AU2006284545B2 - Extracting and purifying limit dextrinases - Google Patents

Abstract

The invention relates to improved methods for extracting and purifying limit dextrinase enzymes from cells, in particular plant cells. The process of the invention includes heating a cell homogenate at 4OºC, in the presence of divalent cations thus increasing the specific activity of the limit dextrinase.

Description

WO 2007/022597 PCT/AU2006/001240 1 Extracting and purifying limit dextrinases Field of the invention The invention relates to extracting and purifying an enzyme from a cell, particularly, but not exclusively, to extracting and purifying a limit dextrinase. 5 Background of the invention Limit dextrinase (EC 3.2.1.142), otherwise known as dextrin a-1,6 glucanohydrolase; R-enzyme; or amylopectin-1-6 glucosidase; is an enzyme that catalyses the hydrolysis of (1-6)-c-D-glycosidic linkages in a- and p-limit dextrins of amylopectin and pullulan. 10 Limit dextrinases have little or no activity on glycogen, incomplete action on amylopectin and complete action on a-limit dextrins. These enzymes release maltose from an -(1-6)- linkage and hence are particularly important in food industries for providing maltose. The processes for obtaining commercial quantities of limit dextrinases tend to be 15 difficult to operate on a commercial scale, in terms of requiring sophisticated fermentation technology, extraction and separation techniques, multiple steps and expensive reagents and equipment. Some processes are characterised by an unacceptable loss or wastage of limit dextrinases. Other processes tend to produce a non purified final product that has a sub-optimal specific activity. 20 In view of the above, there is a need for improved processes for purification of limit dextrinases. Summary of the invention The invention seeks to at least minimise one or more of the above identified problems or limitations and/or to provide an improved process for purification of limit 25 dextrinase. In one aspect, the invention provides a process for purifying a limit dextrinase from a cell. The process includes a step of heating an extract of a cell formed from a WO 2007/022597 PCT/AU2006/001240 2 solution including at least one divalent cation, to increase the specific activity of a limit dextrinase in the extract. In another aspect, the invention provides a process for purifying limit dextrinase from a barley cell. The process includes the following steps: 5 (a) releasing limit dextrinase from a barley cell into a solution including calcium and magnesium to form an extract; (b) heating the extract to increase the specific activity of limit dextrinase in the extract. In another aspect, the invention provides a process for purifying limit dextrinase 10 from a barley cell. The process includes the following steps: (a) releasing limit dextrinase from a barley cell into a solution including calcium and magnesium to form an extract; (b) heating the extract to increase the specific activity of limit dextrinase in the extract; and 15 (c) utilising anion exchange chromatography to purify limit dextrinase from the heated extract. Typically the cell is a barley cell, such as a cell derived from a barley rootlet or grain. In another aspect, the invention provides limit dextrinase produced by the process 20 of the invention. In another aspect, the invention provides a cell including limit dextrinase produced by the process of the invention. Detailed description of the embodiments As described herein, it has been found that creating a suitable buffered 25 environment during extraction and conducting heat treatment of an extract of a barley cell or rootlet in a solution comprising at least one divalent cation permits the specific activity of the extract with respect to limit dextranase contained within it to be increased. For example, the specific activity of a heat treated extract of a barley rootlet formed from a WO 2007/022597 PCT/AU2006/001240 3 example, the specific activity of a heat treated extract of a barley rootlet formed from a solution comprising 50mM Calcium Chloride and 50 mM Magnesium Chloride was observed to increase 2.4 fold over a non heat treated sample (756.54 pimoles/min/mL compared with 312.09 ptmoles/min/mL). Further, a heat treated extract containing 50mM 5 Calcium Chloride and 50mM Magnesium Chloride was observed to have an improved specific activity (385.7 tmoles/min/mL) compared with a heat treated extract containing no Calcium and Magnesium (75.0ptmoles/min/mL). This is a significant finding because it permits heat treatment, a purification step that is relatively simple to operate on a commercial scale, to be implemented with 10 minimal loss of activity of limit dextrinase. Thus in certain embodiments there is provided a process for purifying a limit dextrinase from a cell including the step of heating an extract of a cell formed from a solution including at least one divalent cation, to increase the specific activity of a limit dextrinase in the extract. 15 In other embodiments there is provided a process for increasing the specific activity of a limit dextrinase in a cell extract, said extract being one formed from a solution including at least one divalent cation. The process includes the step of heating the cell extract to increase the specific activity of a limit dextrinase in the extract. It is believed that the specific activity of the extract is increased because the 20 divalent cation protects limit dextrinase from denaturation at temperatures at which other proteins in the extract are degraded. Typically, the at least one divalent cation in the solution may be Calcium and/or Magnesium. For example, the solution may contain Calcium Chloride and/or Magnesium Chloride. 25 Zinc, copper and manganese are also cations. The Calcium and Magnesium ions may be included in the extract in an amount to permit control of the denaturation of limit dextrinase when the extract is heated. Typically, Calcium and Magnesium are included in the extract in an amount to at least limit the denaturation of limit dextrinase when the extract is heated. For example, the WO 2007/022597 PCT/AU2006/001240 4 concentration of Calcium may be less than 100 mM and the concentration of Magnesium may be less than 100 mM. A concentration of Calcium and Magnesium in a range between about 25 to 50 mM is particularly useful as further down stream processing of the extract for further 5 purification, such as anion exchange chromatography, may require removal of Calcium and Magnesium. Accordingly a concentration of Calcium ions of about 50 mM and a concentration of Magnesium ions of about 50 mM is particularly useful. In certain embodiments, the Calcium ions are provided in a concentration selected from the group consisting of 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM 10 and 45 mM. In certain embodiments, the Magnesium ions are provided in a concentration selected from the group consisting of 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM and 45 mM. Typically the solution further includes a reducing agent for denaturing disulfide 15 bonding. Typically the agent is L- cysteine or ascorbic acid, although in appropriate processing circumstances, other reducing agents might be used, including glutathione, 2 mercaptoethanol and dithiothrietol. Further, the solution may be buffered to about pH 7.5 using a suitable solution, such as Tris HCl. In particular, and with reference to the preceding, it has been found that by heating an extract buffered at pH 7.5 with 200mM 20 TrisHCl and containing divalent cations and a reducing agent such as 20mM L-cysteine, the specific activity of the extract can be increased at least 5.14 fold (385.7 ptmoles/min/mL) compared with a heat treated extract containing no reducing agent, Calcium or Magnesium (75.0pmoles/min/mL). Thus in certain embodiments, there is provided a process for purifying a limit 25 dextrinase from a cell. The process includes a step of heating an extract of a cell formed from a solution having a pH of at least about 5 and including at least one divalent cation and a reducing agent, to increase the specific activity of a limit dextrinase in the extract. Typically the reducing agent is L-cysteine. Useful concentrations of L-cysteine include concentrations from about 2 mM to 25 mM, although higher concentrations of L- WO 2007/022597 PCT/AU2006/001240 5 cysteine are contemplated. The concentration of L-cysteine may be