Characterization of the Xylanolytic Activity of Celkulomonas

: Maximum production of xylanolytic enzymes by Cellulomonas CSI-17 required aerobic conditions, a temperature of 300C and a pH between 6.2 and 7.2. Under these conditions, maximum levels of xylanolytic activity were reached within 48 hr; addition of further mineral salts and yeast extract to the growth medium decreased the time to 24 hr. At pH values between 5.4 and 8.4 the xyhase was stableup to a temperature of 300C, for at least 48 hr after harvest; over the same temperature range, theb-xylosidase was stable only at the higher end of this pH range. The culture fltrate was similar to the growing cells in its ability to hydrolyse xylan whilst the growing cells metabolized the bulk of the hydrolysis products,. the latter accumuIated as soluble reducing sugars when the culture fdtrate was used as the enzyme source.

100% saturation; (c) either soluble or insoluble xylan replaced the mixed product in some media; standard medium containing 0.5% xylan was prepared, autoclaved. cooled and centrifuged at 20,000g for 30 min; the supernatant served as the soluble xylan medium (0.34% carbohydrate); the residue was suspended in fresh medium to yield the insoluble xylan medium (0.4% carbohydrate); both media were resterilized; (d) the cellular fraction of the harvested cultures was not homogenized prior to assay of xylanolytic activity; the cells. were collected by centrifugation, washed and suspended in buffer in the standard manner and used as an intact cell preparation.

Viable Cell Counts.
The culture was diluted 1 in lo7 by serial dilution. 0.1 ml of each dilution was spread on nutrient agar plates in duplicate and incubated at 30°C until single colonies appeared. Viable cells were determined by counting the number of colonies on plates containing 50 to 200 colonies and taking the average value.

Assay Procedures.
Xylanase, 8-xylosidase, reducing sugar and total carbohydrate were assayed as described by Peiris.8 All enzyme activities, including those in the cellular fraction, are expressed in milliunits (mu)/ml of culture, where one International Unit releases one pmole of productlmin. Reducing sugar (RS) and total carbohydrate (TC) are expressed in pglml: the percentage hydrolysis was calculated as the (RCITC) x 100.

3.1
Effect of Growth Parameters on Xylanolytic Activity 3.1.1 pH. When the Cellulomonas strain was grown for two days in culture media buffered between pH 5.8 and 7.2, maximum levels of both xylanase and B-xylosidase activity were recorded at pH 7.1 ( Table I  ) . The enzyme levels were, however, virtually constant between pH 6.2 and 7.2, the variation being considered f o be within the limits of accuracy of the enzymic assays. The xylanase activity was less than that recorded in a standard (unbuffered) culture. Other growth parameters were tested in unbuffered cultures, initially adjusted to pH 7.2.

Tempemtare.
When the temperature was varied between 230C and 400C, maximum enzyme activity at the end of the first day was recorded in the culture grown at 35OC (Table 2). However, at the end ofthe second day, maximum xylanase activity was recorded in the culture grown at 300C; similar levels of 8-xylosidase activities were recorded in the cultures grown at 230C and 300C1 . . .

Dissolved Oxygen Tension (DOT).
Growth and enzymic activity was very low in cultures grown under anaerobic conditions (DOT 0% saturation, maintained by bubbling sterile nitrogen gas into the medium) (Table 3). Although high aeration (DOT 60-loo%, maintained by bubbling sterile air into the medium) promoted enhanced growth, xylanase production was maximized at moderate aeration (DOT 10-30% saturation, maintained by regulating the inflow of sterile nitrogen gas and air into the medium). Moderate aeration probably is comparable to standard shake flask conditions. The differences in 8xylosidase production at high and moderate aeration were minimal.

Mineral Salts and Yeast Extract.
Supplementation of the culture medium with a three-fold increase in the yeast extract and all the Dubos mineral salts had a marked effect on the rate of production of the xylanolytic enzymes, which were increased approximately three-fold over their levels in the culture containing the standard medium by the end of the first day ( Table 4). By the end of the second day, however, the activities in the standard culture had virtually equalized with those in the supplemented culture. Choudhury,lfound that increased concentrations of Dubos salts and yeast extract in a growth medium cogtaining 2% pretreated bagasse enhanced the viable count and the hydrolytic activity of CS1-17 cultures. They suggested that the increased activity was a direct result of the increased biomass. In the current study, where the growth substrate was 0.5% xylan, the effect of the salts and yeast extract on the rate of production of xylanolytic activity over the first day appeared not to be dependent on increased biomass but rather a direct effect on enzyme production per unit cell number.
It appears that, the addition of yeast extract and Dubos mineral salts to the growth medium had been effective in improving xylanolytic enzyme production by CS1-17 beyond that obtained in standard cultures. The variation of temperature, pH and DOT virtually had no effect on the enzyme production.
Under all the growth conditions tested, xylanase activity (at its maximum levels) was mainly present in the culture filtrate (extracellular fluid), whilst the Bxylosidase remained associated with the homogenized cells.

