at 35 °C. The sugar solution was subjected to cation and anion exchange resins to remove salt and ions present in buffer solutions.

The sugar solution was concentrated using rotary vacuum evaporator system and subsequently passed through a column packed with activated charcoal, in order to remove the color. The purity of the product was analyzed by HPLC (Fig. 6) and ions contaminations were analyzed in ion chromatography (Dionex). Physico-chemical properties and purity of the product were carried out using standard techniques to confirm the safety aspects of produced isomaltulose in this process. Bioconversion of isomaltulose form sucrose was observed ~83 %.

Advantage of the invention:

The genetically modified genes encoding for sucrose isomerase and isomaltulose synthase is capable of expressing 14% to 19% more of the total cellular protein as compared to native gene.

The enzyme produced by the present process appears to be more active as the enzyme requirement and time required for sugar conversion substantially lower than the native enzymes. Non-patented literature:

Nagai, Y. T., Sugitani, and K. Tsuyuki. 1994, Characterization of alpha- glucosyltransferase from Pseudomonals mesoacidophila producting trehalulose. Biosci. Biotechnol. Biochem. 58:1789-1793.

Watzlawick, H., Mattes, R. 2009. Gene cloning, protein characterization, and alteration of product selectivity for the trehalulose hydrolase and trehalulose synthase from "Pseudomonas mesoacidophila" MX-45. Appl. Environ. Microbiol. 75:7026-7036.

Wu, L, Birch, R.G. 2004. Characterization of Pantoea dispersa UQ.68J: producer of a highly efficient sucrose isomerase for isomaltulose biosynthesis. J. Appl. Microbiol. 97: 93-103.