selected from the group consisting of 4 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 22 mM and 24 mM. Where the reducing agent is other the L-cysteine, such as ascorbic acid, these concentrations of ascorbic acid may also be used, although 5 concentrations of ascorbic acid up to 50 mM can be used. Typically the solution has a pH of at least about 5, although higher ranges to about pH 9.0 are particularly useful for enhancing the specific activity of the enzyme in the extract. A solution having a pH selected from the group consisting of 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5 may be used. A pH of above 9.0 could be used, however above this range 10 the activity of the enzyme tends to be affected. Trizma base buffered in HCl in a concentration of about 200mM is particularly useful to provide the appropriate pH. It is believed that the major constituents of an extract of a barley rootlet include a number of enzymes having activity for various carbohydrate and protein substrates. Thus, the extract is typically heated to a temperature that permits denaturation of unwanted 15 proteases, ancillary enzymes, or otherwise, destruction of activity of these enzymes in the extract. As described herein, temperatures less than 65 0 C are suitable for this purpose. It is particularly advantageous to heat the extract to between about 35 and 60'C because at temperatures approaching 65'C and above, limit dextrinase activity may be lost. Accordingly, a temperature of about 55'C is particularly useful. 20 The inventor has also found that the purification of limit dextrinase from a barley cell extract can be improved by extracting a barley cell homogenate at 40"C in a solution including Calcium and Magnesium. Specifically, as described herein, the specific activity of an extract comprising Calcium and Magnesium after maintenance at 40'C was found to be 303.86-3 pmoles/min/mL as compared with the activity of an extract maintained at 25 40 0 C in the absence of Calcium and Magnesium and L-Cysteine (65.12~3 pLmoles/min/mL). It is believed that maintenance of such an extract at 40'C is important because it permits limit dextrinase to disassociate from solids in the extract, and accordingly, to solubilise into the liquid phase of the extract, prior to further processing of the extract, 30 such as a heat treatment step or a chromatographic separation step. The Calcium and WO 2007/022597 PCT/AU2006/001240 6 Magnesium are believed to be important for limiting hydrolysis of the enzyme during the maintenance of the extract at 40'C. Thus in accordance with the invention, a process for purifying limit dextrinase from a barley cell includes the following steps: (a) releasing limit dextrinase from a barley cell into a solution including 5 Calcium and Magnesium Chloride to form an extract; and (b) heating the extract to increase the specific activity of limit dextrinase in the extract. Typically, the extract is maintained in conditions for promoting stabilization of the limit dextrinase in the extract prior to heating the extract. 10 The extract may be maintained at less than 10 'C for less than 3 days. For example, the extract may be maintained between 0 to about 4 "C for between about 1 to 48 hours. In certain embodiments, the solution further includes a reducing agent as discussed above in concentrations as discussed above. The solution is further buffered to 15 a pH range as discussed above. Thus, in another aspect, the invention provides a process for purifying limit dextrinase from a barley cell. The process includes the following steps: (a) releasing limit dextrinase from a barley cell into a solution having a pH of at least about 5.0, the solution including calcium, magnesium and a reducing agent, to form an extract; 20 (b) heating the extract to increase the specific activity of limit dextrinase in the extract. It is particularly advantageous to maintain the extract for 12 hours at 4 "C prior to extraction at 40'C as this improves the speed of purification protocols that comprise further purification steps. 25 It has further been found that limit dextrinase can be purified to virtual homogeneity from a barley cell extract by a process including the following steps: WO 2007/022597 PCT/AU2006/001240 7 (a) releasing limit dextrinase from a barley cell into a solution including Calcium and Magnesium and a reducing agent, the solution being at least about pH 5.0 to form an extract; (b) heating the extract to increase the specific activity of limit dextrinase in 5 the extract; and (c) utilising chromatography to purify limit dextrinase from the heated extract. As described herein, limit dextrinase can be further purified from a heat treated barley cell extract by anion exchange chromatography. Accordingly, typically, in step (c), anion exchange chromatography is utilised to purify limit dextrinase from the heated 10 extract. It has been found that Calcium and Magnesium ions and L-Cysteine tend to limit binding of limit dextrinase during anion exchange chromatography. Accordingly, typically the extract is desalted before anion exchange chromatography. One way of desalting to remove Calcium and Magnesium ions and L-Cysteine is by ultrafiltration. 15 Alternatively, a preparative de-salting column, such as a Hi Prep 26/10 desalting column can be used. It is particularly advantageous to remove substantially all of the Calcium and Magnesium from the extract prior to anion exchange chromatography for the purpose of maximising the yield of limit dextrinase purified from the anion exchange column. Typically, the extract is maintained in conditions for promoting solubilisation of 20 the limit dextrinase in the extract prior to heating the extract. In the processes of the invention described above, the extract of the barley cell is typically produced by homogenising barley rootlets in an appropriate buffer. One way of homogenizing grains is by use of a blender, such as a Waring blender. Alternatively, the extract may be produced by milling barley grains in an appropriate buffer using a roller 25 mill following a predetermined steeping and germination. As discussed above, the solution into which the limit dextrinase from the cell is released to form an extract is typically a buffer for controlling pH. Solutions prepared from Trisma base are examples of such a solution. A solution having a concentration of WO 2007/022597 PCT/AU2006/001240 8 no more than about 300 mM Tris is suitable, for example, 200 mM Tris is particularly advantageous adjusted and maintained at a pH 7.5 . It will be understood that the processes of the invention are useful for purifying limit dextrinase from cells other than barley cells. Other examples include cells of grains 5 such as rice and wheat, and other vegetable matter. Further, it will be understood that processes of the invention are useful for isolating barley limit dextrinase from cells that contain a recombinant nucleic acid molecule that encodes barley limit dextrinase. Examples of such cells include bacterial cells and yeast cells. Example 1: Materials and equipment. 10 Germinating barley seeds (Schooner variety) were obtained from Barrett Burston Malting, (Thornleigh, NSW, Australia), Calcium Chloride, Magnesium Chloride, Potassium Chloride, Sodium Chloride, Trisma base, Sodium Acetate, Hydrochloric Acid and L-Cysteine were supplied by Sigma Aldrich (Castle Hill, NSW, Australia), Red Pullulan was obtained from Megazyme ( Bray, Ireland) and undenatured Ethanol was 15 purchased from CSR Distilleries (Ingleburn, NSW, Australia). The germinated barley grains were milled on a Kustnel Freres & Cie roller mill to a gap setting of 1mm to crack the grains allowing extraction of enzymes. The crude enzyme extract was coarse filtered though double cheesecloth then centrifuged at 26,800 x g for 30 minutes at 4'C to remove any precipitate. 20 The crude extract was concentrated and buffer exchanged using a MidGee cross flow ultrafiltration unit combining a Masterflex economy drive peristaltic pump and Masterflex Easy load II head, UFP-30-H24LA ultrafiltration cartridge with 30kDa nominal cut off and MidGee starter kit KMDG-1. A flow rate of 17mL per minute at 10 psi pressure was sufficient to separate and concentrate the limit dextranase containing 25 fractions. The buffer used for FPLC gel filtration and ion exchange chromatography was 25mM Sodium Acetate (pH 5.5). The eluent buffer for ion exchange chromatography included 1 M NaCl.

