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4.3.3 Construction of Amylase/Pullulanase Double Clone and its Effect on Starch Utilization.
The 3.9 kb EcoR I fragment from pPTl2
(K ■pneumoniae amylase) was ligated into the EcoR I site of pPT77 in both orientations, thus making amylase/
pullulanase double clones pPT99 and pPTlOO (Figure 4.14). The plasmids were then tested for their ability to
utilize starch and pullulan (Table 4.7) on solid medium. From the results it can be concluded that both activities function normally and that the orientation of the amylase
gene is irrelevant. It is of note that HW87/pPT12 grows
on amylose better than on starch presumably because it
has no a - 1 , 6 linkages to contend with. Also it is
interesting that the double clones do not show this
difference, presumably because the a - 1 , 6 can be
hydrolysed by pullulanase. To determine if the double
clone utilizes starch more efficiently than the amylase clone alone, the doubling times of HW87/pPTl2 and
HW87/pPT99 grown on starch minimal medium were
determined. Figure 4.15 shows the comparison of these
two clones and it can be concluded that the pullulanase gene does not make any significant difference to the growth rate on starch, in fact the double clone has a slightly longer doubling time.
Table 4.7
Growth Characteristics of HW87/pPT99 and HW87/pPTl00 and Controls.
Strain Carbon Source 48hr s 72hrs 120hrs
HW8 7/pPTl2 amylose + + + + + + HW87/pPTl2 starch - + + + + HW87/pPTl2 pullulan - - - HW87/pPTl4 amylose - - - HW87/pPTl4 starch - - - HW87/pPTl4 pullulan + + + + + + + + HW87/pPT99 amylose + + + + + + HW87/pPT99 starch + + + + + + HW87/pPT99 pullulan - + + + +
The medium was M9 + leu containing either amylose, starch or pullulan. - (no significant growth) to +++
(good growth). HW87/pPTl00 gave the same response as
1 1 4 Eco R1 Bgl II
i
Eco R1 DIGESTION Eco R1EcoR1 DIGESTION, ISOLATION OF 3-9kb AMYLASE FRAGMENT
LI GATI ON
Figure 4.14
Contruction of pPT99 and pPTlOO; K.pneumoniae Pullulanase/ Amylase Double Clone.
All manipulations were carried out as described in Chapter
2. Essentially, the 3.9 kb amylase fragment from pPTl2
(open box) was cloned into EcoR I cut pPT77 (subcloned pullulanase fragment, filled box) in both orientations.
1 1 5
a ) H W ff f r t a P I lZ ( k v w t l i o n S t a r c h b i H T B ^pP T & 9 G r o w t h o n S t a r c h 0.7 0.7 a .« A a.a / 1.5 0.5- 1.4- / 1,4 / 8" / 6 " □ / d / Q / d / d /a u / 1.2 / a.a- / /& / f A d . t . 1 8 4 m ln / u d . t . 1 9 0 m l n / / a 60 120 100 240 300 560 430 M | 60 130 160 240 300 360 420 T im e ( m i n ) T im e ( m i n ) Figure 4.15Comparative Growth Rates of H W 8 7 / p P T 1 2 a n d H W 8 7 / p P T 9 9 .
0.5 ml of an overnight culture grown in M9 + starch + leu + Cb was inoculated into 50 ml of the same medium in a
500 ml baffle flask and the OD was followed. Each
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D i s c u s s i o n .
The K.pneumoniae pullulanase gene has been cloned
and expressed in E .coli. Strains carrying pPTl4 produce
a protein of the same molecular weight as purified
pullulanase, can degrade pullulan to maltotriose and can
utilize pullulan as sole carbon source. Extracts made
from malQ strains still retain the ability to degrade maltotriose to maltose and glucose, similar to the result obtained with the K ■pneumoniae amylase (Chapter 2).
Strains containing the pullulanase gene show some very interesting growth characteristics:
a) Insertions over a large area of the insert have an effect on pullulan utilization in that they either lead to a Pul- phenotype, a slow growth phenotype or have no effect on growth on
pullulan.
b) Subcloning of the pullulanase structural gene similarly affect pullulan utilization, in that all the subclones exhibit a slow growth
phenotype on pullulan.
c) In a lamB background the parental plasmid can still grow well on pullulan, whereas the
subclones grow poorly. This contradicts the
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a-amylase and K .pneumoniae maltohexaose
producing amylase where the lamB gene product was essential for growth on starch (Chapters 3 and Chapter 5).
d) In a wild type background strains carrying the subcloned pullulanase plasmids (pPT76, pPT78 and pPT79) grow better than when in a lamB background, but still not as well as the parental plasmid (pPTl4).
e) In a low copy number background the parental plasmid confers pullulan utilization ability, but the subclones do not.