P-Xylosidase Location
It was conceivable that the association of the b-xylosidase in the cellular fraction of the cultures was the result of its adsorption to unutilized insoluble xylan substrate present in this fraction. In order to eliminate this possibility, ar, experiment was conducted where soluble xylan replaced the mixture of soluble and insoluble xylanas the growth substrate. After growth for two days by the standard procedure, the culture was fractionated into its cellular and extracellular (culture filtrate) fractions. The cells were washed twice with saline, resuspended in 25ml of dilute (1: 10) McIlvaine's buffer2 and a sample (intact cells) taken for assay. The remainder was homogenized according to the standard procedure and a sample taken for assay. The remainder of the homogenate was centrifuged at 17,000g for 15 min and the supernatant was designated the intracellular fraction. The pellet, after resuspension in lorn1 of dilute McIlvaine's buffer, was designated the cell wall fraction. The intact cells, homogenized cells, the intracellular fraction, the wall fraction and the culture filtrate were all assayed for 8-xylosidase activity. Results (Table 5) were all corrected for volume changes and in common with other results, are reported in rn units/rnl of original culture.
Only 2% of the activity was located in the extracellular fraction, the remainder being associated with the intact cells. 37% of the cellular activity was 'lost' during homogenization, presumably as a result of enzyme inactivation. 69% of the residual activity in the homogenized cells was associated with the wall fraction while 3 1 % was intracellular. The activity of the intact cells suggests that either the j-xylosidase is located on the outside of the cells or that the synthetic substrate used in this investigation readily passes into the cells and the nitrophenol product is readily excreted. The fact that the cell wall fraction possessed more than twice the activity of the intracellular fraction supports the concept of the enzyme's location on or in the cell 'wall.
As soluble xylan was the substrate, the results minimize the possibility that the location of the enzyme in the standard cellular preparations (containing homogenized cells plus unutilized insoluble xylan) is due to its adsorption on to the insoluble xylan.

Enzyme Stability
This study was part of an overall aim to maximize the ability of preparations from Cellulomonas cultures to hydrolyse pretreated bagasse. It was therefore necessary to ensure that the enzymes, including the xylanolytic enzymes, were stable after harvest. The enzymes tested were the B-xylosidase of intact cell preparations and the extracellular xylanase of a culture of CS1-17 which had been grown for two days on 0.5% xylan under standard conditions.
In order to test the effect of pH on storage stability, portions of the culture filtrate and intact cell preparations were adjusted to specified pH values by the addition of dilute (1:lO) McIlvaine's buffer.2 These were then stored at specified temperatures and the extracellular xylanase and cellular 8-xylosidase activities Initial pH values had been adjusted to 5.0, 6.0, 7.0 and 8.0. During storage, however, pH changes occurred, presumably as a result of certain metabolic reactions which had not been inhibited by 0.2% azide. In all cases mean values between initial and final pH are recorded.
The results, recorded in Table 6, revealed that, at least for two days, the extracellular xylanase was stable at pH values between 5.4 and 8.4 and temperatures up to and including 300C. At 42OC, it was unstable a t all pH values tested and lost at least 50% of its initial activity within one day.
The stability of the cellular 8-xylosidase was pH and temperature dependent. At all pH values tested, it was relatively stable at 40C, but at likely process temperatures (250C, 300C and 420C) it lost more than 50% of its activity during storage for two days at pH 5.3 and 6.1. At the higher pH values tested (6.9 and 7.4) it retained at least 70 0/, of its activity at 250C and 3CoC and 50% at 420C. This suggests that to' maintain this enzyme in an industrial process the pH should not be allowed to decllne below 6.9 (or perhaps a lower, untested, value between 6.1 and 6.9)