WO 2007/022597 PCT/AU2006/001240 9 An Amersham Pharmacia AKAT gradient processing FPLC system complete with a 900 model monitor, lamp and detector (set at 280nm), 920 model pump and Frac 950 fraction collector interfaced to a Compaq Deskpro Pentium III computer supporting Unicorn analytical software was used for all protein purification. The columns used 5 included a Hi Prep 26/10 desalting column connected to a Super loop 50 (to facilitate larger injection volumes), a 16/10 Hi-Prep DEAE FF anion exchange column with a final purification undertaken on a Mono Q HR 5/5 column. Isolation of limit dextranase was identified by the presence of single protein bands on native electrophoresis gels and single absorption peaks by sequential anion exchange 10 chromatography. An LW Scientific UV-Visible spectrophotometer was used to measure enzyme activity operating at 510 nm. The system was controlled by a Celeron processor computer operating a LW Scientific Graphite version 3.1 enzyme kinetics software program. 15 Example 2: Preparation of a standard curve for dye labelled Red Pullulan to determine limit dextrinase activity A standard curve for the identification of Pullulanase activity was supplied by Megazyme utilising a pullulan substrate derived from Bacillus acidopullulyticus. The extracted pullulan is standardised for molecular weight and degree of a (1-6) branching 20 by the action of borohydride and conjugated with Procion Red MX-5B to an extent of one dye molecule per an estimated 30 sugar residues. The Red pullulan substrate (0.5g) is added to 25ml of 0.5M Potassium Chloride and vortexed until completely dissolved. Working standards are prepared in the range of 100 to 800 qM/mL in 25mM Sodium Acetate buffer at pH 5.5 and read spectrophotometrically at 51 Onm. The solution is stored 25 at 4 0 C in a well sealed glass bottle with an overlay of toluene to prevent microbial infection until required. A Example 3: Preparation of a standard curve for protein to determine limit dextrinase specific activity.