These observations suggest that pullulan is
utilized in a different way to starch. Clearly the
growth is lamB-independent, suggesting that the pullulan is broken down extra-cellularly, the maltotriose produced would then be transported via the outer membrane porin
proteins. It also seems likely that the parental plasmid
expresses another gene product or products which function
in the utilization of pullulan. It is tempting to
speculate that this may be in the form of a pullulan
permease. However, as the pullulanase is exported into
the medium in its K ■pneumoniae host (Wallenfels et a l .,
1966) this is unlikely. One could also envisage a
mechanism similar to that of hemolysin, where other gene products are thought to function in its export across the outer membrane (Goebel and Hedgpeth, 1983), however
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pullulanase was not detected in the culture medium in significant amounts with any of the pullulanase clones. However, it has been shown that pullulanase can be either membrane bound or extra-cellular depending on the growth conditions (Wallenfels et a l ., 1966; Hope and Dean, 1974,
1975). As specific culture effects have not been
assessed, the possibility that the parental plasmid
contains information which targets the pullulanase in the outer membrane in such a way that it can act on the
substrate can not be ruled out. The fact that
pullulanase is a lipoprotein (see section 4.1 and below) and is processed and modified correctly in E .coli
(Pugsley et a l ., 1986), but not correctly localised, suggests that at least one other gene product which is absent in E .coli is required for correct localization and
secretion. The large area (=8 kb) in which Bgl II
insertions affect growth on pullulan of strains carrying these plasmids suggests that more than one gene product
may be involved. A candidate for at least one gene
product which may be involved is the the 94,000
protein which was expressed in maxicells (section 4.2.3).
The pullulanase gene from K .pneumoniae has been cloned by two other groups (Takizawa and Murooka, 1985;
Michaelis et a l ., 1985: see section 4.1). It is
interesting that these two clones show strikingly
different restriction maps. When comparing the
restriction map of pPTl4 to these clones it shows
greatest homology with the clone isolated by Takizawa and
cloned pullulanase fragments have been cloned on
relatively small fragments and could not contain all of
the region which affects growth in pPT14. Michaelis et
a l . , 1985 have shown that their pullulanase clone does not allow growth on pullulan in a malT+ background even when preinduced on maltose, but in a malTC background it
can grow, albeit at a slow rate. This suggests that
there is a problem with induction of the pullulanase and the maltose maltodextrin transport system, presumably because the pullulanase can not reach the substrate and thus produce maltotriose which would induce the system. This is similar to the findings in this study when comparing the effect on growth on pullulan of the parental plasmid and the subclones or the insertions. Michaelis et a l ., 1985 has also shown that pullulanase expressed in E .coli is loosely associated with the outer membrane and that at least a small fraction of the fully induced pullulanase expressed in E .coli is accessible to the subtrate.
Taking all this information into consideration it is possible to put forward an explanation for the
pullulan utilization data from this study. On the
assumption that pullulan can not gain access to the periplasm through the lamB porin (this is unknown, but the results above suggest this) then the pullulanase can not get access to its substrate without being either correctly localised in the outer membrane or secreted
into the medium. That the strain carrying the parental
i 2 0
into contact with the substrate, possibly because the pullulanase is correctly orientated in the outer
membrane. In the case of the strains carrying the
subclones and if they are analogous to the clone used by Michaelis et al ■ , 1985, then the enzyme will be
incorrectly orientated in the inner and outer membranes
as well as the periplasm. The slow growth phenotype
could be explained by the over expression of the
subclones causing correct localization of a small number of molecules or by causing the membrane to become leaky (thus explaining the Pul- phenotype in a low copy number
background). It is also possible that the lamB porin may
transport pullulan, but ineffectually. Obviously a lot
more work is needed to determine the molecular events which occur within the cell during growth on pullulan. There are a number of interesting questions which remain
unanswered. Can pullulan gain access to the periplasm in
E . coli ? Is the 94,000 protein involved in export/
localisation of pullulanase? Is it possible to improve
the growth rate of strains carrying the pullulanase subclones or insertions by "complimenting" them with
genes of the putative export apparatus? Is the
pullulanase protein, expressed in strains carrying pPTl4, found in the outer membrane, and is the active site of the enzyme exposed to the extracellular medium?
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CHAPTER 5C L O N I N G A N D C H A R A C T E R I S A T I O N OF THE