Extent of Hydrolysis of Xylan
In order to determine whether non-growing cultures of Cellulomonus were as effective as growing cultures in hydrolysing xylan, in vivo and in vitro tests were conducted. Both soluble and insoluble xylan were used as substrates for digestion in each case. The extent of hydrolysis during growth over two days under standard conditions was compared with' that obtained over a further two days after addition of fresh substrate.
Each in vitro digest contained 15ml of enzyme (culture filtrate from the standard culture) plus 15ml of fresh xylan medium and 0.2% sodium azide. Incubation was carried out at 400C. Zero time samples were prepared by mixing enzyme which had been boiled for half an hour with an equal volume of fresh xylan medium.
In the case of both the in vivo and the in vitro tests, the zero time samples and those collected after incubation for two days were boiled for 15 min and reducing sugar (RS) and tvtal carbohydrate (TC) measured. The percentage hydrolysis of the residual carbohydrate was calculated from the values for the two day samples, using the expression (RS/ TC) x 100. In addition, the difference between the initial and final TC values was considered to be metabolized andtherefore fully hydrolysed carbohydrate. Its percentage of the initial TC was added to the percentage hydrolysis of the residual carbohydrate (corrected for the fraction of the initial carbohydrate which it represented) to give the total percentage hydrolysis. The initial RS was comparatively high in the in vitro tests due to 'carry over' with the enzyme preparation. P. S. Peirzk, Pamela A. D. Rickard and Jan M. Daly The values for percentage hydrolysis can be considered as approximations only. Use of the DNS method for -measurement ofreducingequivalents was based o n , the fact that it is widely used for the determination of reducing sugars liberated by cc -amylase13 and by carboxymethylcellulase.6 Since the reducing power (on a molar basis) of an oligomeric series increases with chain length, falsely high values are given for percentage hydrolysis when dimers and oligomers are present in the digest. The discrepancy does however decrease with chain length; this is evident when the ratio (1 .O: 1.4) of reducing powers of glucose and maltose5 is compared with the ratio of (1 .O: 1.35) of reducing powers of maltose and maltoheptaose.~* The method therefore provides a relatively simple semi-quantitative means of comparing the abilities of different enzyme preparations to hydrolyse polysaccharides.
The results (Tables 7 and 8) reveal that neither the in vivo nor the in vitro system was capable of hydrolysing soluble or insoluble xylan completely. The total percentage hydrolysis values indicated that in vitro the cell free culture filtrate was just as effective as a living culture in hydrolysing about 75% of the soluble xylan. Only when the cell free system was obtained from a culture grown on insoluble xylan was there an apparent decrease (from 67% to 50%) in the ability of the culture filtrate to match that of the living culture to hydrolyse insoluble xyIan. (This may perhaps indicate that a factor (or factors) required for effective hydrolysis of insoluble xylan remained associated with the residual insoluble xylan of the culture; this was a significant proportion of the initial carbohydrate and was discarded when the cell free culture filtrate was separated and used as the enzyme source). Minimization of carbohydrate metabolism in vztro resulted in the products of hydrolysis accumulatihg as soluble reducing sugars.
A further experiment was undertaken to determine whether inclusion of cells in the in vitro enzyme digest would improve the rate and extent of soluble xylan hydrolysis. A standard culture of CS1-17, grown on soluble xylan, was divided into two portions; one was centrifuged and the cell-free culture filtrate collected, whilst th-e other was left untreated. Both preparations served as enzyme preparations for in vitro digestion of a soluble xylan. In contrast to the previous test, the reaction mixture was buffered at pH 7.0 to ensure maximum enzyme stability (see above) and enzyme activity.loEnzyme stability was also improved by incubating the digests at 300C. rather than at the optimum (400C) for enzyme activity10 as in the previous test.
Flasks containing 40ml of soluble xylan, 40ml of McIlvaine's buffer (pH 7.0), 40 ml of enzyme and 0.2% sodium azide, were incubated at 30aC for two days. Samples, taken at various time intervals, were boiled for 15 min and the reducingsugar (RS) and the total carbohydrate (TC) determined (after removal of cells by centrifugation, where appropriate). Controls were prepared using enzyme sources which had been boiled previously for Ihr before mixing them with soluble xylan solution and buffer in the ratio 1 : 1 : 1. The controls were assayed in the same manner as the samples. In all cases, the approximate &gree of polymerization (DP) was calculated by dividing the TC by the RS; the same limitations in accuracy apply to calculation of DP as to calculation of percentage hydrolysis (see above).
Results are recorded in Table 9. Their control values reveal that the introduction of relatively low molecular weight fragments with the enzyme preparations made interpretation difficult. They do demonstrate however that an initial rapid rate of hydrolysis, detected even at zero time and continuing over 5 hiwas followed by minimal further activity. Neither digest was capable of completely hydrolysing the soluble xylan; even at the end of two days, the DP was still 1.5 (67% hydrolysis) in both cases.
Addition of fresh xylan at 5 hr to the culture filtrate resulted in it too being hydrolysed to the same extent as the original material. This indicated that the cessation of activity was not due to enzyme inactivation but rather to resistance ofthe substrate to complete hydrolysis by the enzyme preparation. A possible explanation is that CS1-17 produces xylanolytic activity which is inversely proportional to chain length and which fails to hydrolyse the low molecular weight xylose oligomers.
The investigation suggested that cell-free preparations of Cellulomonas CSl-17 are similar to growing cultures of the strain in their ability t o hydrolyse xylan. In comparsion to the in vivo conditions, where the products were metabolized, relatively high levels of reducing sugars accumulated in the soluble fraction of the in vitro digests. This suggests that the latter system is one which is appropriate for saccharification of the hemicellulose fraction of lignocellulose.   Although the initial in vitro rate was rapid, the reaction ceased before complete hydrolysis was reached. Even when the digestion was carried out under conditions which maximized enzyme stability, only about 67% hydrolysis was attained by the cell-free extracts. As the 8-xylosidase activity is located in the Cellulornonas cells and since this enzyme could be necessary for hydrolysis of the oligomers released by xylanase activity, it was considered that inclusion of cells in the digest may improve the degree of hydrolysis; this proved not to be the case.
Investigations are in progress to identify the products of enzymic digestion of xylan and to improve conditions for their further hydrolysis.