WO 2007/022597 PCT/AU2006/001240 10 Protein was determined using the BioRad micro assay procedure derived from the original method of Bradford utilising a standard curve produced for bovine serum albumin. Each analysis was conducted in duplicate requiring incubation at room temperature for 10 minutes with the absorbance measured at 595 nm. Standards were 5 prepared in the range of 0.2 to 1.4 mg/mL of protein. Example 4: Enzyme kinetics assay. The assay requires lml of the extracted enzyme solution [suspended in 200 mM Sodium Acetate buffer at pH 5 (post buffer exchange)] pre-equilibrated at 40'C for 5 minutes. To this suspension is added 0.5mL Red pullulan substrate [(0.5g) in 25ml of 10 0.5M Potassium Chloride]. The mixture is stirred and incubated at 40C for exactly 10 minutes. The reaction is terminated by the addition of 2.5ml of 95% (v/v) ethanol vortexing for 10 seconds. The reaction tubes are allowed to equilibrate at room temperature for 10 minutes and then centrifuged at 1,000 x g to precipitate the higher molecular weight fractions of the substrate. The supernatant is transferred directly to a 15 curvette and the absorbance read at 510nm. Activity is determined by reference to the standard curve. A reference blank is prepared by adding 1mL of distilled water to 2.5mL ethanol and 0.5mL of the Red Pullulan substrate. Example 6: Preparation of crude limit dextrinase extract. 30 g of 3 to 12 month old stored barley grains were dispersed in 45mL 0.2M Tris 20 HCl (pH7.5) containing 20mM L-Cysteine, 50mM MgCl 2 and 50mM CaCl 2 following a germination period. The germinated grains were firstly milled using smooth rollers at a gap setting of 1mm and speed 440 rpm, feed rate of 1kg per minute prior to extraction at 40'C for 2 hours to facilitate solubilisation of limit dextrinase. The insoluble material was removed from the extract by filtering through double 25 cheese cloth. The filtrate was centrifuged at 15,000 rpm for 30 minutes at 4"C to remove solids and the supernatant was passed through a 0.45pM filter and stored at 4'C in a sterile container with 0.01% sodium azide. This process formed the crude limit dextrinase extract.

WO 2007/022597 PCT/AU2006/001240 11 The activity and specific activity of the crude limit dextrinase extract was then determined according to Examples 2,3 and 4 above. Example 7: Purification of limit dextrinase from the crude limit dextrinase extract. 5 The first stage of the purification process involved the removal of heat labile proteases, inhibitory proteins and any superfluous proteinaceous materials from the crude limit dextrinase extract with the aim of reducing any loss of activity or damage to the structure of limit dextrinase while increasing the specific activity of limit dextranase extract. To inactivate and remove these proteins, the crude extract was heated in a water 10 bath to 55 C and maintained at that temperature for 1 hour. The extract was then cooled to room temperature and buffer exchanged by cross flow ultrafiltration with 25mM Sodium Acetate pH 5.5 to facilitate gel filtration and ion exchange chromatography. The extract was initially centrifuged and filtered through a 0.45ptm filter. The activity and specific activity of the heat treated limit dextrinase extract was 15 then determined according to Examples 2,3 and 4 above. Gel filtration and ion exchange chromatography was then undertaken. A 50 mL sample of the extract was injected into a Super loop 50 column and gel filtered by FPLC on a Hi Prep 26/10 desalting column at a flow rate of 7.0 mL per minute, to remove magnesium , calcium and L-Cysteine. The desalted fractions were then pooled and 20 reloaded onto the Super loop column and passed through a Hi PREP 16/10 DEAE anion exchange column at 3.0 mL per minute to initially fractionate limit dextrinase. The isolated fraction was again desalted to remove the IM NaCl elution buffer and purified by passing the fraction through the Mono Q HR 5/5 column at 1.5 mL per minute. A single peak was obtained and analysed for activity and specific activity according to 25 Examples 2 to 4 above. Example 8: Purification profile for limit dextrinase. The results for the purification of limit dextrinase are shown in Table 1. Table 1 WO 2007/022597 PCT/AU2006/001240 12 Sample Activity Specific activity Purification factor Crude extract 411.96 312.09 1 Heat treatment 416.1 756.54 2.42 Anion 420.05 8738.52 28 exchange Activity: x 10 3 pLmoles/min/mL Specific activity: x 103 ptmoles/min/mL/mg Example 9: Effect of Calcium, Magnesium and L-Cysteine on limit dextranase activity of crude extract during solubilization at 40'C. 5 We sought to determine whether calcium magnesium and L-Cysteine would have an effect on stabilisation of limit dextrinase in the crude extract, or otherwise, on preserving or enhancing limit dextrinase activity of the crude extract, during the step of extracting limit dextrinase at 40"C that follows the milling step described in Example 6. To this end we extracted the enzyme in (i) water, (ii) 0.2M Tris-HCL (pH 7.5), 10 (iii) 0.2M Tris-HCL (pH 7.5) with 50mm Calcium Chloride and 50mm Magnesium Chloride, (iv) 0.2M Tris-HCL (pH 7.5) with 50mm Calcium Chloride and 50mm Magnesium Chloride and 20mM L-Cysteine and maintained the extract at 40'C for 2 hours. We found that the buffer containing 0.2M Tris-HCL maintained at a pH of 7.5 with the addition of 50mM Calcium Chloride and 50mM Magnesium Chloride in the 15 presence of 20mM L-Cysteine, enhanced and indeed stabilised limit dextranase activity compared to water (observed over a decreasing range of pH). The limit dextranase activity was 466% greater than in the sample with no Calcium, Magnesium or L-Cysteine at decreasing pH, (303.86 qmoles/min/mL compared to 65.12 qmoles/min/mL). Example 10. Effect of calcium, magnesium L-Cysteine on limit dextrinase 20 activity maintained at pH 7.5 of crude extract during heat treatment. We sought to determine whether calcium, magnesium and L-Cysteine would have an effect on stabilisation of limit dextrinase in the crude extract or otherwise on WO 2007/022597 PCT/AU2006/001240 13 preserving or enhancing limit dextrinase activity of the crude extract, during the step of heating limit dextrinase as described in Example 7. To this end, we incubated the crude extract at temperatures from 25'C to 65 "C for 1 hour in 0.2M Tris-HCL (pH 7.5) with 50mm Calcium Chloride and 50mm Magnesium 5 Chloride and 20mM L-Cysteine We found that after heating, the activity dropped significantly in samples held above 65'C (13.6 -jmoles/min/mL) compared to extracts held at 25 0 C (168.16 qmoles/min/mL), 40'C (251.47 qmoles/min/mL) and 55'C (242.68 tymoles/min/mL) and as a consequence consolidated the extraction process to 40'C and heating to 55'C.

Claims (20)

1. A process for purifying a limit dextrinase from a grain including the steps of: (a) releasing limit dextrinase from the grain into an alkaline aqueous solution at pH7 pH9, the solution including calcium and magnesium to form an extract; and 5 (b) heating the extract to 35-60C, whereby compounds are selectively precipitated from the extract while retaining limit dextrinase in solution.

2. The process according to claim 1, wherein each of calcium and magnesium is provided in the solution in a concentration of less than about 100mM.

3. The process according to claim 1 or 2, wherein the solution further includes a reducing 10 agent.

4. The process according to claim 3, wherein the reducing agent is L-cysteine.

5. The process according to claim 4, wherein the concentration of L-cysteine in the solution is less than about 25 mM.

6. The process according to any one of claims 1 to 5, wherein the pH of the solution is 7.5. 15

7. The process according to claim 3, wherein the reducing agent is ascorbic acid.

8. The process according to any one of claims I to 7, wherein the extract is heated to 55C.

9. The process according to any one of claims 1 to 8, including further purifying the limit dextrinase from the heated extract.

10. The process according to claim 9, wherein the further purification step utilizes 20 chromatography to purify limit dextrinase from the heated extract.

11. The process according to claim 10, wherein the chromatography is anion exchange chromatography.

12. The process according to claim 9, further comprising the step of desalting the extract prior to purifying the limit dextrinase from the heated extract. 15

13. The process according to any one of claims I to 12, further including the step of maintaining the grain at less than 10*C prior to heating step (b).

14. The process according to claim 1, wherein the releasing step (a) is undertaken at 40*C.

15. The process according to claim 1, further including the step of reducing the pH of the extract 5 after heating step (b) to produce an acidified extract.

16. The process according to any one of claims 1 to 15, wherein the grain is steeped in the solution prior to releasing the limit dextrinase from the grain.

17. The process according to claim 16, wherein the solution includes a reducing agent.

18. The process according to claim 1, wherein heating step (b) involves heating the extract to 10 35-45*C, followed by heating the extract to 50-60*C.

19. The process according to claim 18, wherein the extract is heated to 35-45*C for about 2 hours.

20. A process for purifying limit dextrinase from a grain according to claim 1, substantially as hereinbefore described.